(en)The present invention provides novel polynucleotides encoding plant prenyl protease polypeptides, fragments and homologs thereof. Also provided are vectors, host cells, and recombinant methods for producing said polypeptides. The invention further provides novel polynucleotides encoding plant promoters, polypeptides, fragments and homologs thereof. The invention additionally provides methods of constructing transgenic plants that have altered levels of prenyl protease polynucleotides and polypeptides. Methods for identifying prenyl protease enzymes substrates and inhibitors are also provided. The invention further relates to methods of applying these novel plant polypeptides to the identification, prevention, and/or conferment of resistance to various plant diseases and/or disorders, particularly drought resistance.

1.ApplicationNumber: US-87922607-A
1.PublishNumber: US-2008072350-A1
2.Date Publish: 20080320
3.Inventor: SARRIA-MILLAN RODRIGO
HENKES STEFAN
ALLEN DAMIAN
DA COSTA E SILVA OSWALDO
CHEN RUOYING
WAN JIANGXIN
HUANG YAFAN
MELO DELINA M.
KUZMA MONIKA M.
GILLEY SAMPLE ANGELA P.

4.Inventor Harmonized: SARRIA-MILLAN RODRIGO(US)
HENKES STEFAN(DE)
ALLEN DAMIAN(US)
DA COSTA E SILVA OSWALDO(DE)
CHEN RUOYING(US)
WAN JIANGXIN(CA)
HUANG YAFAN(CA)
MELO DELINA M(CA)
KUZMA MONIKA M(CA)
GILLEY SAMPLE ANGELA P(CA)

5.Country: US
6.Claims:
(en)The present invention provides novel polynucleotides encoding plant prenyl protease polypeptides, fragments and homologs thereof. Also provided are vectors, host cells, and recombinant methods for producing said polypeptides. The invention further provides novel polynucleotides encoding plant promoters, polypeptides, fragments and homologs thereof. The invention additionally provides methods of constructing transgenic plants that have altered levels of prenyl protease polynucleotides and polypeptides. Methods for identifying prenyl protease enzymes substrates and inhibitors are also provided. The invention further relates to methods of applying these novel plant polypeptides to the identification, prevention, and/or conferment of resistance to various plant diseases and/or disorders, particularly drought resistance.

7.Description:
(en)RELATED APPLICATIONS
This application is a continuation-in-part of U.S. application Ser. No. 10/362,902 and a continuation-in-part of U.S. application Ser. No. 10/210,760. U.S. application Ser. No. 10/362,902 is a national stage application (under 37 U.S.C. 371) of PCT/US01/26854 filed Aug. 27, 2001, which claims benefit of U.S. provisional application 60/227,794, filed Aug. 25, 2000. U.S. application Ser. No. 10/210,760, filed Aug. 1, 2002, claims benefit of U.S. provisional application 60/309,396, filed Aug. 1, 2001, and U.S. provisional application 60/337,084, filed Dec. 4, 2001. The disclosure of each of the above-mentioned applications is incorporated herein by reference in its entirety.


SUBMISSION ON COMPACT DISC
The contents of the following submission on compact discs is incorporated herein by reference in its entirety: two copies of the Sequence Listing (COPY 1 and COPY 2) and a computer readable form copy of the Sequence Listing (CRF COPY), all on compact disc, each containing: file name: Final Sequence Listing-15342-00001; date recorded: Jul. 16, 2007; size: 316 KB.
FIELD OF THE INVENTION
The present invention provides novel polynucleotides encoding plant prenyl protease polypeptides, and fragments and homologs thereof. Also provided are vectors, host cells, and recombinant methods for producing said polypeptides. Also included are transgenic plants expressing the novel polynucleotides. The present invention also includes transgenic plant cells, tissues and plants having novel phenotypes resulting from the expression of these polynucleotides in either the sense or antisense orientation. The invention further relates to methods of applying these novel plant polypeptides to the identification, prevention, and/or conferment of resistance, including resistance to various plant diseases and/or disorders, particularly drought resistance.
BACKGROUND OF THE INVENTION
Drought is one of the most limiting factors in plant growth and productivity. Crop and yield losses due to drought spells in crops such as soybeans, corn, rice and cotton represent a significant economic problem. Moreover, drought is also responsible for food shortages in many countries worldwide. Developing crops tolerant to drought is a strategy that has potential to alleviate some of these adverse situations.
Traditional plant breeding strategies to develop new lines of plants that exhibit tolerance to drought are relatively slow and require specific tolerant lines for crossing with the desired commercial lines. Limited germplasm resources for drought tolerance and incompatibility in crosses between distantly related plant species therefore represent significant problems encountered in conventional breeding. In contrast, plant genetic transformation and availability of useful genes subjected to specific expression patterns allow one to generate drought-tolerant plants using transgenic approaches.
Plants are exposed during their entire life cycle to conditions of reduced environmental water content. Most plants have evolved strategies to protect themselves against these conditions of desiccation. However, if the severity and duration of the drought conditions are extensive, the effects on plant development, growth and yield of most crop plants are profound.
The physiology of a drought-stressed plant is dramatically altered as compared with a plant grown under normal conditions. Most of the changes and their causes remain uncharacterized. Abscisic acid (ABA) plays a central role in mediating the processes between desiccation perception and cellular changes. ABA increases readily upon the onset of cell desiccation and exogenously applied ABA mimics many of the responses induced by water-stress. An increase in ABA causes the closure of stomata, thereby decreasing water loss through transpiration.
The identification of genes that transduce ABA into a cellular response opens the possibility of exploiting these regulators to enhance desiccation tolerance in crop species. In principle, these ABA signaling genes can be coupled with the appropriate controlling elements to allow optimal plant growth, development and productivity. Thus, not only would these genes allow the genetic tailoring of crops to withstand transitory environmental stresses, but they should also broaden the environments where traditional crops can be grown.
The regulation of protein phosphorylation by kinases and phosphatases is accepted as a universal mechanism of cellular control (Cohen 1992, Trends Biochem. Sci. 17: 408-413), and Ca 2+ and calmodulin signals are frequently transduced via Ca 2+ and calmodulin-dependent kinases and phosphatases (Roberts & Harmon 1992, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43: 375-414.). Okadaic acid, a protein phosphatase inhibitor, has been found to affect both gibberellic (GA) and absisic acid (ABA) pathways (Kuo et al. 1996, Plant Cell. 8: 259-269). Although the molecular basis of GA and ABA signal transduction remains poorly understood, it seems well established that the two phytohormones are involved in overall regulatory processes in seed development (e.g. Ritchie & Gilroy 1998, Plant Physiol. 116: 765-776; Arenas-Huertero et al. 2000, Genes Dev. 14: 2085-2096). Likewise, the plant hormones ethylene (e.g. Zhou et al. 1998, Proc. Natl. Acad. Sci. USA 95: 10294-10299; Beaudoin et al. 2000, Plant Cell 2000: 1103-1115) and auxin (e.g. Colon-Carmona et al. 2000, Plant Physiol. 124:1728-1738) are involved in controlling plant development as well.
Protein farnesylation, the addition of a C-terminal, 15-carbon chain to proteins and subsequent processing, has been identified as being crucial for the mediating role of ABA in the desiccation-signal transduction chain. In short, protein farnesylation is required for ABA-induced stomata closure, thus for control of water loss.
Protein farnesylation is a three-step enzymatic reaction as shown in FIG. 1 . Potentially, each of these steps could represent a target for genetic manipulation of the prenylation process to generate a desired phenotype such as stress tolerance.
The drought-tolerant phenotype of the era1 Arabidopsis mutant is due to a null mutation in the β-subunit of the enzyme farnesyl transferase (FTase), the first enzyme in the protein farnesylation pathway. Farnesyl transferase is a heterodimeric enzyme that provides the specific addition of a farnesyl pyrophosphate moiety onto the substrate target sequence. The target sequence is defined as a sequence of four amino acids which are present at the carboxy terminus of the protein and is referred to as a CaaX motif in which the “C” is cysteine, “a” is any aliphatic amino acid and “X” is any amino acid. The α subunit is common with a second prenylation enzyme, geranylgeranyl transferase, that has a different β subunit and adds a geranylgeranyl isoprenyl pyrophosphate moiety to the target sequence.
In plants, prenylation has been linked to cell cycle control, meristem development, and phytohormone signal transduction, however, few details of the role of prenylation, the substrate proteins or the extent to which the plant system will be analogous to the mammalian and yeast systems are known. The most characterized substrates for CaaX modification are the Ras and a-factor proteins of yeast. Although there are three steps to complete protein maturation, abolition or modification of any one step does not necessarily result in cessation of target biological activities. Ras function is attenuated if the −aaX tripeptide is not cleaved but not abolished and some proteins retain the −aaX tripeptide after farnesylation.
In Arabidopsis , more than 600 proteins contain a CaaX motif, suggesting a role for the post-translational modification by prenylation in numerous cellular processes. In Arabidopsis , it has been demonstrated that the loss-of-function of the β-subunit of farnesyl transferase will result in an ABA-hypersensitive phenotype. Although it is still not clear why plants lacking the functional β-subunit of farnesyl transferase become more sensitive to ABA, it clearly suggests that protein prenylation is involved in regulation of the homeostasis of ABA sensitivity. The balance of ABA cellular responses, whether more sensitive or less sensitive to ABA, is possibly regulated by the relative activities of prenylated proteins. The changes in Arabidopsis prenyl protease expression and gene activity may affect the activity of two pools of genes, one pool acting as positive regulators (pool A) and the second pool (pool B) as negative regulators, which require prenylation in order to function properly. Pool A may contain genes that can promote ABA sensitivity, and pool B genes that may reduce ABA sensitivity. The homeostasis of ABA sensitivity may therefore be governed by the ratio of activity of pool A to pool B. For example, in the case of up-regulation of Arabidopsis prenyl protease in Arabidopsis , the activity ratio of pool A over pool B may be increased due to differences in substrate affinity of pool A proteins toward Arabidopsis prenyl protease, thus the homeostasis of ABA sensitivity is changed, and the Arabidopsis prenyl protease over-expression plants are more sensitive to ABA.
There is a need in the art to identify new plant genes encoding these protein farnesylation enzymes as another opportunity to generate plants tolerant to environmental stress, such as drought.
BRIEF SUMMARY OF THE INVENTION
The present invention provides novel polynucleotides encoding prenyl protease polypeptides or active fragments thereof from Physcomitrella patens, Arabidopsis thaliana , rapeseed ( Brassica napus ), soybeans ( Glycine max ), and corn ( Zea mays ).
The present invention fulfills a need in the art, in part, by providing the isolated polynucleotide and polypeptide sequences of plant-derived PrPases from five species, namely, the PrPase sequences from moss ( Physcomitrella patens ; PpPrPase1), three PrPase sequences from Arabidopsis thaliana (AtPrPase1, AtPrPase2, and AtCPP), two PrPase from soybeans ( Glycine max ; GmPrPase2 and GmCPP), one PrPase from corn ( Zea mays ; ZmPrPase2), and one from rapeseed ( Brassica napus ; BnCPP). The invention also provides partial PrPase sequences from soybean (GmPrPase1) and corn (ZmPrPase1).
In a first embodiment, an isolated or recombinant polynucleotide is provided which encodes a plant prenyl protease having the amino acid sequence of SEQ ID NO: 4 or a homolog of SEQ ID NO: 4 having prenyl protease activity and having at least 70% identity at the amino acid level to SEQ ID NO: 4, with the proviso that the polynucleotide as set forth in SEQ ID NO: 27 and the polynucleotide as set forth in SEQ ID NO: 29 are excluded.
In a further embodiment, the invention provides an isolated or recombinant polynucleotide wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of a polynucleotide as set forth in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23; a polynucleotide encoding the polypeptide as set forth in SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24; a polynucleotide encoding a polypeptide having at least 95% identity at the amino acid level to SEQ ID NO: 2, 4, 6, 15, 17, 22, or 24; a polynucleotide encoding a polypeptide having at least 99% identity at the amino acid level to SEQ ID NO: 8; a polynucleotide encoding a polypeptide having at least 96% identity at the amino acid level to SEQ ID NO: 12; a polynucleotide encoding a polypeptide having at least 85% identity at the amino acid level to SEQ ID NO: 19; and a polynucleotide complementary to any of these sequences.
The nucleic acid can be, for example, a genomic DNA fragment, a cDNA molecule, or is naturally occurring. In one aspect, the invention provides a nucleic acid that includes the sequence of SEQ ID NO: 84, 86, 88 or 90. The invention also provides a nucleic acid sequence that is complementary to the nucleic acid sequence of SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 18, for example, SEQ ID NO: 9, 13, or 20.
A further embodiment is an isolated polypeptide molecule that comprises the sequence of SEQ ID NO: 85, 87, 89, or 91.
Further described are transformation vectors useful to transform plants, such as Physcomitrella, Arabidopsis , rapeseed, soybeans, and corn plants. Such vectors comprise one or more of the nucleic acid molecules described herein. The invention provides for plants and host cells transformed with a PrPase nucleic acid or a vector comprising a PrPase nucleic acid. For example, a vector may comprise at least one of the nucleic acid sequences of SEQ ID NO: 41, 42, or 52-69.
In further embodiments, the invention provides methods of using the polynucleotides and polypeptides of the invention to create transgenic plants with one or more desirable traits, which include, but are not limited to, enhanced plant defense, increased tolerance to stress, drought tolerance, salt tolerance, ultraviolet (uv) tolerance, enhanced flower development, delayed senescence, increased ABA sensitivity, terpene synthesis, increased formation of seed storage compounds (like oil, sugars and proteins), increased yield, increased productivity and increased biomass compared to a wild type plant, and the identification, prevention, and/or conferment of resistance to various plant diseases and/or disorders by introducing into one or more cells of a plant a compound that alters (e.g., increases or decreases) PrPase expression or activity in the plant. In one aspect, the compound is a PrPase nucleic acid or polypeptide. In one embodiment, the nucleic acid is an inhibitor of farnesylation. Alternatively, the compound is a PrPase double stranded RNA-inhibition hair-pin nucleic acid or PrPase antisense nucleic acid.
The present invention further provides a general method for engineering drought-tolerant plants, said method being generally applicable to all plants.
Moreover, the present invention provides the first results suggesting that modulation of PrPase gene expression in a plant directly correlates with increased drought tolerance as compared to untransformed control plants. The present invention also describes methods of engineering drought-tolerant rapeseed, soybeans, and corn plants strains generated through modulation of PrPase expression.
In a preferred embodiment, the invention provides a method of producing a transgenic plant comprising an isolated or recombinant nucleic acid encoding a prenyl protease wherein the plant has increased tolerance to an environmental stress as compared to a wild type variety of the plant, comprising transforming a plant cell with an expression vector comprising a polynucleotide sequence encoding a prenyl protease and generating from the plant cell the transgenic plant with increased tolerance to stress, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 4 or a homolog of SEQ ID NO: 4 having prenyl protease activity and having at least 70% identity at the amino acid level to SEQ ID NO: 4.
In another preferred embodiment, the transgenic plant produced by the above method has increased tolerance to an environmental stress selected from the group consisting of drought, high temperature, and low temperature.
Further provided is the promoter of the Arabidopsis USP gene. This promoter is expressed most strongly during seed developmental stages, i.e. it is a seed-specific promoter.
Further provided is the promoter of the Arabidopsis FTase gene. This promoter is expressed most strongly in guard-cells, i.e. it is a guard-cell specific promoter. In another aspect of this invention, the promoter of an Arabidopsis PrPase is described. This promoter is guard-cell specific and can be used to engineer traits such as drought tolerance and regulation of gas exchange in the plant.
Moreover, the present invention provides novel polynucleotides encoding plant PrPase polypeptides, including a substantially purified PrPase polypeptide, and fragments and homologs thereof. Also provided are vectors, host cells, and recombinant methods for producing said polypeptides.
The invention further provides a method for producing a PrPase polypeptide by providing a cell containing a PrPase nucleic acid, e.g., a vector that includes a PrPase nucleic acid, and culturing the cell under conditions sufficient to express the PrPase polypeptide encoded by the nucleic acid. The expressed PrPase polypeptide can then be recovered from the cell. Preferably, the cell produces little or no endogenous PrPase polypeptide. The cell can be, e.g., a prokaryotic cell or eukaryotic cell.
Another aspect of this invention provides yeast expression vectors used to produce large quantities of the Arabidopsis PrPase in yeast.
The invention also provides methods for more particularly refining the function of the polynucleotides and/or polypeptides of the present invention.
The invention is also directed to plants transformed with the polynucleotides of the invention, to seed and progeny thereof, and to methods of producing these transgenic plants.


BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS
FIG. 1 —Schematic representation of the protein farnesylation pathway. This figure identifies the known enzymes involved in the protein farnesylation pathway, in addition to their functional relationship. For illustration purposes, a prospective target protein is represented by a dotted line; while the solid line represents the 15-carbon atom chain added to the C-terminus of the target protein at the “CaaX” site. “C” is Cysteine, “a” is any aliphatic amino acid, and “X” is any amino acid. The 15-carbon chain is added to a conserved Cysteine (C) by the enzyme farnesyl transferase (Ftase). The last three amino acid residues (aaX) are cleaved by the enzyme prenyl protease (PrPase). Lastly, the modified Cysteine is methylated by a methylase to create the final active product of the protein farnesylation pathway.
FIG. 2 is a schematic representation of the vector constructs, A) pBI121-AtCPP, B) pBI121-antisense-AtCPP, and C) pBI121-HP-AtCPP.
FIG. 3 -Comparison of the Exon/Intron structure of the computer-predicted ORF of the Arabidopsis PrPase from genomic sequence of GenBank Accession No. AF007269 (SEQ ID NO: 30; labeled as Predicted Ara PrPase; predicted from Genefinder (P. Green and L. Hillier, National Center for Biotechnology Information (“NCBI”) website)) and experimentally demonstrated ORFs of the Arabidopsis PrPases (SEQ ID NO: 4 and 6; labeled as Ara PrPase1-2). The exons are numbered sequentially in both clones. Corresponding exons are placed below each other. The lines connecting the introns have no biological significance. Breaks were introduced in the sequences to make this figure.
FIG. 4A -N-Nucleic acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, default parameters for ClustalW 1.82) of GmCPP (SEQ ID NO: 18), GmPrPase2 (SEQ ID NO: 16), AtCPP (SEQ ID NO: 7), AtPrPase1 (SEQ ID NO: 3), AtPrPase2 (SEQ ID NO: 5), BnCPP (SEQ ID NO: 11), ZmPrPase2 (SEQ ID NO: 23), PpPrPase1 (SEQ ID NO: 1), and three other disclosed Arabidopsis PrPases sequences: AT4g01320 (SEQ ID NO: 27; GenBank Accession NO: AL161491), AF007269 (SEQ ID NO: 29), and AFC1 (SEQ ID NO: 25; GenBank Accession NO: AF353722). The consensus sequence (bottom line) is shown in SEQ ID NO: 84.
FIG. 5A -C—Amino acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, using default parameters) of GmCPP (SEQ ID NO: 19), GmPrPase2 (SEQ ID NO: 17), AtCPP (SEQ ID NO: 8), AtPrPase1 (SEQ ID NO: 4), AtPrPase2 (SEQ ID NO: 6), BnCPP (SEQ ID NO: 12), ZmPrPase2 (SEQ ID NO: 24), PpPrPase1 (SEQ ID NO: 2), three other disclosed Arabidopsis PrPases sequences: AT4g01320 (SEQ ID NO: 28; GenBank Accession NO: AL161491), AF007269 (SEQ ID NO: 30), and AFC1 (SEQ ID NO: 26; GenBank Accession NO: AF353722), and the sequence of the Saccharomyces cerevisiae PrPase (Swiss-Prot Accession No. P47154; SEQ ID NO: 31). The boxed area is the zinc metalloprotease motif (HEXXH). The consensus sequence (bottom line) is shown in SEQ ID NO: 85.
FIG. 6 is a comparison of nucleic acid sequence identities between the sequences of the invention (SEQ ID NOs: 1, 3, 5, 7, 11, 16, 18, 23), and three other disclosed Arabidopsis PrPases sequences (GenBank Accession NOs: AL161491 (AT4g01320; SEQ ID NO: 27), AF007269 (SEQ ID NO: 29), and AF353722 (AFC1; SEQ ID NO: 25)) as determined by EMBOSS Pairwise Alignment algorithms (Parameters: Blosum 62 matrix, 10.0 gap penalty, 0.1 extend penalty). FIG. 6A shows the comparison using the full-length sequences (including the 5′- and 3′-end non-coding sequences). FIG. 6B shows the comparison using the coding region of the sequences (excluding the 5′- and 3′-end non-coding sequences).
FIG. 7 —Amino acid sequence comparison as determined by EMBOSS Pairwise Alignment algorithms (Parameters: Blosum 62 matrix, 10.0 gap penalty, 0.1 extend penalty) of the PrPase polypeptides of the present invention (SEQ ID NOS: 2, 4, 6, 8, 12, 17, 19, 24), other disclosed Arabidopsis PrPases sequences (GenBank Accession NOs: AL161491 (AT4g01320; SEQ ID NO: 28), AF007269 (SEQ ID NO: 30), and AF353722 (AFC1; SEQ ID NO: 26)), and sequence of the Saccharomyces cerevisiae PrPase (Swiss-Prot Accession No. P47154; SEQ ID NO: 31). The percent identity is shown with the percent similarity values in parenthesis.
FIG. 8 is an illustration showing the relative expression of AtCPP mRNA transcript (solid bars) and AtCPP protein levels (stippled bars) in several pBI121-AtCPP transgenic lines.
FIG. 9 is a histogram showing the percentage of lines which were categorized as ABA sensitive, moderately ABA sensitive or ABA insensitive. Seedlings were assessed on agar plates containing 1 μM ABA and scored at 21 days growth. Thirty-six lines of the pBI121-AtCPP over-expression construct were assessed at 21 days by leaf and seedling development. Thirty-two lines of the 35S-HP-AtCPP down-regulation construct were assessed at 21 days for leaf and seedling development. Each line was assessed by plating approximately 100 seeds per plate and the seedlings scored and recorded as the percent insensitive seedlings per plate. Each line was then expressed as a percent of wild type (Wt). Lines were categorized as sensitive (less than 1% of Wt) solid bars, intermediate (1-50% of Wt) diagonally lined or insensitive (greater than 50% of Wt) stippled, based on their relationship to Wt and the percentage of each category plotted as a histogram.
FIG. 10 is a photograph showing the response of wild type and a pRD29A-HP-AtCPP transgenic line to various concentrations of ABA in two week old seedlings.
FIG. 11 is a histogram showing the analysis of transgenic plants containing the pBI121-AtCPP over-expression construct (SEQ ID NO: 41). Water loss is shown per gram of shoot dry weight after four days of water stress treatment. Lines that are marked with a star are those which were strongly ABA sensitive. Lines marked with a triangle are moderately ABA sensitive. Bars represent means of eight replicates. Lines marked with a filled dot above the bar represents lines which were significantly different from control at a p=0.05 value.
FIG. 12 is a histogram showing seed yield in grams of transgenic Arabidopsis lines of pBI121-AtCPP grown under optimal water conditions.
FIG. 13 is a bar chart showing growth and yield of transgenic Arabidopsis lines of pBI121-AtCPP grown under optimal watering conditions plus a biotic stress condition. Yields as a % of wild type, rosette leaf number, rosette leaf fresh weight and shoot dry weight are plotted.
FIG. 14 are photographs showing 12 day old seedling growth of transgenic Arabidopsis lines of pBI121-AtCPP (35S AtCPP) grown on agar plates. Changes to root growth visible (toluidin blue).
FIG. 15 is a bar chart showing growth of transgenic Arabidopsis lines of pRD29A-HP-AtCPP grown under optimal watering conditions. Rosette leaf number, rosette leaf dry weight and shoot dry weight are plotted.
FIG. 16A -K—Nucleic acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, using default parameters) of the PrPase sequences of the invention from dicot plants: GmCPP (SEQ ID NO: 18), GmPrPase2 (SEQ ID NO: 16), AtCPP (SEQ ID NO: 7), AtPrPase1 (SEQ ID NO: 3), AtPrPase2 (SEQ ID NO: 5), BnCPP (SEQ ID NO: 11); and three other disclosed dicot PrPases sequences: GenBank Accession NOs: AL161491 (AT4g01320; SEQ ID NO: 27), AF007269 (SEQ ID NO: 29), and AF353722 (AFC1; SEQ ID NO: 25). The consensus sequence (bottom line) is shown in SEQ ID NO: 86.
FIG. 17A -B—Amino acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, using default parameters) of the PrPase sequences of the invention from dicot plants: GmCPP (SEQ ID NO: 19), GmPrPase2 (SEQ ID NO: 17), AtCPP (SEQ ID NO: 8), AtPrPase1 (SEQ ID NO: 4), AtPrPase2 (SEQ ID NO: 6), BnCPP (SEQ ID NO: 12); and three other disclosed dicot PrPases sequences: GenBank Accession NOs: AL161491 (AT4g01320; SEQ ID NO: 28), AF007269 (SEQ ID NO: 30), and AF353722 (AFC1; SEQ ID NO: 26). The consensus sequence (bottom line) is shown in SEQ ID NO: 87.
FIG. 18A -H—Nucleic acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, using default parameters) of the Arabidopsis PrPase sequences of the invention: AtCPP (SEQ ID NO: 7), AtPrPase1 (SEQ ID NO: 3), AtPrPase2 (SEQ ID NO: 5); and three other disclosed Arabidopsis PrPases sequences: GenBank Accession NOs: AL161491 (AT4g01320; SEQ ID NO: 27), AF007269 (SEQ ID NO: 29), and AF353722 (AFC1; SEQ ID NO: 25). The consensus sequence (bottom line) is shown as SEQ ID NO: 88.
FIG. 19A -B—Amino acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, using default parameters) of the Arabidopsis PrPase sequences of the invention: AtCPP (SEQ ID NO: 8), AtPrPase1 (SEQ ID NO: 4), AtPrPase2 (SEQ ID NO: 6); and other disclosed Arabidopsis PrPases sequences: GenBank Accession NOs: AL161491 (AT4g01320; SEQ ID NO: 28), AF007269 (SEQ ID NO: 30), and AF353722 (AFC1; SEQ ID NO: 26). The consensus sequence (bottom line) is shown in SEQ ID NO: 89.
FIG. 20A -B—Nucleic acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, using default parameters) of the soybean PrPase sequences of the invention: GmCPP (SEQ ID NO: 18) and GmPrPase2 (SEQ ID NO: 16). The consensus sequence (bottom line) is shown in SEQ ID NO: 90.
FIG. 21 -Amino acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, using default parameters) of the soybean PrPase sequences of the invention: GmCPP (SEQ ID NO: 19) and GmPrPase2 (SEQ ID NO: 17). The consensus sequence (bottom line) is shown in SEQ ID NO: 91.


DETAILED DESCRIPTION OF THE INVENTION
The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included herein.
One aspect of this invention pertains to isolated nucleic acid molecules that encode PrPase polypeptides from Physcomitrella patens, Arabidopsis thaliana , soybeans, rapeseed, and corn. Moreover, this invention pertains to nucleic acid fragments originated from the clones mentioned above, as well as to other nucleic acid fragments from other organisms that can be isolated using the described nucleic acid fragments as probes in hybridization experiments.
As explained above, introduction of a PrPase into plants can improve tolerance to environmental stress. This invention also describes modulation of PrPase gene activity in a plant. The only condition necessary to realize either of these embodiments is the isolation of the corresponding PrPase genes from the target plants. The use of the described clones to isolate corresponding PrPase genes from other plants is something appreciated by someone skilled in the art.
Modulation of PrPase gene activity can be accomplished by reduction in PrPase, for example by: (a) antisense gene-expression repression, (b) targeted antibodies to PrPase, and (c) targeted, engineered promoter repression with for example zinc-finger derived transcription factors.
The present invention can make a significant contribution to the art by providing new strategies to engineer drought-tolerance in crop plants, especially the use of the previously unknown PrPase clones from plant origin. The polynucleotides and polypeptides of the present invention have uses which include conferring resistance to or modulating susceptibility to biotic and/or abiotic stresses such as heat, drought, and salt stress in plants.
In one embodiment, over-expression of a PrPase polypeptide of the present invention within a plant using a constitutive promoter (e.g., 35S, or other promoters disclosed herein), preferably not in the guard-cell, improves drought and salt tolerance in a plant.
In another embodiment, overexpression of a PrPase polypeptide of the present invention within a plant using a seed-specific promoter (e.g., unknown seed protein, USP, promoter) increases the amount of seed storage compounds.
Constitutive over-expression in plants of the farnesylation pathway, namely of a prenyl protease, may result in increased cell proliferation and increased plant growth. The polynucleotides and polypeptides of the present invention, including fragments thereof, have uses that include modulating plant growth, and potentially plant yield, preferably increasing plant growth.
The polynucleotides can also be used to express recombinant proteins for analysis, characterization and agronomic use, to express recombinant proteins to raise antibodies directed against polypeptides of the present invention, as markers for tissues in which the corresponding protein is expressed (e.g., preferentially, or non-preferentially), as hybridization markers on Southern gels, as genetic markers for breeding assistance, as RFLP markers, as markers for genotyping (varieties, etc,), and the encoded protein, can, at the very least, be used as a molecular weight marker.
The polynucleotides of the present invention are also useful as chromosome markers or tags (when labeled) to identify chromosomes, to map related gene positions within a chromosome, or as a comparative reference to endogenous DNA sequences of mutant plants to identify allelic variants, and/or spontaneous or biotic mutations.
The polynucleotides of the present invention are also useful for genetic fingerprinting, for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns for particular genes, to differentiate intron and/or exon boundaries, to identify splice and/or allelic variants, and as diagnostic tools for identification of developmental stages, disease states, and/or nutrient levels.
The present invention encompasses polynucleotides that hybridize to the polynucleotides of the present invention under either stringent or non-stringent conditions and their uses as described herein. Such hybidization may be used to identify orthologs, homologs, allelic variants, variants, and/or mutants of the polynucleotides of the present invention. Additionally, the polynucleotides of the present invention may be used to clone orthologs, homologs, alleleic variants, variants, and/or mutants of the polynucleotides of the present by using oligonucleotides directed to polynucleotide sequences of the present invention, and performing PCR on plant cell or tissue samples.
The present invention encompasses the identification of proteins, nucleic acids, or other molecules, that bind to polypeptides and polynucleotides of the present invention (for example, in a receptor-ligand interaction). The polynucleotides of the present invention can also be used in interaction trap assays (such as, for example, that described by Ozenberger and Young (Mol. Endocrinol., 9(10):1321-9, (1995); and Ann. NY Acad. Sci., 7; 766:279-81, (1995)).
Potential uses of polynucleotides and polypeptides of the present invention include nutrition (e.g., as an amino acid supplement), as a carbon source, as nitrogen source, as a carbohydrate source, modulating plant defense activity, modulating signal transduction, modulating metabolite transport (e.g., carbon, nitrogen fluxes, etc.), conferring abiotic stress tolerance and/or resistance (water, drought, cold, salt, etc.), conferring xenobiotic stress tolerance and/or resistance, and development control (for example, yield, flowering time, etc.).
The polynucleotide and polypeptides of the present invention are useful as probes for the identification and isolation of full-length cDNAs and/or genomic DNA which correspond to the polynucleotides of the present invention, as probes to hybridize and discover novel, related DNA sequences, as probes for positional cloning of these or related sequences, as probes to “subtract-out” known sequences in the process of discovering other novel polynucleotides, as probes to quantify gene expression, and as probes for microarrays.
In addition, polynucleotides and polypeptides of the present invention may comprise one, two, three, four, five, six, seven, eight, or more membrane domains.
Also, in preferred embodiments the present invention provides methods for further refining the biological function of the polynucleotides and/or polypeptides of the present invention.
Specifically, the invention provides methods for using the polynucleotides and polypeptides of the invention to identify orthologs, homologs, variants, and/or allelic variants of the invention. Also provided are methods of using the polynucleotides and polypeptides of the invention to identify the entire coding region of the molecules of the invention, non-coding regions of the molecules of the invention, regulatory sequences associated with the molecules of the invention, and secreted, mature, pro-, prepro- and other forms of the molecules of the invention.
In further preferred embodiments, methods are provided for evolving the polynucleotides and polypeptides of the present invention using molecular evolution to create and identify novel variants with desired structural, functional, and/or physical characteristics.
The present invention in further preferred embodiments provides for other experimental methods and procedures currently available to derive functional assignments. These procedures include but are not limited to spotting of clones on arrays, micro-array technology, PCR based methods and other procedures that could use sequence information from our clones to build a primer or a hybrid partner.
As used herein, the term “environmental stress” refers to any sub-optimal growing condition and includes, but is not limited to, sub-optimal conditions associated with salinity, drought, temperature, metal, chemical, pathogenic and oxidative stresses, or combinations thereof. In preferred embodiments, the environmental stress can be salinity, drought, or temperature, or combinations thereof, and in particular, can be high salinity, low water content or low temperature. It is also to be understood that as used in the specification and in the claims, “a” or “an” can mean one or more, depending upon the context in which it is used. Thus, for example, reference to “a cell” can mean that at least one cell can be utilized.
As also used herein, the term “nucleic acid” and “polynucleotide” refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. The term “nucleic acid molecule” is intended to also include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. These terms also encompass untranslated sequences located at both the 3′ and 5′ ends of the coding region of the gene: at least about 1000 nucleotides of sequence upstream from the 5′ end of the coding region and at least about 200 nucleotides of sequence downstream from the 3′ end of the coding region of the gene. Less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2′-hydroxy in the ribose sugar group of the RNA can also be made. The antisense polynucleotides and ribozymes can consist entirely of ribonucleotides, or can contain mixed ribonucleotides and deoxyribonucleotides. The polynucleotides of the invention may be produced by any means, including genomic preparations, cDNA preparations, in vitro synthesis, RT-PCR and in vitro or in vivo transcription.
An “isolated” nucleic acid molecule is one that is substantially separated from other nucleic acid molecules which are present in the natural source of the nucleic acid (e.g., sequences encoding other polypeptides). Preferably, an “isolated” nucleic acid is free of some of the sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in its naturally occurring replicon. For example, a cloned nucleic acid is considered isolated. In various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. In further embodiments, the isolated PrPase nucleic acid molecule can contain less than about 5 mb, 1 mb, 0.5 mb, 0.1 mb, 50 kb, 25 kb, 20 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A nucleic acid is also considered isolated if it has been altered by human intervention, or placed in a locus or location that is not its natural site, or if it is introduced into a cell by agroinfection or other means of transformation. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be free from some of the other cellular material with which it is naturally associated, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
Specifically excluded from the definition of “isolated nucleic acids” are: naturally-occurring chromosomes (such as chromosome spreads), artificial chromosome libraries, genomic libraries, and cDNA libraries that exist either as an in vitro nucleic acid preparation or as a transfected/transformed host cell preparation, wherein the host cells are either an in vitro heterogeneous preparation or plated as a heterogeneous population of single colonies. Also specifically excluded are the above libraries wherein a specified nucleic acid makes up less than 5% of the number of nucleic acid inserts in the vector molecules. Further specifically excluded are whole cell genomic DNA or whole cell RNA preparations (including whole cell preparations that are mechanically sheared or enzymatically digested). Even further specifically excluded are the whole cell preparations found as either an in vitro preparation or as a heterogeneous mixture separated by electrophoresis wherein the nucleic acid of the invention has not further been separated from the heterologous nucleic acids in the electrophoresis medium (e.g., further separating by excising a single band from a heterogeneous band population in an agarose gel or nylon blot).
One aspect of the invention pertains to isolated PrPase proteins, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. In one embodiment, native PrPase proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, PrPase proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a PrPase protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the PrPase protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of PrPase protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of PrPase protein having less than about 30% (by dry weight) of non-PrPase protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-PrPase protein, still more preferably less than about 10% of non-PrPase protein, and most preferably less than about 5% non-PrPase protein. When the PrPase protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
The language “substantially free of chemical precursors or other chemicals” includes preparations of PrPase protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of PrPase protein having less than about 30% (by dry weight) of chemical precursors or non-PrPase chemicals, more preferably less than about 20% chemical precursors or non-PrPase chemicals, still more preferably less than about 10% chemical precursors or non-PrPase chemicals, and most preferably less than about 5% chemical precursors or non-PrPase chemicals.
Polynucleotides and Polypeptides of the Invention
The present invention provides novel polynucleotide and polypeptide sequences of plant-derived prenyl protease (PrPase) from five species namely, the nucleic acid PrPase sequences and the encoded polypeptides from the moss Physcomitrella patens (PpPrPase1, SEQ ID NOs: 1, 2), three from Arabidopsis thaliana (AtPrPase1, SEQ ID NOs: 3, 4; AtPrPase2, SEQ ID NOs: 5, 6; and AtCPP, SEQ ID NOs: 7, 8), two from soybeans Glycine max (GmPrPase2, SEQ ID NOs: 16, 17; and GmCPP, SEQ ID NOs: 18, 19), one from corn Zea mays (ZmPrPase2, SEQ ID NOs: 23, 24), and one from rapeseed Brassica napus (BnCPP, SEQ ID NOs: 11, 12). The invention also provides partial PrPase sequences from soybean (GmPrPase1, SEQ ID NOs: 14, 15) and corn (ZmPrPase1, SEQ ID NOs: 21, 22).
The sequences are collectively referred to as “PrPase nucleic acids”, “PrPase polynucleotides” or “PrPase antisense nucleic acids” and the corresponding encoded polypeptide is referred to as a “PrPase polypeptide” or “PrPase protein”. Unless indicated otherwise, “PrPase” is meant to refer to any of the novel sequences disclosed herein.
The prenyl proteases of the invention have recognizable homology at the nucleotide and amino acid level to both human and yeast prenyl proteases. Additionally, in a BLAST search of public sequence databases, it was found, for example, that the AtCPP nucleic acid sequence has 99.3% identity to an Arabidopsis thaliana CaaX processing zinc-metallo endoprotease (AFC1) mRNA (Genbank Accession No.: AF353722). FIG. 6A . The full amino acid sequence of the AtCPP protein of the invention was found to be 98.8% identical to Arabidopsis thaliana CaaX processing zinc-metallo endoprotease (AFC1) polypeptide (Genbank Accession No.: AAK39514). FIG. 7 . A multiple alignment of the eight polypeptides of the invention (SEQ ID NO: 2, 4, 6, 8, 12, 17, 19, and 24), of yeast, and of three other published Arabidopsis PrPases is illustrated in FIG. 5 with the consensus sequence shown in SEQ ID NO: 85. Similarly, a multiple alignment of the polynucleotides of the invention (SEQ ID NO: 1, 3, 5, 7, 11, 16, 18, and 23) and of three other published Arabidopsis PrPase sequences is illustrated in FIG. 4 with the consensus sequence shown in SEQ ID NO: 84.
The percent identity between the various sequences of FIGS. 4 and 5 are tabulated in FIGS. 6 and 7 . A multiple alignment of the PrPase sequences for dicot plants of the invention and three published PrPase are provided in FIGS. 16 and 17 with the consensus sequences shown in SEQ ID NOs: 86-87. A multiple alignment of the PrPase sequences for Arabidopsis are provided in FIGS. 18 and 19 with the consensus sequences shown in SEQ ID NOs: 88-89. A multiple alignment of the PrPase sequences of the invention for soybean plants are provided in FIGS. 20 and 21 with the consensus sequences shown in SEQ ID NOs: 90-91.
The previously described genomic sequence encoding the Arabidopsis PrPase (GenBank accession number AF007269, gene=“A_IG002N01.21) was predicted by the computer program (Genefinder (P. Green and L. Hillier, National Center for Biotechnology Information (NCBI) website)) to contain an ORF at positions 24979 to 28076. That predicted ORF does not reflect the real ORF for this gene as shown in FIG. 3 .
Based on their structural and functional relatedness to known CaaX prenyl protease proteins, the PrPase proteins of the invention are novel members of the CaaX prenyl protease family of proteins. PrPase nucleic acids, and their encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, the nucleic acids (i.e., sense or antisense PrPase nucleic acids) can be used to produce transgenic plants that have an increased resistance to biotic and abiotic stresses, e.g., chilling stress, salt stress, water stress (e.g., drought), wound healing, pathogen challenge, grazing pests, or herbicides. Additionally, the transgenic plants have an increased productivity during both optimal and suboptimal growth conditions, increased yield, increased biomass, or delayed senescence. Alternatively, the transgenic plants have an increased sensitivity to the phytohormone abscisic acid (ABA). By resistant is meant the plant grows under stress conditions (e.g., high salt, decreased water, low temperatures) or under conditions that normally inhibit, to some degree, the growth of an untransformed plant.
Although it is believed the encoded polypeptides may share at least some biological activities with prenyl proteases, a number of methods of determining the exact biological function of these clones are either known in the art or are described elsewhere herein. Briefly, the function of these clones may be determined, for example, by applying microarray methodology. The clones of the present invention may be arrayed on microchips for expression profiling. Depending on which polynucleotide probe is used to hybridize to the slides, a change in expression of a specific gene may provide additional insight into the function of this gene based upon the conditions being studied. For example, an observed increase or decrease in expression levels when the polynucleotide probe used comes from tissue that has been cold treated might indicate a function in modulating cold tolerance. Tissue deprived of water or stressed by other biotic or abiotic stresses (heat, drought, high light, high salt, etc.) should be used to extract RNA to prepare the probe. Moreover, different stages of seed development (early, middle, late) could be used to extract RNA to prepare the probe.
In addition, the function of the proteins may be assessed by applying quantitative PCR methodology, for example. Real time quantitative PCR would provide the capability of following the expression of specific genes throughout the plant development cycle, for example. Quantitative PCR methodology requires only a nominal amount of tissue from each developmentally important step (3 days germinated seedlings; 1 week old seedlings and their roots, shoots, and stems; roots, leaves and stems before the onset of flowering, flowers and their different parts; and/or developing embryos) is usually used to perform such experiments. Therefore, the application of quantitative PCR methodology to refining the biological function of these polypeptides is encompassed by the present invention. Also encompassed by the present invention are PCR probes corresponding to the polynucleotide sequences provided or portions thereof.
The function of the proteins may also be assessed through complementation assays in yeast. For example, transforming yeast deficient in prenyl protease activity and assessing their ability to grow would provide convincing evidence that the clones of the invention have prenyl protease activity. Additional assay conditions and methods that may be used in assessing the function of the polynucleotides and polypeptides of the present invention are known in the art.
Alternatively, the biological function of the encoded polypeptides may be determined by disrupting a homologue of the polypeptides in Synechosystis . Cyanobacteria (blue-green algae) is considered a precursor to the plant chloroplast. It possesses both photosynthetic systems and many other metabolic processes similar to those of plants. These processes are often targets for many commercial herbicides, and this organism has been widely used in the study of the mode of action of many classes of herbicides. Synechocystis is one of the best-studied cyanobacteria. In addition to most of the features common to cyanobacteria, it offers many other advantages. Synechocystis has a naturally occurring genetic transformation system, thus entailing vigorous and sophisticated genetic and molecular manipulation (e.g. targeted-gene disruption, gene replacement, etc.) applicable to some of the well-characterized systems ( S. cerevisiae, E. coli ). Most importantly, the availability of the complete genomic sequence information of the Synechocystis affords an avenue for the rapid identification and cloning of gene(s) of interest, and elucidation of gene function through genetic and molecular means.
Moreover, the biological function of polypeptides may be determined by the application of antisense and/or sense methodology and the resulting generation of transgenic plants. Expressing a particular gene in either sense or antisense orientation in a transgenic plant can lead to respectively higher or lower expression levels of that particular gene. Altering the endogenous expression levels of a gene can lead to the observation of a particular phenotype that can then be used to derive indications on the function of the gene. The gene can be either over-expressed or under expressed in every cell of the plant at all times using a strong ubiquitous promoter, or it can be expressed in one or more discrete parts of the plant using a well characterized tissue-specific promoter (i.e., a root promoter or a flower specific promoter or a seed-specific promoter), or it can be expressed at a specified time of development using an inducible and/or a developmentally regulated promoter.
In the case of the transgenic plants of the invention, if no phenotype is apparent in normal growth conditions, observing the plants under stress conditions (deprivation of water, presence of high salt, or other biotic or abiotic stresses, such as cold, heat, drought, high light, etc.) may lead to understanding the function of the genes. Therefore, the application of antisense and/or sense methodology to the creation of transgenic plants to refine the biological function of the polypeptides is encompassed by the present invention.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to the sequences of the invention and may have been publicly available prior to conception of the present invention. Such related polynucleotides are specifically excluded from the scope of the present invention.
PpPrPase1
The polynucleotide sequence of the partial PrPase from Physcomitrella patens (Clone ID No: PpPrPase1) of the present invention is provided as SEQ ID NO: 1. The polynucleotide sequence contains a sequence of 1398 nucleotides. The deduced amino acid sequence of SEQ ID NO: 1 is provided as SEQ ID NO: 2 and contains 394 amino acids.
In further embodiments, deletion mutants of PpPrPase1 are encompassed by the present invention, the following N-terminal deletion mutants: L1-D394, K2-D394, L3-D394, S4-D394, N5-D394, L6-D394, P7-D394, A8-D394, P9-D394, L10-D394, K11-D394, G12-D394, I13-D394, V14-D394, S15-D394, Q16-D394, E17-D394, K18-D394, F19-D394, E20-D394, K21-D394, A22-D394, Q23-D394, A24-D394, Y25-D394, S26-D394, L27-D394, D28-D394, K29-D394, S30-D394, R31-D394, F32-D394, H33-D394, F34-D394, V35-D394, H36-D394, A37-D394, A38-D394, V39-D394, N40-D394, I41-D394, V42-D394, E43-D394, E44-D394, S45-D394, A46-D394, I47-D394, L48-D394, L49-D394, L50-D394, G51-D394, L52-D394, L53-D394, P54-D394, W55-D394, A56-D394, W57-D394, D58-D394, K59-D394, S60-D394, G61-D394, S62-D394, L63-D394, V64-D394, G65-D394, K66-D394, L67-D394, G68-D394, F69-D394, D70-D394, E71-D394, K72-D394, S73-D394, E74-D394, I75-D394, L76-D394, Q77-D394, T78-D394, L79-D394, S80-D394, F81-D394, L82-D394, A83-D394, V84-D394, T85-D394, T86-D394, L87-D394, W88-D394, S89-D394, Q90-D394, I91-D394, L92-D394, E93-D394, L94-D394, P95-D394, F96-D394, S97-D394, L98-D394, Y99-D394, S100-D394, T101-D394, F102-D394, V103-D394, I104-D394, E105-D394, A106-D394, R107-D394, H108-D394, G109-D394, F110-D394, N111-D394, K112-D394, Q113-D394, T114-D394, I115-D394, W116-D394, L117-D394, F118-D394, L119-D394, R120-D394, D121-D394, M122-D394, I123-D394, M124-D394, G125-D394, L126-D394, A127-D394, L128-D394, M129-D394, M130-D394, V131-D394, V132-D394, G133-D394, P134-D394, P135-D394, I136-D394, V137-D394, S138-D394, A139-D394, I140-D394, I141-D394, Y142-D394, I143-D394, V144-D394, Q145-D394, N146-D394, G147-D394, G148-D394, P149-D394, Y150-D394, L151-D394, A152-D394, L153-D394, Y154-D394, L155-D394, W156-D394, A157-D394, F158-D394, M159-D394, L160-D394, L161-D394, L162-D394, S163-D394, L164-D394, V165-D394, L166-D394, M167-D394, A168-D394, L169-D394, Y170-D394, P171-D394, V172-D394, L173-D394, I174-D394, A175-D394, P176-D394, L177-D394, F178-D394, N179-D394, T180-D394, F181-D394, T182-D394, P183-D394, L184-D394, P185-D394, E186-D394, G187-D394, Q188-D394, L189-D394, R190-D394, A191-D394, K192-D394, I193-D394, E194-D394, K195-D394, L196-D394, A197-D394, S198-D394, S199-D394, L200-D394, D201-D394, F202-D394, P203-D394, L204-D394, K205-D394, K206-D394, L207-D394, F208-D394, V209-D394, I210-D394, D211-D394, G212-D394, S213-D394, T214-D394, R215-D394, S216-D394, S217-D394, H218-D394, S219-D394, N220-D394, A221-D394, Y222-D394, M223-D394, Y224-D394, G225-D394, F226-D394, Y227-D394, N228-D394, S229-D394, K230-D394, R231-D394, I232-D394, V233-D394, L234-D394, Y235-D394, D236-D394, T237-D394, L238-D394, I239-D394, S240-D394, Q241-D394, C242-D394, K243-D394, N244-D394, E245-D394, E246-D394, E247-D394, V248-D394, V249-D394, A250-D394, V251-D394, I252-D394, A253-D394, H254-D394, E255-D394, L256-D394, G257-D394, H258-D394, W259-D394, K260-D394, L261-D394, S262-D394, H263-D394, T264-D394, M265-D394, Y266-D394, S267-D394, F268-D394, L269-D394, A270-D394, M271-D394, Q272-D394, V273-D394, L274-D394, T275-D394, L276-D394, L277-D394, Q278-D394, F279-D394, G280-D394, G281-D394, Y282-D394, T283-D394, L284-D394, V285-D394, R286-D394, N287-D394, S288-D394, S289-D394, G290-D394, L291-D394, F292-D394, L293-D394, S294-D394, F295-D394, G296-D394, F297-D394, S298-D394, T299-D394, Q300-D394, P301-D394, V302-D394, L303-D394, I304-D394, G305-D394, L306-D394, I307-D394, L308-D394, F309-D394, Q310-D394, H311-D394, T312-D394, I313-D394, M314-D394, P315-D394, F316-D394, H317-D394, H318-D394, L319-D394, V320-D394, S321-D394, F322-D394, A323-D394, L324-D394, N325-D394, L326-D394, L327-D394, S328-D394, R329-D394, A330-D394, F331-D394, E332-D394, F333-D394, Q334-D394, A335-D394, D336-D394, A337-D394, F338-D394, A339-D394, R340-D394, S341-D394, L342-D394, G343-D394, Y344-D394, R345-D394, E346-D394, P347-D394, L348-D394, R349-D394, A350-D394, G351-D394, L352-D394, I353-D394, K354-D394, L355-D394, Q356-D394, E357-D394, E358-D394, N359-D394, L360-D394, S361-D394, A362-D394, M363-D394, N364-D394, T365-D394, D366-D394, P367-D394, W368-D394, Y369-D394, S370-D394, A371-D394, Y372-D394, H373-D394, H374-D394, S375-D394, H376-D394, P377-D394, P378-D394, L379-D394, V380-D394, E381-D394, R382-D394, L383-D394, Q384-D394, A385-D394, L386-D394, D387-D394, E388-D394, of SEQ ID NO: 2. The invention includes the polynucleotide sequences encoding these mutant polypeptides.
In further embodiments, deletion mutants of PpPrPase1 are encompassed by the present invention, including the following C-terminal deletion mutants: L1-D394, L1-T393, L1-K392, L1-K391, L1-S390, L1-T389, L1-E388, L1-D387, L1-L386, L1-A385, L1-Q384, L1-L383, L1-R382, L1-E381, L1-V380, L1-L379, L1-P378, L1-P377, L1-H376, L1-S375, L1-H374, L1-H373, L1-Y372, L1-A371, L1-S370, L1-Y369, L1-W368, L1-P367, L1-D366, L1-T365, L1-N364, L1-M363, L1-A362, L1-S361, L1-L360, L1-N359, L1-E358, L1-E357, L1-Q356, L1-L355, L1-K354, L1-I353, L1-L352, L1-G351, L1-A350, L1-R349, L1-L348, L1-P347, L1-E346, L1-R345, L1-Y344, L1-G343, L1-L342, L1-S341, L1-R340, L1-A339, L1-F338, L1-A337, L1-D336, L1-A335, L1-Q334, L1-F333, L1-E332, L1-F331, L1-A330, L1-R329, L1-S328, L1-L327, L1-L326, L1-N325, L1-L324, L1-A323, L1-F322, L1-S321, L1-V320, L1-L319, L1-H318, L1-H317, L1-F316, L1-P315, L1-M314, L1-I313, L1-T312, L1-H311, L1-Q310, L1-F309, L1-L308, L1-I307, L1-L306, L1-G305, L1-I304, L1-L303, L1-V302, L1-P301, L1-Q300, L1-T299, L1-S298, L1-F297, L1-G296, L1-F295, L1-S294, L1-L293, L1-F292, L1-L291, L1-G290, L1-S289, L1-S288, L1-N287, L1-R286, L1-V285, L1-L284, L1-T283, L1-Y282, L1-G281, L1-G280, L1-F279, L1-Q278, L1-L277, L1-L276, L1-T275, L1-L274, L1-V273, L1-Q272, L1-M271, L1-A270, L1-L269, L1-F268, L1-S267, L1-Y266, L1-M265, L1-T264, L1-H263, L1-S262, L1-L261, L1-K260, L1-W259, L1-H258, L1-G257, L1-L256, L1-E255, L1-H254, L1-A253, L1-I252, L1-V251, L1-A250, L1-V249, L1-V248, L1-E247, L1-E246, L1-E245, L1-N244, L1-K243, L1-C242, L1-Q241, L1-S240, L1-I239, L1-L238, L1-T237, L1-D236, L1-Y235, L1-L234, L1-V233, L1-I232, L1-R231, L1-K230, L1-S229, L1-N228, L1-Y227, L1-F226, L1-G225, L1-Y224, L1-M223, L1-Y222, L1-A221, L1-N220, L1-S219, L1-H218, L1-S217, L1-S216, L1-R215, L1-T214, L1-S213, L1-G212, L1-D211, L1-I210, L1-V209, L1-F208, L1-L207, L1-K206, L1-K205, L1-L204, L1-P203, L1-F202, L1-D201, L1-L200, L1-S199, L1-S198, L1-A197, L1-L196, L1-K195, L1-E194, L1-I193, L1-K192, L1-A191, L1-R190, L1-L189, L1-Q188, L1-G187, L1-E186, L1-P185, L1-L184, L1-P183, L1-T182, L1-F181, L1-T180, L1-N179, L1-F178, L1-L177, L1-P176, L1-A175, L1-I174, L1-L173, L1-V172, L1-P171, L1-Y170, L1-L169, L1-A168, L1-M167, L1-L166, L1-V165, L1-L164, L1-S163, L1-L162, L1-L161, L1-L160, L1-M159, L1-F158, L1-A157, L1-W156, L1-L155, L1-Y154, L1-L153, L1-A152, L1-L151, L1-Y150, L1-P149, L1-G148, L1-G147, L1-N146, L1-Q145, L1-V144, L1-I143, L1-Y142, L1-I141, L1-I140, L1-A139, L1-S138, L1-V137, L1-I136, L1-P135, L1-P134, L1-G133, L1-V132, L1-V131, L1-M130, L1-M129, L1-L128, L1-A127, L1-L126, L1-G125, L1-M124, L1-I123, L1-M122, L1-D121, L1-R120, L1-L119, L1-F118, L1-L117, L1-W116, L1-I115, L1-T114, L1-Q113, L1-K112, L1-N111, L1-F110, L1-G109, L1-H108, L1-R107, L1-A106, L1-E105, L1-I104, L1-V103, L1-F102, L1-T101, L1-S100, L1-Y99, L1-L98, L1-S97, L1-F96, L1-P95, L1-L94, L1-E93, L1-L92, L1-I91, L1-Q90, L1-S89, L1-W88, L1-L87, L1-T86, L1-T85, L1-V84, L1-A83, L1-L82, L1-F81, L1-S80, L1-L79, L1-T78, L1-Q77, L1-L76, L1-I75, L1-E74, L1-S73, L1-K72, L1-E71, L1-D70, L1-F69, L1-G68, L1-L67, L1-K66, L1-G65, L1-V64, L1-L63, L1-S62, L1-G61, L1-S60, L1-K59, L1-D58, L1-W57, L1-A56, L1-W55, L1-P54, L1-L53, L1-L52, L1-G51, L1-L50, L1-L49, L1-L48, L1-I47, L1-A46, L1-S45, L1-E44, L1-E43, L1-V42, L1-I41, L1-N40, L1-V39, L1-A38, L1-A37, L1-H36, L1-V35, L1-F34, L1-H33, L1-F32, L1-R31, L1-S30, L1-K29, L1-D28, L1-L27, L1-S26, L1-Y25, L1-A24, L1-Q23, L1-A22, L1-K21, L1-E20, L1-F19, L1-K18, L1-E17, L1-Q16, L1-S15, L1-V14, L1-I13, L1-G12, L1-K11, L1-L10, L1-P9, L1-A8, L1-P7, of SEQ ID NO: 2. The invention includes the polynucleotide sequences encoding these mutant polypeptides.
AtPrPase1
The nucleotide sequence of the full-length PrPase (AtPrPase1) from Arabidopsis thaliana (Clone ID No: AtPrPase1) of the present invention is provided as SEQ ID NO: 3. The polynucleotide sequence contains a sequence of 1275 nucleotides. The deduced amino acid sequence of SEQ ID NO: 3 is provided as SEQ ID NO: 4 and contains 424 amino acids.
In a further embodiment, the invention also encompasses the promoter of the AtPrPase1 gene (SEQ ID NO: 10). The promoter has uses including, but not limited to, directing expression of a gene of interest in plant guard-cells. The gene of interest may be any gene endogenous to a plant, a non-plant derived gene (e.g., viral, mammalian, human, synthetic, molecularly evolved, bacterial, fungal, etc.,), a reporter gene, a marker gene, a desired input trait, a desired output trait, a gene capable of conferring a specific phenotype in a plant, one or more genes of the present invention, antibody genes, antibody genes directed against the polypeptides of the present invention, antisense genes, in addition to other genes known in the art and/or disclosed herein.
In further embodiments, deletion mutants of AtPrPase1 are encompassed by the present invention, including the following N-terminal deletion mutants: M1-D424, A2-D424, I3-D424, P4-D424, F5-D424, M6-D424, E7-D424, T8-D424, V9-D424, V10-D424, G11-D424, F12-D424, M13-D424, I14-D424, V15-D424, M16-D424, Y17-D424, I18-D424, F19-D424, E20-D424, T21-D424, Y22-D424, L23-D424, D24-D424, L25-D424, R26-D424, Q27-D424, L28-D424, T29-D424, A30-D424, L31-D424, K32-D424, L33-D424, P34-D424, T35-D424, L36-D424, P37-D424, K38-D424, T39-D424, L40-D424, V41-D424, G42-D424, V43-D424, 144-D424, S45-D424, Q46-D424, E47-D424, K48-D424, F49-D424, E50-D424, K51-D424, S52-D424, R53-D424, A54-D424, Y55-D424, S56-D424, L57-D424, D58-D424, K59-D424, S60-D424, Y61-D424, F62-D424, H63-D424, F64-D424, V65-D424, H66-D424, E67-D424, F68-D424, V69-D424, T70-D424, I71-D424, L72-D424, M73-D424, D74-D424, S75-D424, A76-D424, I77-D424, L78-D424, F79-D424, F80-D424, G81-D424, I82-D424, L83-D424, P84-D424, W85-D424, F86-D424, W87-D424, K88-D424, M89-D424, S90-D424, G91-D424, A92-D424, V93-D424, L94-D424, P95-D424, R96-D424, L97-D424, G98-D424, L99-D424, D100-D424, P101-D424, E102-D424, N103-D424, E104-D424, I105-D424, L106-D424, H107-D424, T108-D424, L109-D424, S110-D424, F111-D424, L112-D424, A113-D424, G114-D424, V115-D424, M116-D424, T117-D424, W118-D424, S119-D424, Q120-D424, I121-D424, T122-D424, D123-D424, L124-D424, P125-D424, F126-D424, S127-D424, L128-D424, Y129-D424, S130-D424, T131-D424, F132-D424, V133-D424, I134-D424, E135-D424, S136-D424, R137-D424, H138-D424, G139-D424, F140-D424, N141-D424, K142-D424, Q143-D424, T144-D424, I145-D424, W146-D424, M147-D424, F148-D424, I149-D424, R150-D424, D151-D424, M152-D424, I153-D424, K154-D424, G155-D424, T156-D424, F157-D424, L158-D424, S159-D424, V160-D424, I161-D424, L162-D424, G163-D424, P164-D424, P165-D424, I166-D424, V167-D424, A168-D424, A169-D424, I170-D424, I171-D424, F172-D424, I173-D424, V174-D424, Q175-D424, K176-D424, G177-D424, G178-D424, P179-D424, Y180-D424, L181-D424, A182-D424, I183-D424, Y184-D424, L185-D424, W186-D424, A187-D424, F188-D424, M189-D424, F190-D424, I191-D424, L192-D424, S193-D424, L194-D424, V195-D424, M196-D424, M197-D424, T198-D424, I199-D424, Y200-D424, P201-D424, V202-D424, L203-D424, I204-D424, A205-D424, P206-D424, L207-D424, F208-D424, N209-D424, K210-D424, F211-D424, T212-D424, P213-D424, L214-D424, P215-D424, D216-D424, G217-D424, D218-D424, L219-D424, R220-D424, E221-D424, K222-D424, I223-D424, E224-D424, K225-D424, L226-D424, A227-D424, S228-D424, S229-D424, L230-D424, K231-D424, F232-D424, P233-D424, L234-D424, K235-D424, K236-D424, L237-D424, F238-D424, V239-D424, V240-D424, D241-D424, G242-D424, S243-D424, T244-D424, R245-D424, S246-D424, S247-D424, H248-D424, S249-D424, N250-D424, A251-D424, Y252-D424, M253-D424, Y254-D424, G255-D424, F256-D424, F257-D424, K258-D424, N259-D424, K260-D424, R261-D424, I262-D424, V263-D424, L264-D424, Y265-D424, D266-D424, T267-D424, L268-D424, I269-D424, Q270-D424, Q271-D424, C272-D424, K273-D424, N274-D424, E275-D424, D276-D424, E277-D424, I278-D424, V279-D424, A280-D424, V281-D424, I282-D424, A283-D424, H284-D424, E285-D424, L286-D424, G287-D424, H288-D424, W289-D424, K290-D424, L291-D424, N292-D424, H293-D424, T294-D424, T295-D424, Y296-D424, S297-D424, F298-D424, I299-D424, A300-D424, V301-D424, Q302-D424, I303-D424, L304-D424, A305-D424, F306-D424, L307-D424, Q308-D424, F309-D424, G310-D424, G311-D424, Y312-D424, T313-D424, L314-D424, V315-D424, R316-D424, N317-D424, S318-D424, T319-D424, D320-D424, L321-D424, F322-D424, R323-D424, S324-D424, F325-D424, G326-D424, F327-D424, D328-D424, T329-D424, Q330-D424, P331-D424, V332-D424, L333-D424, I334-D424, G335-D424, L336-D424, I337-D424, I338-D424, F339-D424, Q340-D424, H341-D424, T342-D424, V343-D424, I344-D424, P345-D424, L346-D424, Q347-D424, H348-D424, P349-D424, V350-D424, S351-D424, F352-D424, G353-D424, L354-D424, N355-D424, L356-D424, V357-D424, S358-D424, R359-D424, A360-D424, F361-D424, E362-D424, F363-D424, Q364-D424, A365-D424, D366-D424, A367-D424, F368-D424, A369-D424, V370-D424, K371-D424, L372-D424, G373-D424, Y374-D424, A375-D424, K376-D424, D377-D424, L378-D424, R379-D424, P380-D424, T381-D424, L382-D424, V383-D424, K384-D424, L385-D424, Q386-D424, E387-D424, E388-D424, N389-D424, L390-D424, S391-D424, A392-D424, M393-D424, N394-D424, T395-D424, D396-D424, P397-D424, L398-D424, Y399-D424, S400-D424, A401-D424, Y402-D424, H403-D424, Y404-D424, S405-D424, H406-D424, P407-D424, P408-D424, L409-D424, V410-D424, E411-D424, R412-D424, L413-D424, R414-D424, A415-D424, I416-D424, D417-D424, G418-D424, of SEQ ID NO: 4. The invention includes the polynucleotide sequences encoding these mutant polypeptides.
In further embodiments, deletion mutants of AtPrPase1 are encompassed by the present invention, including the following C-terminal deletion mutants: M1-D424, M1-T423, M1-K422, M1-K421, M1-D420, M1-E419, M1-G418, M1-D417, M1-I416, M1-A415, M1-R414, M1-L413, M1-R412, M1-E411, M1-V410, M1-L409, M1-P408, M1-P407, M1-H406, M1-S405, M1-Y404, M1-H403, M1-Y402, M1-A401, M1-S400, M1-Y399, M1-L398, M1-P397, M1-D396, M1-T395, M1-N394, M1-M393, M1-A392, M1-S391, M1-L390, M1-N389, M1-E388, M1-E387, M1-Q386, M1-L385, M1-K384, M1-V383, M1-L382, M1-T381, M1-P380, M1-R379, M1-L378, M1-D377, M1-K376, M1-A375, M1-Y374, M1-G373, M1-L372, M1-K371, M1-V370, M1-A369, M1-F368, M1-A367, M1-D366, M1-A365, M1-Q364, M1-F363, M1-E362, M1-F361, M1-A360, M1-R359, M1-S358, M1-V357, M1-L356, M1-N355, M1-L354, M1-G353, M1-F352, M1-S351, M1-V350, M1-P349, M1-H348, M1-Q347, M1-L346, M1-P345, M1-I344, M1-V343, M1-T342, M1-H341, M1-Q340, M1-F339, M1-I338, M1-I337, M1-L336, M1-G335, M1-I334, M1-L333, M1-V332, M1-P331, M1-Q330, M1-T329, M1-D328, M1-F327, M1-G326, M1-F325, M1-S324, M1-R323, M1-F322, M1-L321, M1-D320, M1-T319, M1-S318, M1-N317, M1-R316, M1-V315, M1-L314, M1-T313, M1-Y312, M1-G311, M1-G310, M1-F309, M1-Q308, M1-L307, M1-F306, M1-A305, M1-L304, M1-I303, M1-Q302, M1-V301, M1-A300, M1-I299, M1-F298, M1-S297, M1-Y296, M1-T295, M1-T294, M1-H293, M1-N292, M1-L291, M1-K290, M1-W289, M1-H288, M1-G287, M1-L286, M1-E285, M1-H284, M1-A283, M1-I282, M1-V281, M1-A280, M1-V279, M1-I278, M1-E277, M1-D276, M1-E275, M1-N274, M1-K273, M1-C272, M1-Q271, M1-Q270, M1-I269, M1-L268, M1-T267, M1-D266, M1-Y265, M1-L264, M1-V263, M1-I262, M1-R261, M1-K260, M1-N259, M1-K258, M1-F257, M1-F256, M1-G255, M1-Y254, M1-M253, M1-Y252, M1-A251, M1-N250, M1-S249, M1-H248, M1-S247, M1-S246, M1-R245, M1-T244, M1-S243, M1-G242, M1-D241, M1-V240, M1-V239, M1-F238, M1-L237, M1-K236, M1-K235, M1-L234, M1-P233, M1-F232, M1-K231, M1-L230, M1-S229, M1-S228, M1-A227, M1-L226, M1-K225, M1-E224, M1-I223, M1-K222, M1-E221, M1-R220, M1-L219, M1-D218, M1-G217, M1-D216, M1-P215, M1-L214, M1-P213, M1-T212, M1-F211, M1-K210, M1-N209, M1-F208, M1-L207, M1-P206, M1-A205, M1-I204, M1-L203, M1-V202, M1-P201, M1-Y200, M1-I199, M1-T198, M1-M197, M1-M196, M1-V195, M1-L194, M1-S193, M1-L192, M1-I191, M1-F190, M1-M189, M1-F188, M1-A187, M1-W186, M1-L185, M1-Y184, M1-I183, M1-A182, M1-L181, M1-Y180, M1-P179, M1-G178, M1-G177, M1-K176, M1-Q175, M1-V174, M1-I173, M1-F172, M1-I171, M1-I170, M1-A169, M1-A168, M1-V167, M1-I166, M1-P165, M1-P164, M1-G163, M1-L162, M1-I161, M1-V160, M1-S159, M1-L158, M1-F157, M1-T156, M1-G155, M1-K154, M1-I153, M1-M152, M1-D151, M1-R150, M1-I149, M1-F148, M1-M147, M1-W146, M1-I145, M1-T144, M1-Q143, M1-K142, M1-N141, M1-F140, M1-G139, M1-H138, M1-R137, M1-S136, M1-E135, M1-I134, M1-V133, M1-F132, M1-T131, M1-S130, M1-Y129, M1-L128, M1-S127, M1-F126, M1-P125, M1-L124, M1-D123, M1-T122, M1-I121, M1-Q120, M1-S119, M1-W118, M1-T117, M1-M116, M1-V115, M1-G114, M1-A113, M1-L112, M1-F111, M1-S110, M1-L109, M1-T108, M1-H107, M1-L106, M1-I105, M1-E104, M1-N103, M1-E102, M1-P100, M1-D100, M1-L99, M1-G98, M1-L97, M1-R96, M1-P95, M1-L94, M1-V93, M1-A92, M1-G91, M1-S90, M1-M89, M1-K88, M1-W87, M1-F86, M1-W85, M1-P84, M1-L83, M1-I82, M1-G81, M1-F80, M1-F79, M1-L78, M1-I77, M1-A76, M1-S75, M1-D74, M1-M73, M1-L72, M1-I71, M1-T70, M1-V69, M1-F68, M1-E67, M1-H66, M1-V65, M1-F64, M1-H63, M1-F62, M1-Y61, M1-S60, M1-K59, M1-D58, M1-L57, M1-S56, M1-Y55, M1-A54, M1-R53, M1-S52, M1-K51, M1-E50, M1-F49, M1-K48, M1-E47, M1-Q46, M1-S45, M1-I44, M1-V43, M1-G42, M1-V41, M1-L40, M1-T39, M1-K38, M1-P37, M1-L36, M1-T35, M1-P34, M1-L33, M1-K32, M1-L31, M1-A30, M1-T29, M1-L28, M1-Q27, M1-R26, M1-L25, M1-D24, M1-L23, M1-Y22, M1-T21, M1-E20, M1-F19, M1-I18, M1-Y17, M1-M16, M1-V15, M1-I14, M1-M13, M1-F12, M1-G11, M1-V10, M1-V9, M1-T8, M1-E7, of SEQ ID NO: 4. The invention includes the polynucleotide sequences encoding these mutant polypeptides.
AtPrPase2
The nucleotide sequence of a full-length PrPase (AtPrPase2) from Arabidopsis thaliana (Clone ID No: AtPrPase2) of the present invention is provided as SEQ ID NO: 5. The polynucleotide sequence contains a sequence of 1275 nucleotides. SEQ ID NO: 3 and SEQ ID NO: 5 are full-length Arabidopsis AtPrPases with 8 nucleotide differences at nucleotide positions: 276, 504, 1046, 1062, 1068, 1141, 1182, and 1190, which are alternative embodiments of this sequence of the invention. The deduced amino acid sequence of SEQ ID NO: 5 is provided as SEQ ID NO: 6 and contains 424 amino acids. SEQ ID NO: 4 and SEQ ID NO: 6 are full-length Arabidopsis AtPrPases with 3 amino acid differences at amino acid positions: 349, 381, and 397.
In preferred embodiments, deletion mutants of AtPrPase2 are encompassed by the present invention, including the following N-terminal deletion mutants: M1-D424, A2-D424, I3-D424, P4-D424, F5-D424, M6-D424, E7-D424, T8-D424, V9-D424, V10-D424, G11-D424, F12-D424, M13-D424, I14-D424, V15-D424, M16-D424, Y17-D424, I18-D424, F19-D424, E20-D424, T21-D424, Y22-D424, L23-D424, D24-D424, L25-D424, R26-D424, Q27-D424, L28-D424, T29-D424, A30-D424, L31-D424, K32-D424, L33-D424, P34-D424, T35-D424, L36-D424, P37-D424, K38-D424, T39-D424, L40-D424, V41-D424, G42-D424, V43-D424, I44-D424, S45-D424, Q46-D424, E47-D424, K48-D424, F49-D424, E50-D424, K51-D424, S52-D424, R53-D424, A54-D424, Y55-D424, S56-D424, L57-D424, D58-D424, K59-D424, S60-D424, Y61-D424, F62-D424, H63-D424, F64-D424, V65-D424, H66-D424, E67-D424, F68-D424, V69-D424, T70-D424, I71-D424, L72-D424, M73-D424, D74-D424, S75-D424, A76-D424, I77-D424, L78-D424, F79-D424, F80-D424, G81-D424, I82-D424, L83-D424, P84-D424, W85-D424, F86-D424, W87-D424, K88-D424, M89-D424, S90-D424, G91-D424, A92-D424, V93-D424, L94-D424, P95-D424, R96-D424, L97-D424, G98-D424, L99-D424, D100-D424, P101-D424, E102-D424, N103-D424, E104-D424, I105-D424, L106-D424, H107-D424, T108-D424, L109-D424, S110-D424, F111-D424, L112-D424, A113-D424, G114-D424, V115-D424, M116-D424, T117-D424, W118-D424, S119-D424, Q120-D424, I121-D424, T122-D424, D123-D424, L124-D424, P125-D424, F126-D424, S127-D424, L128-D424, Y129-D424, S130-D424, T131-D424, F132-D424, V133-D424, I134-D424, E135-D424, S136-D424, R137-D424, H138-D424, G139-D424, F140-D424, N141-D424, K142-D424, Q143-D424, T144-D424, I145-D424, W146-D424, M147-D424, F148-D424, I149-D424, R150-D424, D151-D424, M152-D424, I153-D424, K154-D424, G155-D424, T156-D424, F157-D424, L158-D424, S159-D424, V160-D424, I161-D424, L162-D424, G163-D424, P164-D424, P165-D424, I166-D424, V167-D424, A168-D424, A169-D424, I170-D424, I171-D424, F172-D424, I173-D424, V174-D424, Q175-D424, K176-D424, G177-D424, G178-D424, P179-D424, Y180-D424, L181-D424, A182-D424, I183-D424, Y184-D424, L185-D424, W186-D424, A187-D424, F188-D424, M189-D424, F190-D424, I191-D424, L192-D424, S193-D424, L194-D424, V195-D424, M196-D424, M197-D424, T198-D424, I199-D424, Y200-D424, P201-D424, V202-D424, L203-D424, I204-D424, A205-D424, P206-D424, L207-D424, F208-D424, N209-D424, K210-D424, F211-D424, T212-D424, P213-D424, L214-D424, P215-D424, D216-D424, G217-D424, D218-D424, L219-D424, R220-D424, E221-D424, K222-D424, I223-D424, E224-D424, K225-D424, L226-D424, A227-D424, S228-D424, S229-D424, L230-D424, K231-D424, F232-D424, P233-D424, L234-D424, K235-D424, K236-D424, L237-D424, F238-D424, V239-D424, V240-D424, D241-D424, G242-D424, S243-D424, T244-D424, R245-D424, S246-D424, S247-D424, H248-D424, S249-D424, N250-D424, A251-D424, Y252-D424, M253-D424, Y254-D424, G255-D424, F256-D424, F257-D424, K258-D424, N259-D424, K260-D424, R261-D424, I262-D424, V263-D424, L264-D424, Y265-D424, D266-D424, T267-D424, L268-D424, I269-D424, Q270-D424, Q271-D424, C272-D424, K273-D424, N274-D424, E275-D424, D276-D424, E277-D424, I278-D424, V279-D424, A280-D424, V281-D424, I282-D424, A283-D424, H284-D424, E285-D424, L286-D424, G287-D424, H288-D424, W289-D424, K290-D424, L291-D424, N292-D424, H293-D424, T294-D424, T295-D424, Y296-D424, S297-D424, F298-D424, I299-D424, A300-D424, V301-D424, Q302-D424, I303-D424, L304-D424, A305-D424, F306-D424, L307-D424, Q308-D424, F309-D424, G310-D424, G311-D424, Y312-D424, T313-D424, L314-D424, V315-D424, R316-D424, N317-D424, S318-D424, T319-D424, D320-D424, L321-D424, F322-D424, R323-D424, S324-D424, F325-D424, G326-D424, F327-D424, D328-D424, T329-D424, Q330-D424, P331-D424, V332-D424, L333-D424, I334-D424, G335-D424, L336-D424, I337-D424, I338-D424, F339-D424, Q340-D424, H341-D424, T342-D424, V343-D424, I344-D424, P345-D424, L346-D424, Q347-D424, H348-D424, L349-D424, V350-D424, S351-D424, F352-D424, G353-D424, L354-D424, N355-D424, L356-D424, V357-D424, S358-D424, R359-D424, A360-D424, F361-D424, E362-D424, F363-D424, Q364-D424, A365-D424, D366-D424, A367-D424, F368-D424, A369-D424, V370-D424, K371-D424, L372-D424, G373-D424, Y374-D424, A375-D424, K376-D424, D377-D424, L378-D424, R379-D424, P380-D424, A381-D424, L382-D424, V383-D424, K384-D424, L385-D424, Q386-D424, E387-D424, E388-D424, N389-D424, L390-D424, S391-D424, A392-D424, M393-D424, N394-D424, T395-D424, D396-D424, L397-D424, L398-D424, Y399-D424, S400-D424, A401-D424, Y402-D424, H403-D424, Y404-D424, S405-D424, H406-D424, P407-D424, P408-D424, L409-D424, V410-D424, E411-D424, R412-D424, L413-D424, R414-D424, A415-D424, I416-D424, D417-D424, G418-D424, of SEQ ID NO: 6. The invention includes the polynucleotide sequences encoding these mutant polypeptides.
In further embodiments, deletion mutants of AtPrPase2 are encompassed by the present invention, including the following C-terminal deletion mutants: M1-D424, M1-T423, M1-K422, M1-K421, M1-D420, M1-E419, M1-G418, M1-D417, M1-I416, M1-A415, M1-R414, M1-L413, M1-R412, M1-E411, M1-V410, M1-L409, M1-P408, M1-P407, M1-H406, M1-S405, M1-Y404, M1-H403, M1-Y402, M1-A401, M1-S400, M1-Y399, M1-L398, M1-L397, M1-D396, M1-T395, M1-N394, M1-M393, M1-A392, M1-S391, M1-L390, M1-N389, M1-E388, M1-E387, M1-Q386, M1-L385, M1-K384, M1-V383, M1-L382, M1-A381, M1-P380, M1-R379, M1-L378, M1-D377, M1-K376, M1-A375, M1-Y374, M1-G373, M1-L372, M1-K371, M1-V370, M1-A369, M1-F368, M1-A367, M1-D366, M1-A365, M1-Q364, M1-F363, M1-E362, M1-F361, M1-A360, M1-R359, M1-S358, M1-V357, M1-L356, M1-N355, M1-L354, M1-G353, M1-F352, M1-S351, M1-V350, M1-L349, M1-H348, M1-Q347, M1-L346, M1-P345, M1-I344, M1-V343, M1-T342, M1-H341, M1-Q340, M1-F339, M1-I338, M1-I337, M1-L336, M1-G335, M1-I334, M1-L333, M1-V332, M1-P331, M1-Q330, M1-T329, M1-D328, M1-F327, M1-G326, M1-F325, M1-S324, M1-R323, M1-F322, M1-L321, M1-D320, M1-T319, M1-S318, M1-N317, M1-R316, M1-V315, M1-L314, M1-T313, M1-Y312, M1-G311, M1-G310, M1-F309, M1-Q308, M1-L307, M1-F306, M1-A305, M1-L304, M1-I303, M1-Q302, M1-V301, M1-A300, M1-I299, M1-F298, M1-S297, M1-Y296, M1-T295, M1-T294, M1-H293, M1-N292, M1-L291, M1-K290, M1-W289, M1-H288, M1-G287, M1-L286, M1-E285, M1-H284, M1-A283, M1-I282, M1-V281, M1-A280, M1-V279, M1-I278, M1-E277, M1-D276, M1-E275, M1-N274, M1-K273, M1-C272, M1-Q271, M1-Q270, M1-I269, M1-L268, M1-T267, M1-D266, M1-Y265, M1-L264, M1-V263, M1-I262, M1-R261, M1-K260, M1-N259, M1-K258, M1-F257, M1-F256, M1-G255, M1-Y254, M1-M253, M1-Y252, M1-A251, M1-N250, M1-S249, M1-H248, M1-S247, M1-S246, M1-R245, M1-T244, M1-S243, M1-G242, M1-D241, M1-V240, M1-V239, M1-F238, M1-L237, M1-K236, M1-K235, M1-L234, M1-P233, M1-F232, M1-K231, M1-L230, M1-S229, M1-S228, M1-A227, M1-L226, M1-K225, M1-E224, M1-I223, M1-K222, M1-E221, M1-R220, M1-L219, M1-D218, M1-G217, M1-D216, M1-P215, M1-L214, M1-P213, M1-T212, M1-F211, M1-K210, M1-N209, M1-F208, M1-L207, M1-P206, M1-A205, M1-I204, M1-L203, M1-V202, M1-P201, M1-Y200, M1-I199, M1-T198, M1-M197, M1-M196, M1-V195, M1-L194, M1-S193, M1-L192, M1-I191, M1-F190, M1-M189, M1-F188, M1-A187, M1-W186, M1-L185, M1-Y184, M1-I183, M1-A182, M1-L181, M1-Y180, M1-P179, M1-G178, M1-G177, M1-K176, M1-Q175, M1-V174, M1-I173, M1-F172, M1-I171, M1-I170, M1-A169, M1-A168, M1-V167, M1-I166, M1-P165, M1-P164, M1-G163, M1-L162, M1-I161, M1-V160, M1-S159, M1-L158, M1-F157, M1-T156, M1-G155, M1-K154, M1-I153, M1-M152, M1-D151, M1-R150, M1-I149, M1-F148, M1-M147, M1-W146, M1-I145, M1-T144, M1-Q143, M1-K142, M1-N141, M1-F140, M1-G139, M1-H138, M1-R137, M1-S136, M1-E135, M1-I134, M1-V133, M1-F132, M1-T131, M1-S130, M1-Y129, M1-L128, M1-S127, M1-F126, M1-P125, M1-L124, M1-D123, M1-T122, M1-I121, M1-Q120, M1-S119, M1-W118, M1-T117, M1-M116, M1-V115, M1-G114, M1-A113, M1-L112, M1-F111, M1-S110, M1-L109, M1-T108, M1-H107, M1-L106, M1-I105, M1-E104, M1-N103, M1-E102, M1-P101, M1-D100, M1-L99, M1-G98, M1-L97, M1-R96, M1-P95, M1-L94, M1-V93, M1-A92, M1-G91, M1-S90, M1-M89, M1-K88, M1-W87, M1-F86, M1-W85, M1-P84, M1-L83, M1-I82, M1-G81, M1-F80, M1-F79, M1-L78, M1-I77, M1-A76, M1-S75, M1-D74, M1-M73, M1-L72, M1-I71, M1-T70, M1-V69, M1-F68, M1-E67, M1-H66, M1-V65, M1-F64, M1-H63, M1-F62, M1-Y61, M1-S60, M1-K59, M1-D58, M1-L57, M1-S56, M1-Y55, M1-A54, M1-R53, M1-S52, M1-K51, M1-E50, M1-F49, M1-K48, M1-E47, M1-Q46, M1-S45, M1-I44, M1-V43, M1-G42, M1-V41, M1-L40, M1-T39, M1-K38, M1-P37, M1-L36, M1-T35, M1-P34, M1-L33, M1-K32, M1-L31, M1-A30, M1-T29, M1-L28, M1-Q27, M1-R26, M1-L25, M1-D24, M1-L23, M1-Y22, M1-T21, M1-E20, M1-F19, M1-I18, M1-Y17, M1-M16, M1-V15, M1-I14, M1-M13, M1-F12, M1-G11, M1-V10, M1-V9, M1-T8, M1-E7, of SEQ ID NO: 6. The invention includes the polynucleotide sequences encoding these mutant polypeptides.
GmPrPase
The nucleotide sequence of the full-length PrPase from soybeans ( Glycine max ) (Clone ID No: GmPrPase2) of the present invention is provided as SEQ ID NO: 16. The deduced amino acid sequence of SEQ ID NO: 16 is provided as SEQ ID NO: 17. A partial PrPase nucleotide sequence from soybean (Clone ID No: GmPrPase1) is provided as SEQ ID NO: 14 with the deduced amino acid sequence as SEQ ID NO: 15.
In further embodiments, deletion mutants of GmPrPase are encompassed by the present invention, including the following N-terminal deletion mutants: M1-C400, A2-C400, F3-C400, P4-C400, Y5-C400, M6-C400, E7-C400, A8-C400, V9-C400, V10-C400, G11-C400, F12-C400, M13-C400, I14-C400, L15-C400, M16-C400, Y17-C400, I18-C400, F19-C400, E20-C400, T21-C400, Y22-C400, L23-C400, D24-C400, V25-C400, R26-C400, Q27-C400, H28-C400, R29-C400, A30-C400, L31-C400, K32-C400, L33-C400, P34-C400, T35-C400, L36-C400, P37-C400, K38-C400, T39-C400, L40-C400, E41-C400, G42-C400, V43-C400, I44-C400, S45-C400, Q46-C400, E47-C400, K48-C400, F49-C400, E50-C400, K51-C400, S52-C400, R53-C400, A54-C400, Y55-C400, S56-C400, L57-C400, D58-C400, K59-C400, S60-C400, H61-C400, F62-C400, H63-C400, F64-C400, V65-C400, H66-C400, E67-C400, F68-C400, V69-C400, T70-C400, I71-C400, V72-C400, T73-C400, D74-C400, S75-C400, T76-C400, I77-C400, L78-C400, Y79-C400, F80-C400, G81-C400, V82-C400, L83-C400, P84-C400, W85-C400, F86-C400, W87-C400, K88-C400, K89-C400, S90-C400, G91-C400, D92-C400, F93-C400, M94-C400, T95-C400, I96-C400, A97-C400, G98-C400, F99-C400, N100-C400, A101-C400, E102-C400, N103-C400, E104-C400, I105-C400, L106-C400, H107-C400, T108-C400, L109-C400, A110-C400, F111-C400, L112-C400, A113-C400, G114-C400, L115-C400, M116-C400, I117-C400, W118-C400, S119-C400, Q120-C400, I121-C400, T122-C400, D123-C400, L124-C400, P125-C400, F126-C400, S127-C400, L128-C400, Y129-C400, S130-C400, T131-C400, F132-C400, V133-C400, I134-C400, E135-C400, A136-C400, R137-C400, H138-C400, G139-C400, F140-C400, N141-C400, K142-C400, Q143-C400, T144-C400, P145-C400, W146-C400, L147-C400, F148-C400, F149-C400, R150-C400, D151-C400, M152-C400, L153-C400, K154-C400, G155-C400, I156-C400, F157-C400, L158-C400, S159-C400, V160-C400, I161-C400, I162-C400, G163-C400, P164-C400, P165-C400, I166-C400, V167-C400, A168-C400, A169-C400, I170-C400, I171-C400, V172-C400, I173-C400, V174-C400, Q175-C400, K176-C400, G177-C400, G178-C400, P179-C400, Y180-C400, L181-C400, A182-C400, I183-C400, Y184-C400, L185-C400, W186-C400, V187-C400, F188-C400, T189-C400, F190-C400, G191-C400, L192-C400, S193-C400, I194-C400, V195-C400, M196-C400, M197-C400, T198-C400, L199-C400, Y200-C400, P201-C400, V202-C400, L203-C400, I204-C400, A205-C400, P206-C400, L207-C400, F208-C400, N209-C400, K210-C400, F211-C400, T212-C400, P213-C400, L214-C400, P215-C400, D216-C400, G217-C400, Q218-C400, L219-C400, R220-C400, E221-C400, K222-C400, I223-C400, E224-C400, K225-C400, L226-C400, A227-C400, S228-C400, S229-C400, L230-C400, N231-C400, Y232-C400, P233-C400, L234-C400, K235-C400, K236-C400, L237-C400, F238-C400, V239-C400, V240-C400, D241-C400, G242-C400, S243-C400, T244-C400, R245-C400, S246-C400, S247-C400, H248-C400, S249-C400, N250-C400, A251-C400, Y252-C400, M253-C400, Y254-C400, G255-C400, F256-C400, F257-C400, K258-C400, N259-C400, K260-C400, R261-C400, I262-C400, V263-C400, L264-C400, Y265-C400, D266-C400, T267-C400, L268-C400, I269-C400, Q270-C400, Q271-C400, C272-C400, K273-C400, D274-C400, D275-C400, E276-C400, E277-C400, I278-C400, V279-C400, A280-C400, V281-C400, I282-C400, A283-C400, H284-C400, E285-C400, L286-C400, G287-C400, H288-C400, W289-C400, K290-C400, L291-C400, N292-C400, H293-C400, T294-C400, V295-C400, Y296-C400, T297-C400, F298-C400, V299-C400, A300-C400, M301-C400, Q302-C400, I303-C400, L304-C400, T305-C400, L306-C400, L307-C400, Q308-C400, F309-C400, G310-C400, G311-C400, Y312-C400, T313-C400, L314-C400, V315-C400, R316-C400, N317-C400, S318-C400, A319-C400, D320-C400, L321-C400, Y322-C400, R323-C400, S324-C400, F325-C400, G326-C400, F327-C400, D328-C400, T329-C400, Q330-C400, P331-C400, V332-C400, L333-C400, I334-C400, G335-C400, L336-C400, I337-C400, I338-C400, F339-C400, Q340-C400, H341-C400, T342-C400, V343-C400, I344-C400, P345-C400, L346-C400, Q347-C400, Q348-C400, L349-C400, V350-C400, S351-C400, F352-C400, G353-C400, L354-C400, N355-C400, L356-C400, V357-C400, S358-C400, R359-C400, S360-C400, F361-C400, E362-C400, F363-C400, Q364-C400, A365-C400, D366-C400, G367-C400, F368-C400, A369-C400, K370-C400, K371-C400, L372-C400, G373-C400, Y374-C400, A375-C400, S376-C400, G377-C400, L378-C400, R379-C400, G380-C400, G381-C400, L382-C400, V383-C400, K384-C400, L385-C400, Q386-C400, E387-C400, E388-C400, N389-C400, L390-C400, S391-C400, A392-C400, M393-C400, N394-C400, of SEQ ID NO: 15 or 17. The invention includes the polynucleotide sequences encoding these mutant polypeptides.
In further embodiments, deletion mutants of GmPrPase are encompassed by the present invention, including the following C-terminal deletion mutants: M1-C400, M1-S399, M1-C398, M1-P397, M1-D396, M1-T395, M1-N394, M1-M393, M1-A392, M1-S391, M1-L390, M1-N389, M1-E388, M1-E387, M1-Q386, M1-L385, M1-K384, M1-V383, M1-L382, M1-G381, M1-G380, M1-R379, M1-L378, M1-G377, M1-S376, M1-A375, M1-Y374, M1-G373, M1-L372, M1-K371, M1-K370, M1-A369, M1-F368, M1-G367, M1-D366, M1-A365, M1-Q364, M1-F363, M1-E362, M1-F361, M1-S360, M1-R359, M1-S358, M1-V357, M1-L356, M1-N355, M1-L354, M1-G353, M1-F352, M1-S351, M1-V350, M1-L349, M1-Q348, M1-Q347, M1-L346, M1-P345, M1-I344, M1-V343, M1-T342, M1-H341, M1-Q340, M1-F339, M1-I338, M1-I337, M1-L336, M1-G335, M1-I334, M1-L333, M1-V332, M1-P331, M1-Q330, M1-T329, M1-D328, M1-F327, M1-G326, M1-F325, M1-S324, M1-R323, M1-Y322, M1-L321, M1-D320, M1-A319, M1-S318, M1-N317, M1-R316, M1-V315, M1-L314, M1-T313, M1-Y312, M1-G311, M1-G310, M1-F309, M1-Q308, M1-L307, M1-L306, M1-T305, M1-L304, M1-I303, M1-Q302, M1-M301, M1-A300, M1-V299, M1-F298, M1-T297, M1-Y296, M1-V295, M1-T294, M1-H293, M1-N292, M1-L291, M1-K290, M1-W289, M1-H288, M1-G287, M1-L286, M1-E285, M1-H284, M1-A283, M1-I282, M1-V281, M1-A280, M1-V279, M1-I278, M1-E277, M1-E276, M1-D275, M1-D274, M1-K273, M1-C272, M1-Q271, M1-Q270, M1-I269, M1-L268, M1-T267, M1-D266, M1-Y265, M1-L264, M1-V263, M1-I262, M1-R261, M1-K260, M1-N259, M1-K258, M1-F257, M1-F256, M1-G255, M1-Y254, M1-M253, M1-Y252, M1-A251, M1-N250, M1-S249, M1-H248, M1-S247, M1-S246, M1-R245, M1-T244, M1-S243, M1-G242, M1-D241, M1-V240, M1-V239, M1-F238, M1-L237, M1-K236, M1-K235, M1-L234, M1-P233, M1-Y232, M1-N231, M1-L230, M1-S229, M1-S228, M1-A227, M1-L226, M1-K225, M1-E224, M1-I223, M1-K222, M1-E221, M1-R220, M1-L219, M1-Q218, M1-G217, M1-D216, M1-P215, M1-L214, M1-P213, M1-T212, M1-F211, M1-K210, M1-N209, M1-F208, M1-L207, M1-P206, M1-A205, M1-I204, M1-L203, M1-V202, M1-P201, M1-Y200, M1-L199, M1-T198, M1-M197, M1-M196, M1-V195, M1-I194, M1-S193, M1-L192, M1-G191, M1-F190, M1-T189, M1-F188, M1-V187, M1-W186, M1-L185, M1-Y184, M1-I183, M1-A182, M1-L181, M1-Y180, M1-P179, M1-G178, M1-G177, M1-K176, M1-Q175, M1-V174, M1-I173, M1-V172, M1-I171, M1-I170, M1-A169, M1-A168, M1-V167, M1-I166, M1-P165, M1-P164, M1-G163, M1-I162, M1-I161, M1-V160, M1-S159, M1-L158, M1-F157, M1-I156, M1-G155, M1-K154, M1-L153, M1-M152, M1-D151, M1-R150, M1-F149, M1-F148, M1-L147, M1-W146, M1-P145, M1-T144, M1-Q143, M1-K142, M1-N141, M1-F140, M1-G139, M1-H138, M1-R137, M1-A136, M1-E135, M1-I134, M1-V133, M1-F132, M1-T131, M1-S130, M1-Y129, M1-L128, M1-S127, M1-F126, M1-P125, M1-L124, M1-D123, M1-T122, M1-I121, M1-Q120, M1-S119, M1-W118, M1-I117, M1-M116, M1-L115, M1-G114, M1-A113, M1-L112, M1-F111, M1-A110, M1-L109, M1-T108, M1-H107, M1-L106, M1-I105, M1-E104, M1-N103, M1-E102, M1-A101, M1-N100, M1-F99, M1-G98, M1-A97, M1-I96, M1-T95, M1-M94, M1-F93, M1-D92, M1-G91, M1-S90, M1-K89, M1-K88, M1-W87, M1-F86, M1-W85, M1-P84, M1-L83, M1-V82, M1-G81, M1-F80, M1-Y79, M1-L78, M1-I77, M1-T76, M1-S75, M1-D74, M1-T73, M1-V72, M1-I71, M1-T70, M1-V69, M1-F68, M1-E67, M1-H66, M1-V65, M1-F64, M1-H63, M1-F62, M1-H61, M1-S60, M1-K59, M1-D58, M1-L57, M1-S56, M1-Y55, M1-A54, M1-R53, M1-S52, M1-K51, M1-E50, M1-F49, M1-K48, M1-E47, M1-Q46, M1-S45, M1-I44, M1-V43, M1-G42, M1-E41, M1-L40, M1-T39, M1-K38, M1-P37, M1-L36, M1-T35, M1-P34, M1-L33, M1-K32, M1-L31, M1-A30, M1-R29, M1-H28, M1-Q27, M1-R26, M1-V25, M1-D24, M1-L23, M1-Y22, M1-T21, M1-E20, M1-F19, M1-I18, M1-Y17, M1-M16, M1-L15, M1-I14, M1-M13, M1-F12, M1-G11, M1-V10, M1-V9, M1-A8, M1-E7, of SEQ ID NO: 15 or 17. The invention includes the polynucleotide sequences encoding these mutant polypeptides.
ZmPrPase
The nucleotide sequence of the full-length PrPase from corn ( Zea mays ) (Clone ID No: ZmPrPase2) of the present invention is provided as SEQ ID NO: 23. The deduced amino acid sequence of SEQ ID NO: 23 is provided as SEQ ID NO: 24. A partial PrPase nucleotide sequence from corn (Clone ID No: ZmPrPase1) is provided as SEQ ID NO: 21 with the deduced amino acid sequence as SEQ ID NO: 22.
In further embodiments, deletion mutants of ZmPrPase are encompassed by the present invention, including the following N-terminal deletion mutants: T1-D329, R2-D329, L3-D329, S4-D329, A5-D329, E6-D329, N7-D329, E8-D329, I9-D329, I10-D329, H11-D329, T12-D329, L13-D329, A14-D329, F15-D329, L16-D329, A17-D329, G18-D329, S19-D329, M20-D329, V21-D329, W22-D329, S23-D329, Q24-D329, I25-D329, T26-D329, D27-D329, L28-D329, P29-D329, F30-D329, S31-D329, L32-D329, Y33-D329, S34-D329, T35-D329, F36-D329, V37-D329, I38-D329, E39-D329, A40-D329, R41-D329, H42-D329, G43-D329, F44-D329, N45-D329, K46-D329, Q47-D329, T48-D329, I49-D329, W50-D329, L51-D329, F52-D329, I53-D329, R54-D329, D55-D329, M56-D329, I57-D329, K58-D329, G59-D329, I60-D329, L61-D329, L62-D329, S63-D329, M64-D329, I65-D329, L66-D329, G67-D329, P68-D329, P69-D329, I70-D329, V71-D329, A72-D329, A73-D329, I74-D329, I75-D329, Y76-D329, I77-D329, V78-D329, Q79-D329, I80-D329, G81-D329, G82-D329, P83-D329, Y84-D329, L85-D329, A86-D329, I87-D329, Y88-D329, L89-D329, W90-D329, G91-D329, F92-D329, M93-D329, F94-D329, V95-D329, L96-D329, A97-D329, L98-D329, L99-D329, M100-D329, M101-D329, T102-D329, I103-D329, Y104-D329, P105-D329, I106-D329, V107-D329, I108-D329, A109-D329, P110-D329, L111-D329, F112-D329, N113-D329, K114-D329, F115-D329, T116-D329, P117-D329, L118-D329, P119-D329, E120-D329, G121-D329, V122-D329, L123-D329, R124-D329, E125-D329, K126-D329, I127-D329, E128-D329, K129-D329, L130-D329, A131-D329, A132-D329, S133-D329, L134-D329, K135-D329, F136-D329, P137-D329, L138-D329, K139-D329, K140-D329, L141-D329, F142-D329, V143-D329, V144-D329, D145-D329, G146-D329, S147-D329, T148-D329, R149-D329, S150-D329, S151-D329, H152-D329, S153-D329, N154-D329, A155-D329, Y156-D329, M157-D329, Y158-D329, G159-D329, F160-D329, F161-D329, K162-D329, N163-D329, K164-D329, R165-D329, I166-D329, V167-D329, L168-D329, Y169-D329, D170-D329, T171-D329, L172-D329, I173-D329, Q174-D329, Q175-D329, C176-D329, S177-D329, N178-D329, E179-D329, D180-D329, E181-D329, I182-D329, V183-D329, S184-D329, V185-D329, I186-D329, A187-D329, H188-D329, E189-D329, L190-D329, G191-D329, H192-D329, W193-D329, K194-D329, L195-D329, N196-D329, H197-D329, T198-D329, V199-D329, Y200-D329, S201-D329, F202-D329, V203-D329, A204-D329, V205-D329, Q206-D329, L207-D329, L208-D329, M209-D329, F210-D329, L211-D329, Q212-D329, F213-D329, G214-D329, G215-D329, Y216-D329, T217-D329, L218-D329, V219-D329, R220-D329, S221-D329, S222-D329, K223-D329, D224-D329, L225-D329, F226-D329, G227-D329, S228-D329, F229-D329, G230-D329, F231-D329, K232-D329, D233-D329, Q234-D329, P235-D329, V236-D329, I237-D329, I238-D329, G239-D329, L240-D329, I241-D329, I242-D329, F243-D329, P244-D329, H245-D329, T246-D329, I247-D329, I248-D329, P249-D329, I250-D329, Q251-D329, H252-D329, L253-D329, L254-D329, S255-D329, F256-D329, R257-D329, L258-D329, N259-D329, L260-D329, V261-D329, S262-D329, R263-D329, A264-D329, F265-D329, E266-D329, F267-D329, Q268-D329, A269-D329, D270-D329, A271-D329, F272-D329, A273-D329, K274-D329, N275-D329, L276-D329, G277-D329, Y278-D329, A279-D329, P280-D329, Q281-D329, L282-D329, R283-D329, A284-D329, A285-D329, L286-D329, V287-D329, K288-D329, L289-D329, Q290-D329, E291-D329, E292-D329, N293-D329, L294-D329, S295-D329, A296-D329, M297-D329, N298-D329, T299-D329, D300-D329, P301-D329, W302-D329, Y303-D329, S304-D329, A305-D329, Y306-D329, H307-D329, Y308-D329, S309-D329, H310-D329, P311-D329, P312-D329, L313-D329, V314-D329, E315-D329, R316-D329, L317-D329, Q318-D329, A319-D329, L320-D329, E321-D329, D322-D329, S323-D329, of SEQ ID NO: 22 or 24. The invention includes the polynucleotide sequences encoding these mutant polypeptides.
In further embodiments, deletion mutants of ZmPrPase are encompassed by the present invention, including the following C-terminal deletion mutants: T1-D329, T1-E328, T1-K327, T1-K326, T1-D325, T1-D324, T1-S323, T1-D322, T1-E321, T1-L320, T1-A319, T1-Q318, T1-L317, T1-R316, T1-E315, T1-V314, T1-L313, T1-P312, T1-P311, T1-H310, T1-S309, T1-Y308, T1-H307, T1-Y306, T1-A305, T1-S304, T1-Y303, T1-W302, T1-P301, T1-D300, T1-T299, T1-N298, T1-M297, T1-A296, T1-S295, T1-L294, T1-N293, T1-E292, T1-E291, T1-Q290, T1-L289, T1-K288, T1-V287, T1-L286, T1-A285, T1-A284, T1-R283, T1-L282, T1-Q281, T1-P280, T1-A279, T1-Y278, T1-G277, T1-L276, T1-N275, T1-K274, T1-A273, T1-F272, T1-A271, T1-D270, T1-A269, T1-Q268, T1-F267, T1-E266, T1-F265, T1-A264, T1-R263, T1-S262, T1-V261, T1-L260, T1-N259, T1-L258, T1-R257, T1-F256, T1-S255, T1-L254, T1-L253, T1-H252, T1-Q251, T1-I250, T1-P249, T1-I248, T1-I247, T1-T246, T1-H245, T1-P244, T1-F243, T1-I242, T1-I241, T1-L240, T1-G239, T1-I238, T1-I237, T1-V236, T1-P235, T1-Q234, T1-D233, T1-K232, T1-F231, T1-G230, T1-F229, T1-S228, T1-G227, T1-F226, T1-L225, T1-D224, T1-K223, T1-S222, T1-S221, T1-R220, T1-V219, T1-L218, T1-T217, T1-Y216, T1-G215, T1-G214, T1-F213, T1-Q212, T1-L211, T1-F210, T1-M209, T1-L208, T1-L207, T1-Q206, T1-V205, T1-A204, T1-V203, T1-F202, T1-S201, T1-Y200, T1-V199, T1-T198, T1-H197, T1-N196, T1-L195, T1-K194, T1-W193, T1-H192, T1-G191, T1-L190, T1-E189, T1-H188, T1-A187, T1-I186, T1-V185, T1-S184, T1-V183, T1-I182, T1-E181, T1-D180, T1-E179, T1-N178, T1-S177, T1-C176, T1-Q175, T1-Q174, T1-I173, T1-L172, T1-T171, T1-D170, T1-Y169, T1-L168, T1-V167, T1-I166, T1-R165, T1-K164, T1-N163, T1-K162, T1-F161, T1-F160, T1-G159, T1-Y158, T1-M157, T1-Y156, T1-A155, T1-N154, T1-S153, T1-H152, T1-S151, T1-S150, T1-R149, T1-T148, T1-S147, T1-G146, T1-D145, T1-V144, T1-V143, T1-F142, T1-L141, T1-K140, T1-K139, T1-L138, T1-P137, T1-F136, T1-K135, T1-L134, T1-S133, T1-A132, T1-A131, T1-L130, T1-K129, T1-E128, T1-I127, T1-K126, T1-E125, T1-R124, T1-L123, T1-V122, T1-G121, T1-E120, T1-P119, T1-L118, T1-P117, T1-T116, T1-F115, T1-K114, T1-N113, T1-F112, T1-L111, T1-P110, T1-A109, T1-I108, T1-V107, T1-I106, T1-P105, T1-Y104, T1-I103, T1-T102, T1-M101, T1-M100, T1-L99, T1-L98, T1-A97, T1-L96, T1-V95, T1-F94, T1-M93, T1-F92, T1-G91, T1-W90, T1-L89, T1-Y88, T1-I87, T1-A86, T1-L85, T1-Y84, T1-P83, T1-G82, T1-G81, T1-I80, T1-Q79, T1-V78, T1-I77, T1-Y76, T1-I75, T1-I74, T1-A73, T1-A72, T1-V71, T1-I70, T1-P69, T1-P68, T1-G67, T1-L66, T1-I65, T1-M64, T1-S63, T1-L62, T1-L61, T1-I60, T1-G59, T1-K58, T1-I57, T1-M56, T1-D55, T1-R54, T1-I53, T1-F52, T1-L51, T1-W50, T1-I49, T1-T48, T1-Q47, T1-K46, T1-N45, T1-F44, T1-G43, T1-H42, T1-R41, T1-A40, T1-E39, T1-I38, T1-V37, T1-F36, T1-T35, T1-S34, T1-Y33, T1-L32, T1-S31, T1-F30, T1-P29, T1-L28, T1-D27, T1-T26, T1-I25, T1-Q24, T1-S23, T1-W22, T1-V21, T1-M20, T1-S19, T1-G18, T1-A17, T1-L16, T1-F15, T1-A14, T1-L13, T1-T12, T1-H11, T1-I10, T1-I9, T1-E8, T1-N7, of SEQ ID NO: 22 or 24. The invention includes the polynucleotide sequences encoding these mutant polypeptides.
TABLE 1 cDNA NT Total 5′ NT AA Clone SEQ ID NT Seq. of Start 3′ NT SEQ ID Total AA of ID Vector NO: X of Clone Codon of ORF of ORF NO: Y ORF PpPrPase1 pCR2.1 1 1398 33 1214 2 394 AtPrPase1 pCR2.1 3 1275 1 1272 4 424 AtPrPase2 pCR2.1 5 1275 1 1272 6 424 AtCPP 7 1275 1 1275 8 424 BnCPP 11 1275 1 1275 12 424 GmPrPase1 pCR2.1 14 1434 233 1432 15 400 GmPrPase2 16 1405 39 1313 17 424 GmCPP 18 1275 1 1275 19 424 ZmPrPase1 pCR2.1 21 1301 1 987 22 329 ZmPrPase2 23 1518 166 1443 24 424

Table 1 summarizes the information corresponding to each “cDNA Clone ID.” The nucleotide sequences identified as NT SEQ ID NO: 1, 3, 5, 14, and 21 were assembled from partially homologous (“overlapping”) sequences obtained from the corresponding “cDNA clone ID” identified in Table 1 and, in some cases, from additional related DNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually several overlapping sequences at each nucleotide position), resulting in a final sequence identified under “NT SEQ ID NO:X.”
“Vector” in Table 1 refers to the type of vector contained in the cDNA Clone ID. pCR2.1 was obtained from Invitrogen, Inc.
“Total NT Seq. Of Clone” in Table 1 refers to the total number of nucleotides in the clone identified by “cDNA Clone ID.” The nucleotide position of a sequence under NT SEQ ID NO:X of the putative start codon (methionine) is identified as “5′ NT of Start Codon of ORF.”
The translated amino acid sequence, beginning with the methionine, is identified in Table 1 as “AA SEQ ID NO:Y,” although other reading frames can also be easily translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.
The total number of amino acids within the open reading frame of a sequence under AA SEQ ID NO:Y is identified in Table 1 as “Total AA of ORF”.
DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides may cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
For those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides the nucleotide sequence identified as SEQ ID NOs: 1, 3, 5, 7, 11, 14, 16, 18, 21, and 23 and the predicted translated amino acid sequence identified as SEQ ID NOs: 2, 4, 6, 8, 12, 15, 17, 19, 22, and 24. Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the cDNA, collecting the protein, and determining its sequence. Thus, minor errors in the nucleotide sequence can be recognized by persons skilled in the art.
The present invention also relates to the genes corresponding to SEQ ID NOs: 1, 3, 5, 7, 11, 14, 16, 18, 21, and 23, or SEQ ID NOs: 2, 4, 6, 8, 12, 15, 17, 19, 22, and 24. The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
Also provided in the present invention are species homologs, allelic variants, and/or orthologs. The skilled artisan could, using procedures well-known in the art, obtain the polynucleotide sequence corresponding to full-length genes (including, but not limited to the full-length coding region), allelic variants, splice variants, orthologs, and/or species homologs of genes corresponding to SEQ ID NOs: 1, 3, 5, 7, 11, 14, 16, 18, 21, and 23, or SEQ ID NOs: 2, 4, 6, 8, 12, 15, 17, 19, 22, and 24, relying on the sequences disclosed herein. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers which correspond to the 5′, 3′, or internal regions of the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
The polypeptides may be in the form of the protein, or may be a part of a larger protein, such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence to confer stability during recombinant production.
The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide can be substantially purified using techniques known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using protocols known in the art.
The present invention provides a polynucleotide comprising, or alternatively consisting of, the sequence identified as SEQ ID NOs: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23. The present invention also provides a polypeptide comprising, or alternatively consisting of, the sequence identified as SEQ ID NOs: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24. The present invention also provides polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NOs: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24.
The present invention encompasses polynucleotides which are complementary to the PrPase polynucleotides disclosed herein. Such sequences may be complementary to the sequence disclosed as SEQ ID NOs: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, and/or the nucleic acid sequence encoding the sequence disclosed as SEQ ID NOs: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24.
In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, or a portion of these nucleotide sequence. A nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NO:1, 4, 17, 76, 78, 80, 82, or 84 is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23 that it can hydrogen bond with few or no mismatches to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, thereby forming a stable duplex. Exemplary complement nucleic acid sequences include the sequences of SEQ ID NO: 9, I3, and 20.
As used herein, the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotide units of a nucleic acid molecule, and the term “binding” means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, Von der Waals, hydrophobic interactions, etc. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
Hybridization
The present invention also encompasses polynucleotides capable of hybridizing, preferably under reduced stringency conditions, more preferably under stringent conditions, and most preferably under highly stingent conditions, to polynucleotides described herein. Nucleic acid hybridization techniques are well known in the art. Examples of stringency conditions are shown in Table 2 below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
TABLE 2 Hyridization Wash Stringency Polynucleotide Hybrid Length Temperature Temperature Condition Hybrid†† (bp)‡ and Buffer† and Buffer† A DNA:DNA > or equal to 50 65° C.; 1xSSC -or- 65° C.; 0.3xSSC 42° C.; 1xSSC, 50% formamide B DNA:DNA <50 Tb*; 1xSSC Tb*; 1xSSC C DNA:RNA > or equal to 50 67° C.; 1xSSC 67° C.; 0.3xSSC -or- 45° C.; 1xSSC, 50% formamide D DNA:RNA <50 Td*; 1xSSC Td*; 1xSSC E RNA:RNA > or equal to 50 70° C.; 1xSSC 70° C.; 0.3xSSC -or- 50° C.; 1xSSC, 50% formamide F RNA:RNA <50 Tf*; 1xSSC Tf*; 1xSSC G DNA:DNA > or equal to 50 65° C.; 4xSSC 65° C.; 1xSSC -or- 45° C.; 4xSSC, 50% formamide H DNA:DNA <50 Th*; 4xSSC Th*; 4xSSC I DNA:RNA > or equal to 50 67° C.; 4xSSC 67° C.; 1xSSC -or- 45° C.; 4xSSC, 50% formamide J DNA:RNA <50 Tj*; 4xSSC Tj*; 4xSSC K RNA:RNA > or equal to 50 70° C.; 4xSSC 67° C.; 1xSSC -or- 40° C.; 6xSSC, 50% formamide L RNA:RNA <50 Tl*; 2xSSC Tl*; 2xSSC M DNA:DNA > or equal to 50 50° C.; 4xSSC 50° C.; 2xSSC -or- 40° C. 6xSSC, 50% formamide N DNA:DNA <50 Tn*; 6xSSC Tn*; 6xSSC O DNA:RNA > or equal to 50 55° C.; 4xSSC 55° C.; 2xSSC -or- 42° C.; 6xSSC, 50% formamide P DNA:RNA <50 Tp*; 6xSSC Tp*; 6xSSC Q RNA:RNA > or equal to 50 60° C.; 4xSSC 60° C.; 2xSSC -or- 45° C.; 6xSSC, 50% formamide R RNA:RNA <50 Tr*; 4xSSC Tr*; 4xSSC ‡The “hybrid length” in Table 2 is the anticipated length for the hybridized region(s) of the hybridizing polynucleotides. When hybridizing a polynucletotide of unknown sequence, the hybrid is assumed to be that of the hybridizing polynucleotide of the present invention. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or # regions of optimal sequence complementarity. Methods of aligning two or more polynucleotide sequences and/or determining the percent identity between two polynucleotide sequences are well known in the art (e.g., MegAlign program of the DNA*Star suite of programs, etc). †SSPE in Table 2 (1xSSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is 0.15M NaCl anmd 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. The hydridizations and washes may additionally include 5X Denhardt's reagent, .5-1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide. *Tb − Tr in Table 2. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature Tm of the hybrids there Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(° C.) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between # 18 and 49 base pairs in length, Tm(° C.) = 81.5 + 16.6(log 10 [Na+]) + 0.41(% G + C) − (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([NA+] for 1xSSC = .165 M). ††The present invention encompasses the substitution of any one, or more DNA or RNA hybrid partners with either a peptide nucleic acid (“PNA”), or a modified polynucleotide. Such modified polynucleotides are known in the art.

Additional examples of stringency conditions for polynucleotide hybridization are known to those skilled in the art and are provided, for example, in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel et al., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, which are hereby incorporated by reference herein.
Preferably, such hybridizating polynucleotides have at least 70% sequence identity (more preferably, at least 80% identity; and most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which they hybridize, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps. The determination of identity is well known in the art.
In another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500 or 750 nucleotides in length. In another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
Homologs (i.e., nucleic acids encoding PrPase proteins derived from species other than Arabidopsis thaliana, Physcomitrella patens, Brassica napus, Zea mays , or Glycine max ) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
Alternatively, the phrase “stringent hybridization conditions” can refer to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different depending upon circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60° C. for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65° C. This hybridization is followed by one or more washes in 0.2×SSC, 0.01% BSA at 50° C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the nucleic acid sequences of the invention can correspond to a naturally occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In another embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55° C., followed by one or more washes in 1×SSC, 0.1% SDS at 37° C. Other conditions of moderate stringency that may be used are well known in the art. See, e.g., Ausubel et al. (eds.), 1993, C URRENT P ROTOCOLS IN M OLECULAR B IOLOGY , John Wiley & Sons, NY, and Kriegler, 1990, G ENE T RANSFER AND E XPRESSION , A L ABORATORY M ANUAL , Stockton Press, NY.
In yet another embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40° C., followed by one or more washes in 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50° C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel et al. (eds.), 1993, C URRENT P ROTOCOLS IN M OLECULAR B IOLOGY , John Wiley & Sons, NY, and Kriegler, 1990, G ENE T RANSFER AND E XPRESSION , A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981, Proc Natl Acad Sci USA 78: 6789-6792.
The invention encompasses the application of PCR methodology to the polynucleotide sequences of the present invention and/or the cDNA encoding the polypeptides of the present invention. PCR techniques for the amplification of nucleic acids are described in U.S. Pat. No. 4,683,195 and Saiki et al., Science, 239:487-491 (1988). PCR, for example, may include the following steps, of denaturation of template nucleic acid (if double-stranded), annealing of primer to target, and polymerization. The nucleic acid probed or used as a template in the amplification reaction may be genomic DNA, cDNA, RNA, or a PNA. PCR may be used to amplify specific sequences from genomic DNA, specific RNA sequence, and/or cDNA transcribed from mRNA. References for the general use of PCR techniques, including specific method parameters, include Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR Technology, Stockton Press, NY, 1989; Ehrlich et al., Science, 252:1643-1650, (1991); and “PCR Protocols, A Guide to Methods and Applications”, Eds., Innis et al., Academic Press, New York, (1990).
The present invention includes mature forms of the polypeptide of SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24, and the polynucleotides which encode them. As used herein, a “mature” form of a polypeptide or protein described herein relates to the product of a naturally occurring polypeptide or precursor form or proprotein or any precursor or proprotein which is or can be processed to mature form. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an open reading frame.
Polynucleotide and Polypeptide Variants
The present invention also encompasses variants (e.g., allelic variants, orthologs, etc.) of the polynucleotide sequence disclosed herein in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, and 23, and/or the complementary strand thereto.
The present invention also encompasses variants of the polypeptide sequence, and/or fragments thereof, disclosed in SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24.
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23 due to the degeneracy of the genetic code. These nucleic acids thus encode the same PrPase protein as that encoded by the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, e.g., the polypeptide of SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24.
“Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.
The present invention is also directed to polynucleotide sequences which comprise, or alternatively consist of, a polynucleotide sequence which is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the following non-limiting examples, the polynucleotide sequence of the coding region of the sequence in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, the complementary strand of said coding region, a polynucleotide sequence encoding the polypeptide identified as SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24. The invention also encompasses polynucleotide fragments of any of the polynucleotide sequences provided herein.
The present invention encompasses polypeptide sequences which comprise, or alternatively consist of, an amino acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the following non-limited examples, the polypeptide sequence identified as SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24, and/or polypeptide fragments of any of the polypeptides provided herein.
Preferably, the present invention is directed to an isolated or recombinant polynucleotide wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of a polynucleotide as set forth in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23; or a polynucleotide encoding the polypeptide as set forth in SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24.
The term “sequence identity” refers to the degree to which two polynucleotide or polypeptide sequences are identical on a nucleotide-by-nucleotide or residue-by-residue basis over a particular region of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over that region of comparison (e.g., gaps can be introduced in either of the sequences being compared for optimal alignment between the sequences). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. The number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) or amino acid residue occurs in both sequences is determined to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. As used herein amino acid or nucleic acid “homology” is equivalent to amino acid or nucleic acid “identity.”
As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence or amino acid sequence of the present invention can be determined conventionally using known computer programs. Homology or identity determination using computer programs is well known in the art. The skilled artisan will further appreciate that results may vary depending on the parameters and computer program used.
A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the EMBOSS Pairwise Alignment Algorithms program (publicly available online at EMBL-EBI (European Bioinformatics Institute)). This program uses the Needleman-Wunsch global alignment algorithm to find the optimum alignment (including gaps) of two sequences when considering their entire length (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Preferred parameters used in an EMBOSS Pairwise Alignment of DNA or protein sequences to calculate percent identity are: Matrix=BLOSUM62, Gap Open Penalty=10.0, Gap Extension Penalty=0.1.
The CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2):189-191, (1992), can also be used. In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA or protein sequences to calculate percent identity are: Matrix=BLOSUM, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0, Scoring Method=Percent, Window Size=5 or the length of the subject sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results. This is because the CLUSTALW program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This corrected score is what may be used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALW alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
For example, by a nucleic acid having a nucleotide sequence at least 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence of one of the sequences of the invention, the ORF (open reading frame), or any fragment specified as described herein.
As another example, by a polypeptide having an amino acid sequence at least, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
In another embodiment, derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid. Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., C URRENT P ROTOCOLS IN M OLECULAR B IOLOGY , John Wiley & Sons, New York, N.Y., 1993, and below. An exemplary program is the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, Wis.) using the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2: 482-489, which is incorporated herein by reference in its entirety).
A “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of a PrPase polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. Exemplary homologous nucleic acid sequences include the nucleic acid sequences of SEQ ID NO: 84, 86, 88, and 90. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions in sequences of the invention, as well as a polypeptide having PrPase activity, e.g. substrate binding.
The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter substantially the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the mRNA to those preferred by a bacterial host such as E. coli ).
Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
In addition to the Arabidopsis thaliana, Physcomitrella patens, Brassica napus, Zea mays , or Glycine max PrPase nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of PrPase may exist within a population (e.g., of the plant). Such genetic polymorphism in the PrPase gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a PrPase protein, preferably a plant PrPase protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the PrPase gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in PrPase that are the result of natural allelic variation and that do not alter the functional activity of PrPase are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding PrPase proteins from other species, and thus that have a nucleotide sequence that differs from the sequences of the invention are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the PrPase polynucleotides of the invention can be isolated based on their homology to the Arabidopsis thaliana, Physcomitrella patens, Brassica napus, Zea mays , or Glycine max PrPase nucleic acids disclosed herein using the cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
In addition to naturally-occurring allelic variants of the PrPase sequence that may exist, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of the invention, thereby leading to changes in the amino acid sequence of the encoded PrPase protein, without significantly altering the function of the PrPase protein. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of PrPase without substantially altering the biological activity, whereas an “essential” amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the PrPase proteins of the present invention are predicted to be less amenable to alteration.
In general, a PrPase-like variant that preserves PrPase-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as explained above.
Mutations can be introduced into the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Following mutagenesis of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined. For example, a mutant PrPase protein can be assayed for (1) the ability to form protein:protein interactions with other PrPase proteins, other cell-surface proteins, or biologically active portions thereof, (2) complex formation between a mutant PrPase protein and a PrPase receptor; (3) the ability of a mutant PrPase protein to bind to an intracellular target protein or biologically active portion thereof; (e.g., avidin proteins); (4) the ability to bind PrPase protein; or (5) the ability to specifically bind an anti-PrPase protein antibody.
Besides conservative amino acid substitution, variants of the present invention include, but are not limited to, the following: (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).)
Moreover, the invention further includes polypeptide variants created through the application of molecular evolution (“DNA Shuffling”) methodology to the polynucleotide disclosed as SEQ ID NO:X, and/or the cDNA encoding the polypeptide disclosed as SEQ ID NO:Y. Such DNA Shuffling technology is known in the art (e.g., WPC, Stemmer, PNAS, 91:10747, (1994)).
A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of the present invention having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of the present invention, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In specific embodiments, the number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention or fragments thereof (e.g., the mature form and/or other fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions are preferable.
Polynucleotide and Polypeptide Fragments
The present invention is directed to polynucleotide fragments of the polynucleotides of the invention, in addition to polypeptides encoded therein by said polynucleotides and/or fragments.
In the present invention, a “polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence which is a portion of that shown in SEQ ID NO:X (wherein X is any polynucleotide of the invention) or the complementary strand thereto, or is a portion of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:Y (wherein Y is any polypeptide of the invention). The nucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the nucleotide sequence shown in SEQ ID NO:X. In this context “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus, or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers. Of course, larger fragments (e.g., 50, I50, 500, 600, 2000 nucleotides) are preferred.
Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, I951-2000, or 2001 to the end of SEQ ID NO:X, or the complementary strand thereto. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers. Also encompassed by the present invention are polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions, as are the polypeptides encoded by these polynucleotides.
In the present invention, a “polypeptide fragment” refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:Y. Protein (polypeptide) fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context “about” includes the particularly recited ranges or values, and ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Preferred polypeptide fragments include the full-length protein. Further preferred polypeptide fragments include the full-length protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of the full-length polypeptide. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the full-length protein. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
Alternatively, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, e.g., a fragment that can be used as a probe or primer, or a fragment encoding a biologically active portion of PrPase. Fragments provided herein can also be defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide fragments of SEQ ID NO:Y falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotides encoding these domains are also contemplated.
Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention, which can include mature forms, as measured in a particular biological assay, with or without dose dependency. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention. A nucleic acid fragment encoding a “biologically active portion of PrPase” can be prepared by isolating a portion of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23 that encodes a polypeptide having a PrPase biological activity, expressing the encoded portion of PrPase protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of PrPase. In another embodiment, a nucleic acid fragment encoding a biologically active portion of PrPase includes one or more regions.
In a preferred embodiment, the functional activity displayed by a polypeptide encoded by a polynucleotide fragment of the invention may be one or more biological activities typically associated with the full-length polypeptide of the invention. Illustrative of these biological activities includes the fragments ability to interact with at lease one of the same proteins which bind to the full-length, the fragments ability to bind to at least one of the same polynucleotides as the full-length protein, the fragments ability to bind to a receptor of the full-length protein, the fragments ability to bind to a ligand of the full-length protein, and the fragments ability to multimerize with the full-length protein. However, the skilled artisan would appreciate that some fragments may have biological activities which are desirable and directly inapposite to the biological activity of the full-length protein. The functional activity of polypeptides of the invention, including fragments, variants, derivatives, and analogs thereof can be determined by numerous methods available to the skilled artisan.
Biologically active portions of a PrPase protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the PrPase protein, e.g., the amino acid sequence shown in SEQ ID NO: 8 that include fewer amino acids than the full length PrPase proteins, and exhibit at least one activity of a PrPase protein, e.g. substrate binding. Typically, biologically active portions comprise a domain or motif with at least one activity of the PrPase protein. A biologically active portion of a PrPase protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
A biologically active portion of a PrPase protein of the present invention may contain at least one of the above-identified domains conserved between the PrPase proteins. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native PrPase protein.
A biologically active portion or a PrPase protein can be the N-terminal domain of the PrPase polypeptide. Alternatively, a biologically active portion or a PrPase protein can be the C-terminal domain of the PrPase polypeptide. Preferably, the biologically active portion comprises at least 75 amino acids of the C-terminal domain. More preferably, the biologically active portion comprises at least 25 amino acids of the C-terminal domain. Most preferably, the biologically active portion comprises at least 10 amino acids of the C-terminal.
In an embodiment, the PrPase protein has an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24. In other embodiments, the PrPase protein is substantially homologous to SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24 and retains the functional activity of the protein of SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24, yet differs in amino acid sequence due to natural allelic variation or mutagenesis. Accordingly, in another embodiment, the PrPase protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24 and retains the functional activity of the PrPase proteins of SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24.
Exemplary homologous PrPase polypeptides include for example the polypeptide sequences of SEQ ID NO: 85, 87, 89, and 91.
Fusion Proteins
The invention also provides PrPase chimeric or fusion proteins, i.e. a PrPase polypeptide (all or portion) fused-in-frame to be linked to a non-PrPase polypeptide. The non-PrPase polypeptide can be fused to the N-terminus or C-terminus of the PrPase polypeptide. A PrPase chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. Any polypeptide of the present invention can be used to generate fusion proteins. Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. The polypeptides of the present invention can also be fused to marker sequences (also referred to as “tags”).
Polypeptides of the present invention, including fragments and/or variants thereof, may be fused to any of a number of known, and yet to be determined, toxins, such as ricin, saporin (Mashiba H, et al., Ann N Y Acad. Sci. 1999; 886:233-5), HC toxin (Tonukari N J, et al., Plant Cell. 2000 February; 12(2):237-248), BT endotoxin, or pseudomonas endotoxin. Such fusions could be used to deliver the toxins to desired tissues for which a ligand or a protein capable of binding to the polypeptides of the invention exists.
Polypeptide Libraries
In a further embodiment, libraries of fragments of the PrPase protein coding sequence can be used to generate a variegated population of PrPase fragments for screening and subsequent selection of variants of a PrPase protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a PrPase coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the PrPase protein.
Variants of the PrPase protein that function as either PrPase agonists (mimetics) or as PrPase antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the PrPase protein for PrPase protein agonist or antagonist activity. In one embodiment, a variegated library of PrPase variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of PrPase variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential PrPase sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of PrPase sequences therein. There are a variety of methods which can be used to produce libraries of potential PrPase variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential PrPase sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 11:477.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of PrPase proteins and can be amenable to high throughput analysis.
Vectors, Host Cells, and Protein Production
The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
The vectors of the invention can be recombinant expression vectors which comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
The term “regulatory sequence” is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, G ENE E XPRESSION T ECHNOLOGY : M ETHODS IN E NZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
The polynucleotide insert of the present invention should be operatively linked to an appropriate promoter, such as the 35S promoter, the 34S promoter, CMV promoter, phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters, promoters of retroviral LTRs, constitutive promoters, and ABA inducible promoters to name a few. In addition, it may be desirable, or required, in some instances to direct expression of the nucleotide sequence only in certain host cells by having tissue-specific or cell type-specific promoters operably linked to a polynucleotide of the present invention. Examples of suitable plant-expressible promoters selectively expressed in particular tissues or cell types are well known in the art and include, but are not limited to, guard cell specific promoters, seed specific promoters (e.g., WO 89/03887), organ-primordia specific promoters (An et al., Plant Cell, 8:15-30, (1996)), stem-specific promoters (Keller et al., EMBO J., 7:3625-3633, (1988)), leaf specific promoters (Hudspeth et al., Plant. Mol. Biol., 12:579-589, (1989)), mesophyl-specific promoters (such as the light inducible Rubisco promoters), root-specific promoters (Keller et al., Genes Devel., 3:1639-1646, (1989)), tuber-specific promoters (Keil et al., EMBO J., 8:1323-1330, (1989)), vascular tissue specific promoters (Peleman et al., Gene, 84:359-369, (1989)), meristem specific promoters (such as the promoter of the SHOOTMERISTEMLESS (STM) gene, Long, et al., Nature, 379:66-69, (1996)), primodia specific promoter (such as the Antirrhinum CycD3a gene promoter, Doonan et al., in “Plant Cell Division” (Francis, Duditz, and Inze, Eds), Portland Press, London, (1998)), another specific promoters (WO 89/10396, WO 92/13956, and WO 92/13957), stigma-specific promoters (WO 91/02068), degiscence-zone specific promoters (WO 97/13865), seed-specific promoters (WO 89/03887), etc. Organ-specific promoters are also well known. For example, the patatin class I promoter is transcriptionally activated only in the potato tuber and can be used to target gene expression in the tuber (Bevan, M., 1986, Nucleic Acids Research 14:4625-4636). Another potato-specific promoter is the granule-bound starch synthase (GBSS) promoter (Visser, R. G. R, et al., 1991, Plant Molecular Biology 17:691-699). Other organ-specific promoters appropriate for a desired target organ can be isolated using known procedures. These control sequences are generally associated with genes uniquely expressed in the desired organ. In a typical higher plant, each organ has thousands of mRNAs that are absent from other organ systems (reviewed in Goldberg, P., 1986, Trans. R. Soc. London B 314:343).
Further examples of suitable promoters include promoters from genes such as rice actin (McElroy, et al., Plant Cell, 163-171 (1990)); ubiquitin (Christensen, et al., Plant Mol. Biol., 12: 619-632 (1992); and Christensen, et al., Plant Mol. Biol., 18: 675-689 (1992)); pEMU (Last, et al., Theor. Appl. Genet., 81: 581-588 (1991)); MAS (Velten, et al., EMBO J., 3: 2723-2730 (1984)); maize H3 histone (Lepetit, et al., Mol. Gen. Genet., 231: 276-285 (1992); and Atanassvoa, et al., Plant Journal, 2(3): 291-300 (1992)), the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the rubisco promoter, the GRP1-8 promoter, ALS promoter, (WO 96/30530), a synthetic promoter, such as, Rsyn7, SCP and UCP promoters, ribulose-1,3-diphosphate carboxylase, fruit-specific promoters, heat shock promoters. Additional promoters that may be operably linked to a polynucleotide of the present invention may be found in McElroy and Brettel, Tibtech, Vol. 12, February, 1994. Moreover, a number of promoters are currently being used for transformation of dicotyledonous plants. These promoters come from a variety of different sources. One group of commonly used promoters were isolated from Agrobacterium tumefaciens , where they function to drive the expression of opine synthase genes carried on the T-DNA segment that is integrated into the plant genome during infection. These promoters include the octopine synthase (ocs) promoter (L. Comai et al., 1985; C. Waldron et al., 1985), the mannopine synthase (mas) promoter (L. Comai et al., 1985; K. E. McBride and K. R. Summerfelt, 1990) and the nopaline synthase (nos) promoter (M. W. Bevan et al., 1983; L. Herrera-Estrella et al., 1983, R. T. Fraley et al., 1983, M. De Block et al., 1984; R. Hain et al., 1985). These promoters are active in a wide variety of plant tissue.
In addition, the promoters disclosed in the following publications may also be operably linked to a polynucleotide of the present invention: U.S. Pat. Nos. 5,623,067; 5,683,439; 5,712,112; 5,723,751; 5,723,754; 5,723,757; 5,744,334; 5,750,385; 5,750,399; 5,767,363; 5,783,393; 5,789,214; 5,792,922; 5,792,933; 5,801,027; 5,804,694; 5,814,618; 5,824,857; 5,824,863; 5,824,865; 5,824,866; 5,824,872; and 5,929,302; and International Publication Nos. WO 96/30530, WO 97/49727, WO 98/00533, WO 98/03655, WO 98/07846, WO 98/08961, WO 98/08962, WO 98/10734, WO 98/16634, WO 98/22593, WO 98/38295, and WO 98/44097; and European Patent Application No. EP 0 846 770.
Several viral promoters are also used to, drive heterologous gene expression in dicots (J. C. Kridl and R. M. Goodman, 1986) and may be operably linked to a polynucleotide of the present invention. The Cauliflower Mosaic Virus 35S promoter is one of the promoters used most often for dicot transformation because it confers high levels of gene expression in almost all tissues (J. Odell et al., 1985; D. W. Ow et al., 1986; D. M. Shah et al., 1986). Modifications of this promoter are also used, including a configuration with two tandem 35S promoters (R. Kay et al., 1987) and the mas-35S promoter (L. Comai et al., 1990), which consists of the mannopine synthase promoter in tandem with the 35S promoter. Both of these promoters drive even higher levels of gene expression than a single copy of the 35S promoter. Other viral promoters that have been used include the Cauliflower Mosaic Virus 19S promoter (J. Paszkowski et al., 1984; E. Balazs et al.; Mogen, et al., 1990, Plant Cell, 2: 1261-1272) and the 34S promoter from the figwort mosaic virus (M. Sanger et al., 1990).
Alternatively, the polynucleotide insert of the present invention could be operatively linked to any of a number of inducible promoters known in the art, which include, but are not limited to: tetracycline inducible promoters, small-molecule inducible promoters, light inducible promoters, chemical compounds (e.g., safeners, herbicides, glucocorticoids, etc.), abiotic stress inducible promoters (e.g., wounding, heavy metals, cold-sensitive promoters, heat-sensitive promoters, salt sensitive promoters, drought sensitive promoters, hypoxia indicuble (such as those disclosed in EP 1012317), etc.), biotic stress promoters (e.g., pathogen or pest infection including infection by fungi, viruses, bacteria, insects, nematodes, mycoplasms, and mycoplasma-like organisms, etc.). Examples of plant-expressible inducible promoters suitable for the invention are: nematode inducible promoters (such as those disclosed in WO 92/21757 and/or EP1007709), fungus inducible promoters (WO 93/19188, WO 96/28561), chemically inducible Arabidopsis PR-1 promoter (WO 98/03536), the inducible promoters disclosed in WO 98/45445, the inducible promoters disclosed in U.S. Pat. No. 5,804,693, the tomato soft fruit inducible promoter disclosed in U.S. Pat. No. 5,821,398, promoters inducible after application of glucocorticoids such as dexamethasone, or promoters repressed or activated after application of tetracyclin (Gatz et al., PNAS USA, 85:1394-1397, (1988)). Other suitable inducible promoters will be known to the skilled artisan.
Exemplary expression vector constructs include for example the constructs of SEQ ID NO: 41, 42, 52, 53, 55, 56, 57, 58, 60, 61, 63, 64, 66, 67, and 69. Additional exemplary expression vector constructs include constructs comprising PrPase anti-sense nucleic acid such as SEQ ID NO: 51, 54, 59, 62, 65, and 68.
In addition, the polynucleotide insert of the present invention could be operatively linked to “artificial” or chimeric promoters and transcription factors. Specifically, the artificial promoter could comprise, or alternatively consist, of any combination of cis-acting DNA sequence elements that are recognized by trans-acting transcription factors. Preferably, the cis acting DNA sequence elements and trans-acting transcription factors are operable in plants. Further, the trans-acting transcription factors of such “artificial” promoters could also be “artificial” or chimeric in design themselves and could act as activators or repressors to said “artificial” promoter. For example, a chimeric promoter of the invention could comprise one or more, upstream activating sequences from the Octopine Synthase gene (OCS), matrix attachment regions (MAR), etc.
One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication). Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors or plant transformation vectors, binary or otherwise, which serve equivalent functions.
The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
The expression constructs may additionally comprise 5′ leader sequences in the expression constructs. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, 0., Fuerst, T. R., and Moss, B. (1989) PNAS USA, 86:6126-6130); polyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986)); MDMV leader (Maize Dwarf Mosaic Virus) (Virology, 154:9-20); and human immunoglobulin heavy-chain binding protein (BiP), (Macejak, D. G., and Sarnow, P. (1991) Nature, 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L., (1987) Nature, 325:622-625); tobacco mosaic virus leader (TW), (Gallie, D. R. et al. (1989) Molecular Biology of RNA, pages 237-256); and maize chlorotic mottle virus leader (MCNW) (Lommel, S. A. et al. (1991) Virology, 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiology., 84:965-968. Other methods known to enhance translation can also be utilized, for example, introns, and the like.
Additional regulatory elements that may be connected to a PrPase encoding nucleic acid sequence for expression in plant cells include terminators, polyadenylation sequences, and nucleic acid sequences encoding signal peptides that permit localization within a plant cell or secretion of the protein from the cell. Such regulatory elements and methods for adding or exchanging these elements with the regulatory elements PrPase gene are known, and include, but are not limited to, 3′ termination and/or polyadenylation regions such as those of the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan, et al., Nucl. Acids Res., 12: 369-385 (1983)); the potato proteinase inhibitor II (PINII) gene (Keil, et al., Nucl. Acids Res., 14: 5641-5650 (1986) and hereby incorporated by reference); and An, et al., Plant Cell, 1: 115-122 (1989)); and the CaMV 19S gene (Mogen, et al., Plant Cell, 2: 1261-1272 (1990)).
As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include, but are not limited to, dihydrofolate reductase, G418 or neomycin resistance, kanamycin resistance, hygromycin resistance, bialaphos resistance, sulfonoamide resistance, stretomycin resistance, spectinomycin resistance, chlorosulfuron resistance, glyphosphate resistance, and methotrexate resistance, for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Markers may also include resistance to bleomycin and gentamicin. For Example, after transforming plant cells, those cells having the vector will be identified by their ability to grow on a medium containing the particular antibiotic/marker. Replication sequences, of bacterial or viral origin, are generally also included to allow the vector to be cloned in a bacterial or phage host, preferably a broad host range prokaryotic origin of replication is included. A selectable marker for bacteria should also be included to allow selection of bacterial cells bearing the desired construct.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, Bacillus subtilis , and Salmonella typhimurium cells (or any bacterial strain capable of expressing heterologous polypeptides); fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae, Pichia pastoris (ATCC Accession No. 201178), Schizosaccharomyces pombe, Kluyveromyces strains, Candida , or any yeast strain capable of expressing heterologous proteins); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; plant cells, and specifically plant cells and/or tissues derived from any of the plants listed in Table 3. Plant host cells include, for example, plant cells that could function as suitable hosts for the expression of a polynucleotide of the invention include epidermal cells, mesophyll and other ground tissues, and vascular tissues in leaves, stems, floral organs, and roots. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
The polynucleotides and polypeptides of the present invention can be targeted to the chloroplast or amyloplast for expression. In this manner, the expression construct will additionally contain a polynucleotide sequence encoding a transit peptide operably linked to a polynucleotide of the present invention to direct the polynucleotide of the present invention to the chloroplasts. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481.
The expression construct may also comprise any other necessary regulators such as nuclear localization signals (Kalderon et al. (1984) Cell 39:499-509; and Lassner et al. (1991) Plant Molecular Biology 17:229-234); plant translational consensus sequences (Joshi, C. P. (1987) Nucleic Acids Research 15:6643 6653), introns (Luehrsen and Walbot (1991) Mol. Gen. Genet. 225:81-93) and the like, operably linked to a polynucleotide of the present invention.
Plant signal sequences, including, but not limited to, signal-peptide encoding DNA/RNA sequences which target proteins to the extracellular matrix of the plant cell (Dratewka-Kos, et al., J. Biol. Chem., 264: 4896-4900 (1989)) and the Nicotiana plumbaginifolia extension gene (DeLoose, et al., Gene, 99: 95-100 (1991)), or signal peptides which target proteins to the vacuole like the sweet potato sporamin gene (Matsuka, et al., Proc. Nat'l Acad. Sci. (USA), 88: 834 (1991)) and the barley lectin gene (Wilkins, et al., Plant Cell, 2: 301-313 (1990)), or signals which cause proteins to be secreted such as that of PRIb (Lind, et al., Plant Mol. Biol., 18: 47-53 (1992)), or those which target proteins to the plastids such as that of rapeseed enoyl-ACP reductase (Verwaert, et al., Plant Mol. Biol., 26: 189-202 (1994)) are useful in the invention.
It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., PrPase proteins, mutant forms of PrPase proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of PrPase proteins in prokaryotic or eukaryotic cells. For example, PrPase proteins can be expressed in bacterial cells such as Escherichia coli , insect cells (using baculovirus expression vectors) yeast cells, plant cells or mammalian cells. Suitable host cells are discussed further in Goeddel, G ENE E XPRESSION T ECHNOLOGY : M ETHODS IN E NZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
The polynucleotide sequences encoding the proteins or polypeptides of the present invention may be particularly useful in the genetic manipulation of plants. In this manner, the polynucleotides of the invention are provided in expression cassettes for expression in the plant of interest. Where appropriate, the gene(s) may be optimized for increased expression in the transformed plant. That is, the polynucleotides can be synthesized using plant preferred codons for improved expression specific to a particular species. Methods are available in the art for synthesizing plant preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
Depending upon the species in which the DNA sequence of interest is to be expressed, it may be desirable to synthesize the sequence with plant preferred codons, or alternatively with chloroplast preferred codons. The plant preferred codons may be determined from the codons of highest frequency in the proteins expressed in the largest amount in the particular plant species of interest. See, EPA 0359472; EPA 0385962; WO 91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids
Research. In this manner, the polynucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used.
Additionally, it may be desirable to selectively express a polypeptide of the present invention in a specific target cell or tissue of a plant by synthesizing the encoding polynucleotide sequence to contain codons optimized for high translational efficiency within the particular target cell or tissue. Such methods are known in the art and are specifically provided in PCT International Publication No. WO 00/42190 (which is hereby incorporated herein by reference).
Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence may be modified to avoid predicted hairpin secondary mRNA structures.
For in situ production of the antisense mRNA of GST, those regions of the glutathione S-transferase (“GST”) gene which are transcribed into GST mRNA, including the untranslated regions thereof, are inserted into the expression vector under control of the promoter system in a reverse orientation. The resulting transcribed mRNA is then complementary to that normally produced by the plant.
Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., G ENE E XPRESSION T ECHNOLOGY : M ETHODS IN E NZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, G ENE E XPRESSION T ECHNOLOGY : M ETHODS IN E NZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlbad, Calif.).
In another embodiment, the PrPase expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisiae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
Alternatively, PrPase can be expressed in insect cells using baculovirus expression vectors.
In a preferred embodiment, a nucleic acid of the invention is expressed in plants cells using a plant expression vector. Examples of plant expression vectors systems include tumor inducing (Ti) plasmid or portion thereof found in Agrobacterium , cauliflower mosaic virus (CAMV) DNA and vectors such as pBI121. Preferred expression vectors in plant systems include, but are not limited to, Bin 19 (ATCC Deposit No: 37327), GA437 (ATCC Deposit No: 37350), pAK1003 (ATCC Deposit No: 37425), pAS2022 (ATCC Deposit No: 37426), pAS2023 (ATCC Deposit No: 37427), pAP2034 (ATCC Deposit No: 37428), pC22 (ATCC Deposit No: 37493), pHS24 (ATCC Deposit No: 37841), pHS85 (ATCC Deposit No: 37842), pPM1 (ATCC Deposit No: 40172), pGV3111SE (ATCC Deposit No: 53213), pCGN978 (ATCC Deposit No: 67064), pFL61 (ATCC Deposit No: 77215), pGPTV-KAN (ATCC Deposit No: 77388), pGPTV-HPT (ATCC Deposit No: 77389), pGPTV-DHFR (ATCC Deposit No: 77390), pGPTV-BAR (ATCC Deposit No: 77391), pGPTV-BLEO (ATCC Deposit No: 77392), and/or pPE1000 (ATCC Deposit No: 87573). The skilled artisan would appreciate that any of the above vectors could easily be modified to either include or delete specific elements as may be required for operability. Other suitable vectors will be readily apparent to the skilled artisan.
For expression in plants, the recombinant expression cassette will contain in addition to the PrPase nucleic acids, a plant promoter region, a transcription initiation site (if the coding sequence to transcribed lacks one), and a transcription termination/polyadenylation sequence. The termination/polyadenylation region may be obtained from the same gene as the promoter sequence or may be obtained from different genes. Unique restriction enzyme sites at the 5′ and 3′ ends of the cassette are typically included to allow for easy insertion into a pre-existing vector.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell.
Introduction of the construct into the host cell can be effected by biolistic transformation (Klein et al., Nature, 327:70-73 (1987)), PEG-mediated transfection (Paskowski, et al., EMBO J., 3:2717, (1984)), calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation (Fromm, et al., PNAS, USA, 82:5824 (1985)), transduction, infection, Agrobacterium tumefaciens -directed infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986).
A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods.
Alternatively, a polypeptide or protein may also be expressed in a form which will facilitate purification. Polypeptides of the present invention, can also be recovered from: products purified from natural sources, including tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, and higher plant cells.
The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M. Egholm, R. H. Berg and O. Buchardt, Science 254, I497 (1991); and M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the stronger binding characteristics of PNA:DNA hybrids. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.
In addition, polypeptides, analogs, derivatives, and/or fragments of the invention can be chemically synthesized.
Each of the polynucleotides identified herein can be used in numerous ways as reagents, including for chromosome identification and mapping, as a diagnostic or prognostic marker, for control of gene expression through triple helix formation or antisense DNA or RNA, for producing a new trait in the host cell, for identifying organisms from minute biological samples, as polymorphic markers, for identifying the source of a particular tissue.
In preferred embodiments, the polynucleotides are used for modulating, inhibiting, increasing, decreasing, or introducing traits in a plant including one or more of drought tolerance, UV tolerance, flower development, terpene synthesis, abiotic stressed tolerance, heat stress tolerance, cold stress tolerance, nutritional stress tolerance, xenobiotic stress tolerance, protein storage capability, oil storage capability, amino acid content, amino acid composition, carbohydrate storage capability, oil content, oil composition, carbohydrate content, carbohydrate composition, fiber content, fiber composition, metabolite content, metaboliter composition, vitamin content, and/or vitamin composition. The polynucleotides of the invention, are useful in modulating plant yield, plant development, plant differentiation, root growth, root morphology, plant color, plant aroma, plant flavor, palatability of plant tissue, plant organoleptic properties, may be useful in phytoremediation, and/or plant defense. Moreover, the polypeptides of the invention may also be useful in modulating the plants ability to serve as a plant neutriceutical, pharmaceutical, or phytoceutical. Alternatively, polypeptides of the invention may also be useful in modulating the plants ability to produce plant neutriceuticals, pharmaceuticals, or phytoceuticals of either endogenous or exogenous origin (e.g., from another plant species, a human, a mammal, an animal, or other organism). In these contexts, the term “plant” may be applied to mean any plant cell, plant tissue, plant fluid, or plant feature, and includes plant infection structures, which may include, but are not limited to an appressorium, a gall, a canker, and/or nodules. In these contexts, the term “modulate” may be applied to mean the qualitative or quantitative increase, decrease, introduction of, inhibition of, complete loss of, or over-expression of a specific trait or characteristic.
Each of the polypeptides identified herein can be used in numerous ways appreciated by those skilled in the art, including assaying protein levels in a biological sample using antibody-based techniques and in vivo imaging.
The PrPase proteins can be used to screen compounds that modulate the PrPase protein activity or expression. In addition, anti-PrPase antibodies can be made and used to detect and isolate PrPase proteins and modulate PrPase activity.
Another aspect of the present invention relates to methods for the introduction of nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an organism, preferably a plant, to achieve expression of a polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the invention operatively linked to a promoter and other genetic elements necessary for the expression of the polypeptide by the organism or a target tissue. Such transgenic and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.
The polynucleotide constructs can be delivered by any method that delivers materials to the cells of an organism, such as, biolistic injection into the plant tissues (apical meristem, root, flower, stem, and the like). The polynucleotide constructs may be delivered in an acceptable liquid or aqueous carrier.
In one embodiment, the polynucleotide of the invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.
The polynucleotide vector constructs of the invention may integrate into the host genome and may replicate. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.
Any strong promoter known to those skilled in the art can be used for driving the expression of polynucleotide sequence of the invention. Suitable promoters include the 35S, 34S, and actin promoters, in addition to any other promoter known in the art and/or described elsewhere herein. The promoter also may be the native promoter for the polynucleotides of the invention.
The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct injection, direct needle injection at the delivery site, topical administration, and so-called “gene guns”. These delivery methods are known in the art.
The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.
In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA that comprises a sequence encoding polypeptides of the invention. Retroviral integration has been detected to occur in plants based upon the identification of a pararetrovirus sequences within the tobacco genome. Since such integration was determined to occur at very limited integration sites, such a pararetrovirus may represent a desirable genetic transformation vehicle for the polynucleotides of the present invention (Jakowitsch, J., et al., PNAS 96(23):13241-6 (1999).
The present invention also encompasses the application of retrotransposons to the genetic transformation of plants. The retrotransposons would preferably represent retrotransposons with known plant host range and would comprise polynucleotides encoding polypeptides of the present invention. Many retrotransposons are known in the art, some of which are described by Bennetzen J L, Trends Microbiol., 4(9):347-53 (1996) which is hereby incorporated herein by reference.
Polynucleotide constructs are made using standard techniques known in the art.
Preferably, the polynucleotide encoding a polypeptide of the invention contains a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5′ end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transformed. Additionally, the signal sequence may be chemically synthesized using methods known in the art.
Plant Transformation
The invention includes protoplast, plants cells, plant tissue and plants (e.g., monocots and dicots) transformed with a PrPase nucleic acid, a vector containing a PrPase nucleic acid or an expression vector containing a PrPase nucleic acid (i.e., sense or antisense). As used herein, “plant” is meant to include not only a whole plant but also a portion thereof (i.e., cells, and tissues, including for example, leaves, stems, shoots, roots, flowers, fruits and seeds and the progeny derived therefrom).
The plant can be any plant type including, for example, species from the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Gossypium, Picea, Caco , and Populus.
Preferred plants, may include the following non-limiting examples, including barley, oats, rye, sorghum, pea, sunflower, tobacco, cotton, petunia, tomato, broccoli, lettuce, apple, plum, orange, and lemon, and more preferrably rice, maize, conola, wheat, sugerbeet, sugercane, and soybean, in addition to other plants known in the art and referenced more particularly elsewhere herein (e.g., Table 3).
Non-limiting examples of suitable recipient plants for introducing polynucleotides of the invention, polynucleotides encoding the polypeptides of the invention, and/or fragments, and variants therein, are listed in Table 3 below:
TABLE 3 RECIPIENT PLANTS COMMON NAME FAMILY LATIN NAME Maize Gramineae Zea mays Maize, Dent Gramineae Zea mays dentiformis Maize, Flint Gramineae Zea mays vulgaris Maize, Pop Gramineae Zea mays microsperma Maize, Soft Gramineae Zea mays amylacea Maize, Sweet Gramineae Zea mays amyleasaccharata Maize, Sweet Gramineae Zea mays saccharate Maize, Waxy Gramineae Zea mays ceratina Wheat, Dinkel Pooideae Triticum spelta Wheat, Durum Pooideae Triticum durum Wheat, English Pooideae Triticum turgidum Wheat, Large Spelt Pooideae Triticum spelta Wheat, Polish Pooideae Triticum polonium Wheat, Poulard Pooideae Triticum turgidum Wheat, Singlegrained Pooideae Triticum monococcum Wheat, Small Spelt Pooideae Triticum monococcum Wheat, Soft Pooideae Triticum aestivum Rice Gramineae Oryza sativa Rice, American Wild Gramineae Zizania aquatica Rice, Australian Gramineae Oryza australiensis Rice, Indian Gramineae Zizania aquatica Rice, Red Gramineae Oryza glaberrima Rice, Tuscarora Gramineae Zizania aquatica Rice, West African Gramineae Oryza glaberrima Barley Pooideae Hordeum vulgare Barley, Abyssinian Pooideae Hordeum irregulare Intermediate, also Irregular Barley, Ancestral Pooideae Hordeum spontaneum Tworow Barley. Beardless Pooideae Hordeum trifurcatum Barley, Egyptian Pooideae Hordeum trifurcatum Barley, fourrowed Pooideae Hordeum vulgare polystichon Barley, sixrowed Pooideae Hordeum vulgare hexastichon Barley, Tworowed Pooideae Hordeum distichon Cotton, Abroma Dicotyledoneae Abroma augusta Cotton, American Malvaceae Gossypium hirsutum Upland Cotton, Asiatic Tree, also Malvaceae Gossypium arboreum Indian Tree Cotton, Brazilian, also, Malvaceae Gossypium barbadense Kidney, and, brasiliense Pernambuco Cotton, Levant Malvaceae Gossypium herbaceum Cotton, Long Silk, also Malvaceae Gossypium barbadense Long Staple, Sea Island Cotton, Mexican, also Malvaceae Gossypium hirsutum Short Staple Soybean, Soya Leguminosae Glycine max Sugar beet Chenopodiaceae Beta vulgaris altissima Sugar cane Woody-plant Arenga pinnata Tomato Solanaceae Lycopersicon esculentum Tomato, Cherry Solanaceae Lycopersicon esculentum cerasiforme Tomato, Common Solanaceae Lycopersicon esculentum commune Tomato, Currant Solanaceae Lycopersicon pimpinellifolium Tomato, Husk Solanaceae Physalis ixocarpa Tomato, Hyenas Solanaceae Solanum incanum Tomato, Pear Solanaceae Lycopersicon esculentum pyriforme Tomato, Tree Solanaceae Cyphomandra betacea Potato Solanaceae Solanum tuberosum Potato, Spanish, Sweet Convolvulaceae Ipomoea batatas potato Rye, Common Pooideae Secale cereale Rye, Mountain Pooideae Secale montanum Pepper, Bell Solanaceae Capsicum annuum grossum Pepper, Bird, also Solanaceae Capsicum annuum minimum Cayenne, Guinea Pepper, Bonnet Solanaceae Capsicum sinense Pepper, Bullnose, also Solanaceae Capsicum annuum grossum Sweet Pepper, Cherry Solanaceae Capsicum annuum cerasiforme Pepper, Cluster, also Red Solanaceae Capsicum annuum fasciculatum Cluster Pepper, Cone Solanaceae Capsicum annuum conoides Pepper, Goat, also Spur Solanaceae Capsicum frutescens Pepper, Long Solanaceae Capsicum frutescens longum Pepper, Oranamental Solanaceae Capsicum annuum abbreviatum Red, also Wrinkled Pepper, Tabasco Red Solanaceae Capsicum annuum conoides Lettuce, Garden Compositae Lactuca sativa Lettuce, Asparagus, also Compositae Lactuca sativa asparagina Celery Lettuce, Blue Compositae Lactuca perennis Lettuce, Blue, also Compositae Lactuca pulchella Chicory Lettuce, Cabbage, also Compositae Lactuca sativa capitata Head Lettuce, Cos, also Compositae Lactuca sativa longifolia Longleaf, Romaine Lettuce, Crinkle, also Compositae Lactuca sativa crispa Curled, Cutting, Leaf Celery Umbelliferae Apium graveolens dulce Celery, Blanching, also Umbelliferae Apium graveolens dulce Garden Celery, Root, also Umbelliferae Apium graveolens rapaceum Turniprooted Eggplant, Garden Solanaceae Solanum melongena Sorghum Sorghum All crop species Alfalfa Leguminosae Medicago sativum Carrot Umbelliferae Daucus carota sativa Bean, Climbing Leguminosae Phaseolus vulgaris vulgaris Bean, Sprouts Leguminosae Phaseolus aureus Bean, Brazilian Broad Leguminosae Canavalia ensiformis Bean, Broad Leguminosae Vicia faba Bean, Common, also Leguminosae Phaseolus vulgaris French, White, Kidney Bean, Egyptian Leguminosae Dolichos lablab Bean, Long, also Leguminosae Vigna sesquipedalis Yardlong Bean, Winged Leguminosae Psophocarpus tetragonolobus Oat, also Common, Side, Avena Avena sativa Tree Oat, Black, also Bristle, Avena Avena strigosa Lopsided Oat, Bristle Avena Pea, also Garden, Green, Leguminosae Pisum, sativum sativum Shelling Pea, Blackeyed Leguminosae Vigna sinensis Pea, Edible Podded Leguminosae Pisum sativum axiphium Pea, Grey Leguminosae Pisum sativum speciosum Pea, Winged Leguminosae Tetragonolobus purpureus Pea, Wrinkled Leguminosae Pisum sativum medullare Sunflower Compositae Helianthus annuus Squash, Autumn, Winter Dicotyledoneae Cucurbita maxima Squash, Bush, also Dicotyledoneae Cucurbita pepo melopepo Summer Squash, Turban Dicotyledoneae Cucurbita maxima turbaniformis Cucumber Dicotyledoneae Cucumis sativus Cucumber, African, also Momordica charantia Bitter Cucumber, Squirting, Ecballium elaterium also Wild Cucumber, Wild Cucumis anguria Poplar, California Woody-Plant Populus trichocarpa Poplar, European Black Populus nigra Poplar, Gray Populus canescens Poplar, Lombardy Populus italica Poplar, Silverleaf, also Populus alba White Poplar, Western Balsam Populus trichocarpa Tobacco Solanaceae Nicotiana Arabidopsis thaliana Cruciferae Arabidopsis thaliana Turfgrass Lolium Turfgrass Agrostis Other families of turfgrass Clover Leguminosae

Numerous methods for introducing foreign genes into plants are known and can be used to insert a gene into a plant host, including biological and physical plant transformation protocols. See, for example, Miki et al., (1993) “Procedure for Introducing Foreign DNA into Plants”, In: Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson, eds., CRC Press, Inc., Boca Raton, pages 67-88 and Andrew Bent in, Clough S J and Bent A F, 1998. Floral dipping: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana . The methods chosen vary with the host plant, and include chemical transfection methods such as calcium phosphate, polyethylene glycol (PEG) transformation, microorganism-mediated gene transfer such as Agrobacterium (Horsch, et al., Science, 227: 1229-31 (1985)), electroporation, protoplast transformation, micro-injection, flower dipping and biolistic bombardment.
Also included in the invention are methods of producing a transgenic plant. The method includes introducing into one or more plant cells a compound that alters PrPase expression or activity in the plant to generate a transgenic plant cell and regenerating a transgenic plant from the transgenic cell. In some aspects the compound increases alters PrPase expression or activity. Alternatively, the compound decrease alters PrPase expression or activity. The compound can be, e.g., (i) a PrPase polypeptide; (ii) a nucleic acid encoding a PrPase polypeptide; (iii) a nucleic acid that increases expression of a nucleic acid that encodes a PrPase polypeptide; (iv) a nucleic acid that decreases the expression of a nucleic acid that encodes a PrPase polypeptide; (v) a PrPase antisense nucleic acid and derivatives, fragments, analogs and homologs thereof. A nucleic acid that increases expression of a nucleic acid that encodes a PrPase polypeptide includes, e.g., promoters, enhancers. The nucleic acid can be either endogenous or exogenous. Preferably, the “compound” is a nucleic acid encoding a PrPase polypeptide of the invention. For example, the compound comprises the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, or a fragment thereof. Alternatively, the compound is a PrPase antisense nucleic acid. For example, the compound comprises the nucleic acid sequence of SEQ ID NO: 9, I3, or 20.
In various aspects the transgenic plant has an altered phenotype as compared to a wild type plant (i.e., untransformed). By altered phenotype is meant that the plant has a one or more characteristic that is different from the wild type plant. For example, the transgenic plant has an increased resistance to stress. An “increased” stress means that the transgenic plant can grow under stress conditions (e.g., high salt, decreased water, low temperatures, high temperatures) or under conditions that normally inhibit the growth of an untransformed plant. Stresses include, for example, chilling stress, heat stress, heat shock, salt stress, water stress (i.e., drought), nutritional stress, disease, grazing pests, wound healing, pathogens such as for example fungi, bacteria, nematodes, viruses or parasitic weed and herbicides. Alternatively, the transformed plant has an increased (i.e., enhanced) ABA sensitivity. The enhanced ABA sensitivity is at the seedling growth stage. Alternatively, the enhanced ABA sensitivity is at the mature plant stage. Additional altered phenotypes include for example, enhanced vegetative growth (e.g., increased leaf number, thickness and overall biomass), delayed reproductive growth (e.g., flowering later); enhanced seedling vigor (e.g., increased root biomass and length), enhanced lateral root formation and therefore soil penetration more extensive vascular system resulting in an enhanced transport system. In preferred embodiments, the growth and/or yield of transgenic plants is improved relative to a wild-type under a stress condition.
The polynucleotides of the invention are introduced into plant cells using, for example, Agrobacterium -mediated transformation, microprojectile-mediated transformation, sonication of target cells, liposome or spheroplast fusion, direct uptake into protoplasts by CaCl 2 precipitation, electroporation of protoplasts and whole cells and tissues, wounding by particle bombardment followed by use of Agrobacterium for DNA delivery, intact meristem transformation, split meristem method.
Once a single transformed plant has been obtained, conventional plant breeding methods can be used to transfer the gene and associated regulatory sequences via crossing and backcrossing.
Methodologies to determine plant growth or response to stress include for example, height measurements, weight measurements, leaf area, ability to flower, water use, transpiration rates and yield.
Infectious Agents and Pests
A polynucleotide or polypeptide of the present invention can be used to detect, prevent, and/or confer resistance to infectious agents and/or pests which may, for example, inhibit the plants ability to control cellular division, differentiation, and development; absorption of water and minerals from the soil and the translocation of these substances throughout the plant; photosynthesis and translocation of the photosynthetic products to areas of use or storage; metabolism of synthesized compounds; reproduction; and storage of plant food-stuffs for overwintering or reproduction. For example, infection of the root (e.g., root rot), interferes with absorption of water and nutrients from the soil; infection of the xylem vessels (e.g., vascular wilts, cankers, etc.) interferes with translocation of water and minerals to the crown of the plant; infection of the foliage (e.g., leaf spots, blights, mosaics, etc.) interferes with photosynthesis; infection of the cortex (e.g., cortical canker, viral and mycoplasmal infections of phloem, etc.) interferes with the downward translocation of photosynthetic products; flower infections (e.g., bacterial and fungal blights, viral, mycoplasmal, and fungal infections of flowers, etc.) interfere with reproduction; and infections of fruit (e.g., fruit rot, etc.) interfere with reproduction or storage of reserve food stuffs for the new plant.
Infectious agents and pests include, but are not limited to, viruses, bacteria, fungi, parasitic agents (i.e. parasitic weeds), herbaceous species, nematodes, and insects.
Defense Mechanisms and Plant Hormones
A polypeptide or polynucleotide and/or agonist or antagonist of the present invention can be used to increase a plants defense mechanisms against either environmental or pathogenic stresses (e.g., viral, fungal, mycoplasma, bacterial, nematode, herbicidal, insecticidal, acid rain, drought, chemical, etc.). Such defense mechanisms may be a combination of structural characteristics (i.e., to serve as a physical barrier to inhibit a pathogen, for example, from entering or spreading throughout the plant), and biochemical reactions either on the scale of the whole plant or of individual cells (e.g., producing substances that are either toxic to the pathogen, or create an environment that is non-permissive for pathogen survival, etc.).
A polynucleotide or polypeptide and/or agonist or antagonist of the present invention can be used to modulate the hormone levels within a plant (including any of its cells, tissues, and/or organs, etc.). Examples of hormones that may be modulated by the present invention, either directly or indirectly, generally include, but are not limited to, auxins, indoleacetic acid, gibberellins, cytokinins, ethylene, abscisic acid, polyamines, jasmonates, salicylic acid, and brassinolides (see, for example, Davies, P. J., in “Plant Hormones: Physiology, Biochemistry, and Molecular Biology”, Kluwer Academic Publishers, Boston, 1995; which is hereby incorporated by reference in its entirety herein).
Regeneration
The development or regeneration of plants from either single plant protoplasts or various explants is well known in the art (Weissbach and Weissbach, 1988, Plant Molecular Biology, Methods in Enzymology, Vol. 118, Academic Press, Orlando, Fla.). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
The development or regeneration of plants containing a foreign, exogenous gene that encodes a polypeptide of interest introduced by Agrobacterium from leaf explants can be achieved by methods well known in the art such as described (Horsch et al., 1985). In this procedure, transformants are cultured in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant strain being transformed as described (Fraley et al., 1983). In particular, U.S. Pat. No. 5,349,124 (specification incorporated herein by reference) details the creation of genetically transformed lettuce cells and plants resulting therefrom which express hybrid crystal proteins conferring insecticidal activity against Lepidopteran larvae to such plants.
This procedure typically produces shoots within two to four months and those shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Shoots that rooted in the presence of the selective agent to form plantlets are then transplanted to soil or other media to allow the production of roots. These procedures vary depending upon the particular plant strain employed, such variations being well known in the art.
Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants, or pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important, preferably inbred lines. Conversely, pollen from plants of those important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.
A preferred transgenic plant is an independent segregant and can transmit the PrPase gene and its activity to its progeny. A more preferred transgenic plant is homozygous for the gene, and transmits that gene to all of its offspring on sexual mating. Seed from a transgenic plant may be grown in the field or greenhouse, and resulting sexually mature transgenic plants are self-pollinated to generate true breeding plants. The progeny from these plants become true breeding lines that are evaluated for increased expression of the PrPase transgene.
A polynucleotide or polypeptide of the present invention can be used to modulate a plants nutritional status.
A polynucleotide or polypeptide of the present invention may increase the plants ability, either directly or indirectly, to initiate and/or maintain biotic associations with other organisms. Such associations may be symbiotic, nonsymbiotic, endosymbiotic, macrosymbiotic, and/or microsymbiotic in nature.
In another embodiment, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the plants ability to synthesize and/or release a pheromone.
A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may have chemotaxis activity.
A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
Antisense, Ribozymes, PNA Moieties
In further embodiments, the invention encompasses antagonists which correspond to the polynucleotide sequences of the invention, and in particular, the complementary strand of the PrPase polynucleotides.
Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, or fragments, analogs or derivatives thereof. An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire PrPase coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a PrPase protein of SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24, or antisense nucleic acids complementary to a PrPase nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23 are additionally provided. Exemplary PrPase anti-sense nucleic acid include the nucleic acid sequences of SEQ ID NO: 9, I3, and 20.
The antisense nucleic acids may be transiently generated within the organism (e.g., sequence contained within an inducible or constitutively expressed vector introduced into the cells of an organism), stably generated within the organism (e.g., sequence contained within an inducible or constitutively expressed vector introduced into the cells of an organism using transgenic methods, including viral integration, etc.) or may be exogenously administered. For a nucleic acid to serve an antisense role, it is only necessary that it has sequence homology to the sense RNA product of the gene of interest. A number of methods of administering antisense nucleic acids, their compositions, and designs are known in the art and encompassed by the invention (see for example, Agrawal S, et al., Mol Med. Today. 2000 February; 6(2):72-81; Yacyshyn B R, et al, Can J Gastroenterol. 1999 November; 13(9):745-51; Mrsny R J., J Drug Target. 1999; 7(1):1-10; Toulme J J, et al, Nucleic Acids Symp Ser. 1997; (36):39-41.), Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988); and Cooper S R, et al., Pharmacol Ther. 1999 May-June; 82(2-3):427-35). Likewise, a number of methods have been developed regarding the application of triple helix antisense technology to modulating gene expression (see, for example, Gowers D M, et al, Nucleic Acids Res. 1999 Apr. 1; 27(7):1569-77; and Chan P P, et al., J Mol. Med. 1997 April; 75(4):267-82).
Antisense technology has wide-ranging applications in plants. For example, antisense RNA has been shown to effectively downregulate a variety of plant genes as described by Shimada, et al., Theor. Appl. Genet., 86:665-672, (1993); Kull, et al., J. Genet. Breed., 49:67-76, (1995)., Slabas and Elborough, WO 97/07222; Knutzon et al., Proc. Natl. Acad. Sci. USA, 89:2624-2628, (1992), and Baulcombe D C., Plant Mol. Biol. 1996 October; 32(1-2):79-88).
The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of interest. However, absolute complementarity, although preferred, is not required. A sequence “complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids of the invention, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence of the invention it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
Antisense oligonucleotides that are complementary to the 5′ end of the message, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3′ untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., Nature, 372:333-335 (1994). Thus, oligonucleotides complementary to either the 5′- or 3′-non-translated, non-coding regions of a polynucleotide sequence of the invention could be used in an antisense approach to inhibit translation of endogenous mRNA. Oligonucleotides complementary to the 5′ untranslated region of the mRNA should include the complement of the AUG start codon.
Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the protein coding region of Physcomitrella patens, Arabidopsis thaliana, Brassica napus, Zea mays , or Glycine max PrPase corresponds to SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24). The antisense nucleic acid molecule can be complementary to the entire coding region of PrPase mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of PrPase mRNA. Whether designed to hybridize to the 5′-, 3′- or coding region of mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides. In another specific aspect, an antisense oligonucleotide can be, for example, about 5, I0, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
The antisense oligonucleotide may be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide may include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO 88/09810, published Dec. 15, I988) or blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134, published Apr. 25, 1988), hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549).
The oligonucleotide can also be a peptido-nucleic acid (“PNA”) based on a linked N-(2-aminoethyl)glycine backbone to which normal DNA bases have been attached (Egholm et al., 1993, Nature 365:566-67). This PNA obeys specific Watson-Crick base pairing, but with greater free energy of binding and correspondingly higher melting temperatures. Suitable oligomers may be constructed entirely from PNAs or from mixed PNA and DNA and/or RNA oligomers. In fact, PNA:DNA chimeras have increased solubility characteristics, as compared to DNA:DNA or DNA:RNA chimeras of the same sequence. Most notably, PNAs have the unique ability to displace one strand of a DNA double-helix thus making them highly suitable in antisense applications (Uhlmann E., Biol. Chem. 1998 August-September; 379(8-9):1045-52).
In various embodiments, the nucleic acids of PrPase can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” can also refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above;
Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.

PNAs of PrPase can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of PrPase can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe (1996), above).
In another embodiment, PNAs of PrPase can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of PrPase can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al. (1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al. (1996) above). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.
In a further embodiment, the oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
In another embodiment, the anti-sense oligonucleotide of the invention may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.
In another embodiment, the anti-sense oligonucleotide comprises at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the anti-sense oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
The anti-sense oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligos may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate oligos can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. USA 85:7448-7451), etc.
An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a PrPase protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In a specific embodiment, the oligonucleotide comprises catalytic RNA, or a ribozyme (see, e.g., PCT International Publication WO 90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science 247:1222-1225; Hasselhoff, et al., Nature 342:76-79 (1988)). Ribozymes have been used to downregulate gene expression, and more recently in the downregulation of plant proteins (seem e.g., PCT International Publication WO 97/10328).
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave PrPase mRNA transcripts to thereby inhibit translation of PrPase mRNA. A ribozyme having specificity for a PrPase-encoding nucleic acid can be designed based upon the nucleotide sequence of a PrPase DNA disclosed herein (i.e., SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 18). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a PrPase-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, PrPase mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, PrPase gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the PrPase (e.g., the PrPase promoter and/or enhancers) to form triple helical structures that prevent transcription of the PrPase gene in target cells. See generally, Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14: 807-15.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). In another embodiment, the oligonucleotide is a 2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in applications.
Double Stranded RNA Inhibition (RNAi) by Hairpin Nucleic Acids
Another aspect of the invention pertains to the use of post transcriptional gene silencing (PTGS) to repress gene expression, including use of the sequences disclosed herein to produce small interfering RNAs. Double stranded RNA can initiate the sequence specific repression of gene expression in plants and animals. Double stranded RNA is processed to short duplex oligomers of 21-23 nucleotides in length. These small interfering RNAs suppress the expression of endogenous and heterologous genes in a sequence specific manner (Fire et al. Nature 391:806-811, Carthew, Curr. Opin. in Cell Biol., 13:244-248, Elbashir et al., Nature 411:494-498). An RNAi suppressing construct can be designed in a number of ways, for example, transcription of a inverted repeat which can form a long hair pin molecule, inverted repeats separated by a spacer sequence that could be an unrelated sequence such as GUS or an intron sequence. Transcription of sense and antisense strands by opposing promoters or cotranscription of sense and antisense genes.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
EXAMPLES
Example 1
Growth of Physcomitrella patens Cultures
For this study, plants of the species Physcomitrella patens (Hedw.) B.S.G. from the collection of the genetic studies section of the University of Hamburg were used. They originated from the strain 16/14 collected by H.L.K. Whitehouse in Gransden Wood, Huntingdonshire (England), which was subcultured from a spore by Engel (1968, Am J Bot 55, 438-446). Proliferation of the plants was carried out by means of spores and by means of regeneration of the gametophytes. The protonema developed from the haploid spore as a chloroplast-rich chloronema and chloroplast-low caulonema, on which buds formed after approximately 12 days. These grew to give gametophores bearing antheridia and archegonia. After fertilization, the diploid sporophyte with a short seta and the spore capsule resulted, in which the meiospores mature.
Culturing was carried out in a climatic chamber at an air temperature of 25° C. and light intensity of 55 micromol s −1 m −2 (white light; Philips TL 65W/25 fluorescent tube) and a light/dark change of 16/8 hours. The moss was either modified in liquid culture using Knop medium according to Reski and Abel (1985, Planta 165, 354-358) or cultured on Knop solid medium using 1% oxoid agar (Unipath, Basingstoke, England).
The protonemas used for RNA and DNA isolation were cultured in aerated liquid cultures. The protonemas were comminuted every 9 days and transferred to fresh culture medium.
Example 2
Isolation of Total RNA and poly-(A) + RNA and cDNA Library Construction from Physcomitrella patens
For the investigation of transcripts, both total RNA and poly-(A) + RNA were isolated. The total RNA was obtained from wild-type 9 d old protonemata following the GTC-method (Reski et al. 1994, Mol. Gen. Genet., 244:352-359).
The Poly(A)+ RNA was isolated using Dyna Beads® (Dynal, Oslo, Norway) following the instructions of the manufacturer's protocol. After determination of the concentration of the RNA or of the poly(A)+ RNA, the RNA was precipitated by addition of 1/10 volumes of 3 M sodium acetate pH 4.6 and 2 volumes of ethanol and stored at −70 degree C.
RNA preparation from Arabidiopsis seeds—“hot” extraction:
1. Buffers, Enzymes and Solutions




2M KCl
Proteinase K
Phenol (for RNA)
Chloroform:Isoamylalcohol
(Phenol:choloroform 1:1; pH adjusted for RNA)
4 M LiCl, DEPC-treated
DEPC-treated water
3M NaOAc, pH 5, DEPC-treated
Isopropanol
70% ethanol (made up with DEPC-treated water)
Resuspension buffer: 0.5% SDS, 10 mM Tris pH 7.5, 1 mM EDTA made up with DEPC-treated water as this solution can not be DEPC-treated
Extraction Buffer:
0.2M Na Borate
30 mM EDTA
30 mM EGTA
1% SDS*(250 μl of 10% SDS-solution for 2.5 ml buffer)
1% Deoxycholate (25 mg for 2.5 ml buffer)
2% PVPP (unsoluable—50 mg for 2.5 ml buffer)
2% PVP 40K (50 mg for 2.5 ml buffer)
10 mM DTT*
100 mM-Mercaptoethanol*(fresh, handle under fume hood—use 35 μl of 14.3M solution for 5 ml buffer)

2. Extraction





Heat extraction buffer up to 80° C. Grind tissue in liquid nitrogen-cooled mortar, transfer tissue powder to 1.5 ml tube. Tissue should kept frozen until buffer is added so transfer the sample with precooled spatula and keep the tube in liquid nitrogen all time. Add 350 μl preheated extraction buffer (here for 100 mg tissue. Buffer volume can be as much as 500 μl for bigger samples) to tube, vortex and heat tube to 80° C. for ˜1 min. Keep then on ice. Vortex sample, grind additionally with electric mortar.
3. Digestion
Add Proteinase K (0.15 mg/100 mg tissue), vortex and keep at 37° C. for one hour.
4. First Purification
Add 27 μl 2M KCl. Chill on ice for 10 min. Centrifuge at 12.000 rpm for 10 minutes at room temperature. Transfer supernatant to fresh, RNAase-free tube and do one phenol extraction, followed by a choloroform:isoamylalcohol extraction. Add 1 vol. isopropanol to supernatant and chill on ice for 10 min. Pellet RNA by centrifugation (7000 rpm for 10 min at RT). Resolve pellet in 1 ml 4M LiCl by 10 to 15 min vortexing. Pellet RNA by 5 min centrifugation.
5. Second Purification
Resuspend pellet in 500 μl Resuspension buffer. Add 500 μl phenol and vortex. Add 250 μl chloroform:isoamylalcohol and vortex. Spin for 5 min. and transfer supernatant to fresh tube. Repeat choloroform:isoamylalcohol extraction until interface is clear. Transfer supernatant to fresh tube and add 1/10 vol 3M NaOAc, pH 5 and 600 μl isopropanol. Keep at −20 for 20 min or longer. Pellet RNA by 10 min centrifugation. Wash pellet once with 70% ethanol. Remove all remaining alcohol before resolving pellet with 15 to 20 μl DEPC-water. Determine quantity and quality by measuring the absorbance of a 1:200 dilution at 260 and 280 nm. 40 μg RNA/ml=1OD260
For cDNA library construction first strand synthesis was achieved using Murine Leukemia Virus reverse transcriptase (Roche, Mannheim, Germany) and oligo-d(T)-primers, second strand synthesis by incubation with DNA polymerase I, Klenow enzyme and RNAseH digestion at 12 degree C. (2 h), 16 degree C. (1 h) and 22 degree C. (1 h). The reaction was stopped by incubation at 65 degree C. (10 min) and subsequently transferred to ice. Double stranded DNA molecules were blunted by T4-DNA-polymerase (Roche, Mannheim) at 37 degree C. (30 min). Nucleotides were removed by phenol/chloroform extraction and Sephadex G50 spin columns. EcoRI adapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends by T4-DNA-ligase (Roche, 12 degree C., overnight) and phosphorylated by incubation with polynucleotide kinase (Roche, 37 degree C., 30 min). This mixture was subjected to separation on a low melting agarose gel. DNA molecules larger than 300 basepairs were eluted from the gel, phenol extracted, concentrated on Elutip-D-columns (Schleicher and Schuell, Dassel, Germany) and were ligated to vector arms and packed into lambda ZAPII phages or lambda ZAP-Express phages using the Gigapack Gold Kit (Stratagene, Amsterdam, Netherlands) using material and following the instructions of the manufacturer.
Example 3
Sequencing and Function Annotation of Physcomitrella patens ESTs
cDNA libraries as described in Example 2 were used for DNA sequencing according to standard methods, in particular by the chain termination method using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany). Random Sequencing was carried out subsequent to preparative plasmid recovery from cDNA libraries via in vivo mass excision, retransformation, and subsequent plating of DH10B on agar plates (material and protocol details from Stratagene, Amsterdam, Netherlands. Plasmid DNA was prepared from overnight grown E. coli cultures grown in Luria-Broth medium containing ampicillin (see Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6)) on a Qiagene DNA preparation robot (Qiagen, Hilden) according to the manufacturers protocols. Sequencing primers with the following nucleotide sequences were used:
Qiagen1: 5′-CAGGAAACAGCTATGACC-3′ (SEQ ID NO: 32) Qiagen2: 5′-CTAAAGGGAACAAAAGCTG-3′ (SEQ ID NO: 33) Qiagen3: 5′-TGTAAAACGACGGCCAGT-3′ (SEQ ID NO: 34)

Sequences were processed and annotated using the software package EST-MAX commercially provided by Bio-Max (Munich, Germany). The program incorporates practically all bioinformatics methods important for functional and structural characterization of protein sequences.
The most important algorythms incorporated in EST-MAX are:
FASTA: Very sensitive sequence database searches with estimates of statistical significance; Pearson W. R. (1990) Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol. 183:63-98.
BLAST: Very sensitive sequence database searches with estimates of statistical significance. Altschul S. F., Gish W., Miller W., Myers E. W., and Lipman D. J. Basic local alignment search tool. Journal of Molecular Biology 215:403-10.
PREDATOR: High-accuracy secondary structure prediction from single and multiple sequences. Frishman, D. and Argos, P. (1997) 75% accuracy in protein secondary structure prediction. Proteins, 27:329-335.
CLUSTALW: Multiple sequence alignment. Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680.
TMAP: Transmembrane region prediction from multiply aligned sequences. Persson, B. and Argos, P. (1994) Prediction of transmembrane segments in proteins utilising multiple sequence alignments. J. Mol. Biol. 237:182-192.
ALOM2: Transmembrane region prediction from single sequences. Klein, P., Kanehisa, M., and DeLisi, C. Prediction of protein function from sequence properties: A discriminant analysis of a database. Biochim. Biophys. Acta 787:221-226 (1984). Version 2 by Dr. K. Nakai.
PROSEARCH: Detection of PROSITE protein sequence patterns. Kolakowski L. F. Jr., Leunissen J. A. M., Smith J. E. (1992) ProSearch: fast searching of protein sequences with regular expression patterns related to protein structure and function. Biotechniques 13, 919-921.
BLIMPS: Similarity searches against a database of ungapped blocks. J. C. Wallace and Henikoff S., (1992)
PATMAT: A searching and extraction program for sequence, pattern and block queries and databases, CABIOS 8:249-254. Written by Bill Alford.
Example 4
Identification of Arabidopsis , soybean and corn ORFs corresponding to PrPase
The Physcomitrella patens , PpPrPase (SEQ ID NO: 1), was identified in EST-MAX through BLAST analysis. The top hit of the BLAST is an Arabidopsis unknown ORF. The second and the third hits are human and yeast caax prenyl protease. Further analysis of the unknown Arabidopsis ORF revealed that it is a predicted ORF from computer analysis, using the program Genefinder (P. Green and L. Hillier, National Center for Biotechnology Information). The ORF is located on the complementary strand of the BAC clone AF007269 (GenBank accession number, gene=“A_IG002N01.21) from 24979 to 28076.
Using this computer predicted Arabidopsis AtPrPase cDNA as a query, BLAST search in various maize and soybean databases has identified one corn ZmPrPase EST (SEQ ID NO: 21) and one soybean GmPrPase EST (SEQ ID NO: 14).
Example 5
Cloning of Arabidopsis cDNAs Encoding for PrPase
Total RNA isolation from Arabidopsis thaliana
The total RNA was obtained from wild-type 14 day old Arabidopsis thaliana following the Van Slogteren (1983 Plant Mol. Biol. 2: 321-333.) method with slight modifications. Tissue (200 mg) was frozen with liquid nitrogen and ground to a fine powder with a mortar and pestle. The powder was placed in a microfuge tube and the RNA was extracted with 500 ul of extraction buffer (phenyl: 0.1 M LiCl, 100 mM Tris-HCl [pH8.0], 10 mM EDTA, 1% SDS (w/v) [1:1]) preheated to 90° C. The mixture was heated further for 1 min at 90° C. and then vortexed for 5 mins. Proteins were extracted by adding 250 ul of chloroform: isoamyl:alcohol (24:1) and the mixture was vortexed for 5 mins and centrifuged for 15 mins at 13,000 rpm in an Eppendorf centrifuge 5414 at 4° C. The aqueous layer was removed and the protein extraction was repeated twice more. One volume of 4 mM LiCl was added and the RNA was allowed to precipitate overnight at 4° C. To collect the RNA, the mixture was centrifuged for 15 min at 4° C. at 13,000 rpm in an Eppendorf centrifuge 5414. The pellet was resuspended in 250 ul sterile, deionized water. To precipitate the RNA, 0.1 vols of 3M sodium acetate (pH5.2) and 2 vols 100% ethanol were added. An aliquote was taken and centrifuged for 20 mins at 4° C. at 13,000 rpm in an Eppendorf centrifuge 5414. The pellet was washed with 70% ethanol to remove salts from the pellet and dried using a speed vac. The pellet was resuspended in 25 ul DEPC H 2 O and analyzed for integrity via electrophoresis. The RNA was stored at −70° C.
RT-PCR and Cloning of Arabidopsis AtPrPase
The synthetic oligonucleotide primers (MWG-Biotech) were designed based on the BAC clone sequence (GenBank accession number AF007269, gene=“A_IG002N01.21”, complement 24979 . . . 28076).
APP forward: (SEQ ID NO: 35) 5′ CCGTTAACAGCCATGGCGATTCCTTTCATGGAA 3′ APP reverse: (SEQ ID NO: 36) 5′ GTCCCGGGACTTAATCTGTCTTCTTGTCTT 3′

The primers designed contained a HpaI site in the 5′ region and an XmaI site in the 3′ region for cloning purpose.
The synthesis of the first strand cDNA was achieved using AMV Reverse Transcriptase (Roche, Mannheim, Germany). The resulting single-stranded cDNA was amplified via Polymerase Chain Reaction (PCR) utilizing the two gene specific primers. The conditions for the reaction were standard conditions with Expand High Fidelity PCR system (Roche). The parameters for the reaction were: five minutes at 94° C. followed by five cycles of 40 seconds at 94° C., 40 seconds at 50° C. and 1.5 minutes at 72° C. This was followed by thirty cycles of 40 seconds at 94° C., 40 seconds at 65° C. and 1.5 minutes at 72° C. The fragment generated under these RT-PCR conditions was 1.3 kilobase long.
The fragment was extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) and ligated into the TOPO pCR 2.1 vector (Invitrogen) following manufacture's instructions. Recombinant vectors were transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells were selected for on LB agar containing 100 μg/ml carbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 0.8 mg IPTG (isopropylthio-β-D-galactoside) grown overnight at 37° C. White colonies were selected and used to inoculate 3 ml of liquid LB containing 100 μg/ml ampicillin and grown overnight at 37° C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacture's instructions.
The RT-PCR cloned Arabidopsis AtPrPase1 and AtPrPase2 were sequenced to obtain their complete cDNA sequences (SEQ ID NO: 3 and SEQ ID NO: 5).
Example 6
In Vivo Complementation of the SM3614 Yeast (PrPase) Mutant
The fragment containing the Arabidopsis AtPrPase1 cDNA was excised from the recombinant PCR2.1 TOPO vector by digestion with EcoRI (Roche) according to manufacture's instructions. The subsequent fragment was excised from agarose gel with a QIAquick Gel Extraction Kit (QIAgen) according to manufacture's instructions and ligated into the yeast expression vector pYES2 (Invitrogen), also cleaved with EcoRI and dephosphorylated prior to ligation.
The recombinant expression pYES2 vector containing the Arabidopsis AtPrPase1 cDNA in the sense orientation under the yeast GAL1 promoter was transformed into the yeast mutant SM3614 (MATa rcel Δ::TRP1 step24D::LEU2) (Tam et al. 1998) following Invitrogen's protocol. The transformed cells were selected for on Complete Supplement Mixture (CSM) minus Uracil 0.8% agar (Bio 101, Inc.) grown at 30° C. for two days. The transformed colonies were selected to make master plates containing patches of the transformed SM3614 on CSM plates minus Uracil supplemented with 2% galactose for induction of the expression of the Arabidopsis AtPrPase1. The plates were grown at 30° C. for two days. The master plates were replica plated onto a lawn of wild type yeast SM 1068 (MATα lysl) (Tam et al. 1998, The Journal of Cell Biology, 142, 635-649) on SD plates supplemented with 2% galactose under various mating conditions and incubated at 30° C. for two days.
Example 7
Cloning of Soybean and Corn cDNAs Encoding for PrPase
Construction of cDNA Libraries of Soybean and Corn
To isolate the clones encoding PrPase from soybean and corn the cDNA libraries were created with SMART RACE cDNA Amplification kit (Clontech Laboratories) following manufacture's instructions. Total RNA created as described in Example 5 was used as the template. Three-week old corn leaves and stems and soybean leaves were used for total RNA preparation respectively.
Cloning of Soybean and Corn cDNAs Encoding for PrPase
The EST sequences for ZmPrPase and GmPrPase identified from the database search as described in Example 4 were used to design oligos for RACE. The extended partial sequences for ZmPrPase and GmPrPase were obtained by performing Rapid Amplification of cDNA Ends polymerase chain reaction (RACE PCR) using the Advantage 2 PCR kit (Clontech Laboratories) and the SMART RACE cDNA amplification kit (Clontech Laboratories) using a Biometra T3 Thermocycler following the manufacture's instructions. The gene specific synthetic oligonucleotide primer (MWG-Biotech) used were:
For ZmPrPase:
(SEQ ID NO: 37) 5′ RACE oligo: 5′ AGCAGCCACGATTGGTGGCCCCAAT 3′ (SEQ ID NO: 38) 3′ RACE oligo: 5′ GGGCCACCAATCGTGGCTGCTATCA 3′

For GmPrPase:
(SEQ ID NO: 39) 5′ RACE oligo: 5′ CGCAGCCAGTCCTCATTGGGCTCATC 3′ (SEQ ID NO: 40) 3′ RACE oligo: 5′ CGGATAGTTGAGGGAGGAAGCAAG 3′

The sequences obtained from the RACE reactions were compiled to give the nucleotide sequences for the partial GmPrPase (SEQ ID NO: 14) and the partial ZmPrPase (SEQ ID NO: 21).
Having the partial sequences for soybean and corn, full-length sequences were obtained. The nucleotide sequence of the full-length PrPase from soybean ( Glycine max ) (Clone ID No: GmPrPase2) is provided as SEQ ID NO: 16 with the deduced amino acid sequence as SEQ ID NO: 17. The nucleotide sequence of the full-length PrPase from corn ( Zea mays ) (Clone ID No: ZmPrPase2) is provided as SEQ ID NO: 23 with the deduced amino acid sequence as SEQ ID NO: 24.
Example 8
Engineering Drought-Tolerant Arabidopsis Plants
Binary Vector Construction: pGMSG and pGMGG
The pLMNC53 vector (Mankin, 2000, PHD thesis) was digested with HindIII (Roche) and blunt-end filled with Klenow enzyme and 0.1 mM dNTPs (Roche) according to manufacture's instructions. This fragment was extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) according to manufacture's instructions. The purified fragment was then digested with EcoRI (Roche) according to manufacture's instructions. This fragment was extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) according to manufacture's instructions. The resulting 1.4 kilobase fragment, the gentamycin cassette, included the nos promoter (Becker et al., 1992 Plant Molecular Biology 20: 1195-7), aacCI gene (Hajdukiewicz et al., 1994 Plant Molecular Biology 25: 989-94), and the g7 terminator (Becker et al., 1992).
The vector pBlueScript was digested with EcoRI and SmaI (Roche) according to manufacture's instructions. The resulting fragment was extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) according to manufacture's instructions. The digested pBlueScript vector and the gentamycin cassette fragments were ligated with T4 DNA Ligase (Roche) according to manufacture's instructions, joining the two respective EcoRI sites and joining the blunt-ended HindIII site with the SmaI site.
The recombinant vector (pGMBS) was transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells were selected for on LB agar containing 100 g/ml carbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 0.8 mg IPTG (isopropylthio-β-D-galactoside), grown overnight at 37° C. White colonies were selected and used to inoculate 3 ml of liquid LB containing 100 μg/ml ampicillin and grown overnight at 37° C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacture's instructions. Analyses of subsequent clones and restriction mapping were performed according to standard molecular biology techniques (Sambrook et al. 1989).
Both the pGMBS vector and plbxSuperGUS vector were digested with XbaI and KpnI (Roche) according to manufacture's instructions, excising the gentamycin cassette from pGMBS and producing the backbone from the plbxSuperGUS vector. The resulting fragments were extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) according to manufacture's instructions. These two fragments were ligated with T4 DNA ligase (Roche) according to manufacture's instructions.
The resulting recombinant vector (pGMSG) was transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells were selected for on LB agar containing 100 μg/ml carbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 0.8 mg IPTG (isopropylthio-β-D-galactoside), grown overnight at 37° C. White colonies were selected and used to inoculate 3 ml of liquid LB containing 100 μg/ml ampicillin and grown overnight at 37° C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacture's instructions. Analyses of subsequent clones and restriction mapping were performed according to standard molecular biology techniques (Sambrook et al. 1989).
Another example for a plant binary vector is the pGMGG vector, where both the pBinK vector containing the guard cell-specific promoter KST1 (Bernd Muller-Rober, 1999) and pGMSG vector were digested with XbaI and SmaI according to manufacture's instructions, excising KST1 from pBinK and producing the backbone from the pGMSG vector. The resulting fragments were extracted from agarose gel with QIAquick Gel Extraction Kit (Qiagen) according to manufacture's instructions. These two fragments were ligated with T4 DNA ligase (Roche) according to manufacture's instructions.
The resulting recombinant vector (pGMGG) was transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells were selected for on LB agar containing 100 μg/ml carbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 0.8 mg IPTG (isopropylthio-β-D-galactoside), grown overnight at 37° C. White colonies were selected and used to inoculate 3 ml of liquid LB containing 100 μg/ml ampicillin and grown overnight at 37° C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacture's instructions. Analyses of subsequent clones and restriction mapping were performed according to standard molecular biology techniques (Sambrook et al. 1989).
Subcloning of Arabidopsis AtPrPases into the Binary Vectors
The fragment containing the Arabidopsis PrPase cDNA was excised from the recombinant PCR2.1 TOPO vector by digestion with HpaI and XmaI (Roche) according to manufacture's instructions. The subsequent fragment was excised from agarose gel with a QIAquick Gel Extraction Kit (QIAgen) according to manufacture's instructions and ligated into the binary vectors pGMSG, cleaved with XmaI and Ecl136II and dephosphorylated prior to ligation.
The clones AtPrPase1 (SEQ ID NO: 3) and AtPrPase2 (SEQ ID NO: 5) were cloned into pGMSG vectors in sense orientation. The resulting recombinant pGMSG vectors contained the Arabidopsis prenyl protease under the control of the constitutive super promoter.
Agrobacterium Transformation
The recombinant vectors were transformed into Agrobacterium tumefaciens C58C1 and PMP90 according to standard conditions (Hoefgen and Willmitzer, 1990).
Plant Transformation
Arabidopsis thaliana ecotypes C24 and Col-2 were grown and transformed according to standard conditions (Bechtold 1993, Acad. Sci. Paris. 316:1194-1199, Bent et al. 1994, Science 265:1856-1860).
Screening of Transformed Plants
Seeds were sterilized according to standard protocols (Xiong et al. 1999, Plant Molecular Biology Reporter 17: 159-170). Seeds were plated on ½ MS 0.6% agar supplemented with 1-3% sucrose and 50-150 μg/ml gentamycin. Seeds on plates were vernalized for two days at 4° C. The seeds were germinated in a climatic chamber at an air temperature of 22° C. and light intensity of 55 micromols −1 m −2 (white light; Philips TL 65W/25 fluorescent tube) and 24 hour light. Transformed seedlings were selected after 7-14 and transferred to ½ MS 0.6% agar plates supplemented with 1% sucrose and allowed to recover for 1-5 days.
Drought Tolerance Screening
The transgenic plants were screened for their improved drought tolerance. Seedlings were transferred to dry, sterile filter paper and allowed to desiccate for 4 hours. Seedlings were then removed and placed on ½MS 0.6% agar plates and scored after two days.
Salt Tolerance Screening
The transgenic plants are screened for the improved salt tolerance. Seedlings are transferred to ½ MS liquid supplemented with 600 mM NaCl and allowed to incubate for 2-4 hours. Seedlings are then removed and placed on ½ MS 0.6% agar plates and scored for surviving seedlings after two days.
Example 9
Engineering Drought-Tolerant Soybean Plants
The GmPrPase clones (SEQ ID NO: 14 and 16) are cloned into pGMSG vectors in sense orientation. These constructs are used to transform soybean as described below.
Seeds of soybean are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) Tween for 20 minutes with continuous shaking. Then, the seeds are rinsed 4 times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 6 to 39 hours. The seed coats are peeled off, and cotyledons are detached from the embryo axis. The embryo axis is examined to make sure that the meristematic region is not damaged. The excised embryo axes are collected in a half-open sterile Petri dish and air dried to a moisture content less than 20% (fresh weight) in a sealed Petri dish until further use.
Agrobacterium tumefaciens culture is prepared from a single colony in LB solid medium plus appropriate antibiotics (e.g. 100 mg/l streptomycin, 50 mg/l kanamycin) followed by growth of the single colony in liquid LB medium to an optical density at 600 nm of 0.8. Then, the bacteria culture is pelleted at 7000 rpm for 7 minutes at RT, and resuspended in MS (Murashige and Skoog, 1962) medium supplemented with 100 μM acetosyringone. Bacteria cultures are incubated in this pre-induction medium for 2 hours at RT before use. The axis of soybean zygotic seed embryos at approximately 15% moisture content are imbibed for 2 hours at RT with the pre-induced Agrobacterium suspension culture. The embryos are removed from the imbibition culture and are transferred to Petri dishes containing solid MS medium supplemented with 2% sucrose and incubated for 2 days, in the dark at RT. Alternatively, the embryos are placed on top of moistened (liquid MS medium) sterile filter paper in a Petri dish and incubated under the same conditions described above. After this period, the embryos are transferred to either solid or liquid MS medium supplemented with 500 mg/L carbenicillin or 300 mg/L cefotaxime to kill the agrobacteria . The liquid medium is used to moisten the sterile filter paper. The embryos are incubated during 4 weeks at 25° C., under 150 μmol m −2 sec −1 and 12 hours photoperiod. Once the seedlings have produced roots, they are transferred to sterile metromix soil. The medium of the in vitro plants is washed off before transferring the plants to soil. The plants are kept under a plastic cover for 1 week to favor the acclimatization process. Then the plants are transferred to a growth room where they are incubated at 25° C., under 150 μmol m −2 sec −1 light intensity and 12 hours photoperiod for about 80 days.
The transgenic plants are screened for their improved drought tolerance according to the screening method described in Example 8 to demonstrate that transgene expression confers drought tolerance.
Example 10
Engineering Drought-Tolerant Rapeseed Plants with the AtPrPase Clones
The AtPrPase constructs of Example 8 are used to transform rapeseed as described below.
The method of plant transformation described in Example 8 is also applicable to Brassica and other crops. Seeds of canola are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) Tween for 20 minutes, at RT with continuous shaking. Then, the seeds are rinsed 4 times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 18 hours. Then the seed coats are removed and the seeds are air dried overnight in a half-open sterile Petri dish. During this period the seeds lose approx. 85% of its water content. The seeds are then stored at room temperature in a sealed Petri dish until further use. DNA constructs and embryo imbibitions are as described in Example 8. Samples of the primary transgenic plants (T0) are analyzed by PCR to confirm the presence of T-DNA. These results are confirmed by Southern hybridization in which DNA is electrophoresed on a 1% agarose gel and transferred to a positively charged nylon membrane (Roche Diagnostics). The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare a digoxigenin-labelled probe by PCR, and used as recommended by the manufacturer.
The transgenic plants are screened for their improved drought tolerance according to the screening method described in Example 8 to demonstrate that transgene expression confers drought tolerance.
Example 11
Engineering Drought-Tolerant Corn Plants
The ZmPrPase clones (SEQ ID NO: 21 and 23) are cloned into pGMSG vectors in sense orientation. These constructs are used to transform corn.
The imbibition of dry embryos with a culture of Agrobacterium is also applicable to maize embryo axes. The experimental protocol is the same as described in Example 8 but using maize seeds as the source of embryos.
The transgenic plants are screened for their improved drought tolerance according to the screening method described in Example 8 to demonstrate that transgene expression confers drought tolerance.
Example 12
Guard-Cell Specific Expression of the AtPrPase1 Promoter
The promoter region of AtPrPase (SEQ ID NO: 10) is cloned into pGMSG in place of the superpromoter, driving the reporter gene GUS (Jefferson et al., 1987). The resulting construct is transformed into Arabidopsis plants as described in Example 8.
The transgenic plants are screened for their guard-cell specific staining to demonstrate that the transgene expression confers guard-cell specific promoter activity.
Example 13
Over-Expression of PrPase in Plants Leads to Increased Stress Tolerance and Plant Growth
The AtPrPase clones (SEQ ID NO: 3 and 5) used to transform Arabidopsis , as described in Example 8 are used to transform soybean, rapeseed, and corn as described in Examples 9, 10, and 11, respectively.
The transgenic plants are screened for their improved stress tolerance according to the screening method described in Example 8 to demonstrate that transgene expression confers stress tolerance.
The transgenic plants are further screened for their growth rate to demonstrate that transgene expression confers increased growth rates.
Specifically, transgenic Arabidopsis plants transformed with AtPrPase1 (SEQ ID NO: 3 encoding SEQ ID NO: 4) were assessed for stress tolerance and plant growth. The transgenic Arabidopsis plants were grown for three weeks in a growth chamber at a temperature of 22° C., 55% relative humidity and light intensity of 40 micromols −1 m −2 (white light; Philips TL 65W/25 fluorescent tube) and 16 hours light and 8 hours dark day length cycle. Soil moisture was maintained throughout this time at approximately 50% of the maximum water-holding capacity of soil. After 3 weeks, the entire above-group plant biomass was collected, dried at 65° C. for 2 days and weighed.
Three independent experiments were conducted with data shown in Table 4. In each experiment, 10 transgenic events were selected, 10-12 transgenic plants were assayed for each event (including the control event). Different control plants were used for comparison in each experiment. The dry weight was calculated by averaging plants of the same transformation event (given in grams in Table 4). The analysis was run using a Least Square Mean (LSMEAN).
The first experiment used Arabidopsis ecotype C24 as control. The vector control plants were generated by transforming into the wild type C24 plants the T-DNA of a binary vector containing between the left and right borders a selectable marker cassette (Promoter-selectable marker-Terminator) and a cassette with no gene of interest (promoter-terminator).
The second experiment used both MTXC24 and BPSC24 as controls, where MTXC24 and BPSC24 were single seed descent variants of the Arabidopsis C24 ecotype.
The third experiment used Arabidopsis ecotype Col-0 as control.
In all three experiments, AtPrPase1 (SEQ ID NO: 3 encoding SEQ ID NO: 4) transgenic plants in each of the corresponding control background, under limited water condition, accumulated more dry weight than the corresponding control plants as shown in Table 4.
TABLE 4 Control Control Dry Weight AtPrPase1 Dry Weight Experiment used LSMean Std. Error LSMean Std. Error 1 SC24 0.098269 0.0045776 0.141823 0.0043416 2 MTX C24 0.0547 0.0035 0.1047 0.0028 BPS C24 0.0581 0.0035 3 Col-0 0.189 0.009 0.213 0.003

Example 14
Transformation of Dicotyledons
The polynucleotides of the present invention, including the polynucleotides encoding the polypeptides of the present invention, may be used to transform dicotyledons in an effort to confer specific traits into the plant. Such polynucleotides may be either the full-length polynucleotide, fragments, the complementary strand, or variants thereof, and may be either by themselves or operably fused to heterologous polynucleotides.
Transformation techniques for dicotyledons are well known in the art and include Agrobacterium -based techniques and techniques which do not require Agrobacterium . Non- Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al., EMBO J. 3: 2717-2722 (1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich et al., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.
Agrobacterium -mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species. The many crop species which are routinely transformable by Agrobacterium include tobacco, tomato, sunflower, cotton, oilseed rape, potato, soybean, alfalfa and poplar (EP 0 317 511 (cotton), EP 0 249 432 (tomato, to Calgene), WO 87/07299 (Brassica, to Calgene), U.S. Pat. No. 4,795,855 (poplar)). Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which may depend of the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). The transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hofgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).
Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.
Other methods for the transformation of dicotyledons are known in the art. Thus, this example should be not construed as limiting the scope of the invention to only those examples illustrated above or elsewhere herein.
Example 15
Transformation of Monocotyledons
The polynucleotides of the present invention, including the polynucleotides encoding the polypeptides of the present invention, may be used to transform monocotyledons in an effort to confer specific traits into the plant. Such polynucleotides may be either the full-length polynucleotide, fragments, the complementary strand, or variants thereof, and may be either by themselves or operably fused to heterologous polynucleotides as described in more detail elsewhere herein.
Transformation of most monocotyledon species has now also become routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation) and both these techniques are suitable for use with this invention. Co-transformation may have the advantage of avoiding complex vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al. Biotechnology 4: 1093-1096 (1986)).
Patent Applications EP 0 292 435 (to Ciba-Geigy), EP 0 392 225 (to Ciba-Geigy) and WO 93/07278 (to Ciba-Geigy) describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordon-Kamm et al., Plant Cell 2: 603-618 (1990) and Fromm et al., Biotechnology 8: 833-839 (1990) have published techniques for transformation of A188-derived maize line using particle bombardment. Furthermore, application WO 93/07278 (to Ciba-Geigy) and Koziel et al., Biotechnology 11: 194-200 (1993) describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistics device for bombardment.
Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protoplast-mediated transformation has been described for Japonica-types and Indica-types (Zhang et al., Plant Cell Rep 7: 379-384 (1988); Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology 8: 736-740 (1990)). Both types are also routinely transformable using particle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).
Patent Application EP 0 332 581 (to Ciba-Geigy) describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation was been described by Vasil et al., Biotechnology 10: 667-674 (1992) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al., Biotechnology 11: 1553-1558 (1993) and Weeks et al., Plant Physiol. 102: 1077-1084 (1993) using particle bombardment of immature embryos and immature embryo-derived callus. A preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, any number of embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashige & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos which is allowed to proceed in the dark. On the chosen day of bombardment, embryos are removed from the induction medium and placed onto the osmoticum (i.e. induction medium with sucrose or maltose added at the desired concentration, typically 15%). The embryos are allowed to plasmolyze for 2-3 h and are then bombarded. Twenty embryos per target plate is typical, although not critical. An appropriate gene-carrying plasmid (such as pCIB3064- or pSG35) is precipitated onto micrometer size gold particles using standard procedures. Each plate of embryos is shot with the DuPont Biolistics, helium device using a burst pressure of about 1000 psi using a standard 80 mesh screen. After bombardment, the embryos are placed back into the dark to recover for about 24 h (still on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration. Approximately one month later the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS+1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35). After approximately one month, developed shoots are transferred to larger sterile containers known as “GA7s” which contained half-strength MS, 2% sucrose, and the same concentration of selection agent.
An additional method of transforming monocots is found in International Publication No. WO 00/12734, and describes the application of the Ac-Ds transposon system to the insertion of transgenes into plants.
Other methods for the transformation of monocotyledons are known in the art. Thus, this example should be not construed as limiting the scope of the invention to only those examples illustrated above or elsewhere herein.
Example 16
Method of Transforming Plants Using Vacuum Infiltration
Transformation of plants may serve as a vital tool in assessing the biological function of a particular polynucleotide or polypeptide. For example, a plant may be transformed with a vector capable of downregulating a particular gene via anti-sense regulation (i.e., the vector may express a transcript of the gene of interest in the anti-sense direction), or the vector may simply be capable of overexpressing a particular polypeptide, for example. By observing the resulting phenotypes of the transformant, one may derive protein function using techniques known in the art and described elsewhere herein.
The following method of transforming plant material may be applicable to any plant species, though is particularly suited for use in Arabidopsis.
Arabidopsis is grown at 20° C., 8 hr light, 18° C. 16 dark until needed for transformation and is fertilized once a week from below. Plants are thinned to ˜1 per square inch and are used immediately upon bolting. Short days allow stronger vegetative plant growth and increase seed yield.
Plants are transferred to 20° C., 16 hr light, 18° C. 8 hr dark. The plants should bolt quickly, and they are ready to infiltrate when the primary inflorescences are 10-15 cm tall and the secondary inflorescences are appearing at the rosette.
In the meantime, constructs are transformed into Agrobacterium tumefaciens strain EHA105 (Hood et al., Transgenic Research 2:208-218 (1993)) (see Direct Agrobacterium Transformation:Freeze-Thaw Method below). When the plants are ready to transform, 1 ml of an overnight culture is used to innoculate a 500 ml culture of YEB medium (2 L flask) containing the appropriate antibiotic for the construct and 50 ug/mL rifampicin (C58 Agro and or 25 mg/mL gentamycin (pMP90)). Cultures are grown for 2 days at 28° C., ˜275 rmp. YEB media is described below.
When OD 600 is greater than 2.0, the culture is centrifuged for 30 min, at 3500 rpm and resuspended in 0.5-1.0 ml of infiltration medium described below.
The resuspended culture is placed in a container with a large bell jar, and pots containing plants to be infiltrated are inverted into the infiltration medium so that the entire plant is covered (including the rosette, but not too much soil). Any large air bubbles under the plants are removed. A vacuum (˜700 Hg) is drawn, the suction is closed, and the plants are allowed to sit under vacuum for 5 min. The vacuum pressure is quickly released and the pots are briefly drained.
Infiltrated plants are grown as before at 20° C., 16 hr light, and 18° C. 8 hr dark. Plants are staked as the bolts grow. When the plants are finished flowering, the T 0 seeds are harvested.
Seeds are sterilized and screened for transformants on the selective medium described below. Dark green (resistant) plants are transferred to secondary selection plate a week after germination, then to soil after 6-10 days. New transplants are kept covered for several days.
Plant media: Vacuum Infiltration Selection For 1 L Medium Medium MS Salts 2.2 g 4.3 g B5Vitamins, 1000X 1.0 mL 1.0 mL Sucrose 50 g 10 g MES, 200 mg/mL pH 5.7 with KOH 2.5 mL 2.5 mL Benzylamonipurine (BAP, 1 mg/mL) 44 μL — Silweet L-77 200 μL — Phytagar — 8 g Pursuit (1 mM) 100 μL















Bacterial Media
YEP





Yeast extract
1.0 g


Beef Extract
5.0 g


Peptone
5.0 g


Sucrose
5.0 g


MgSO 4
0.5 g






















The skilled artisan would appreciate that the above transformation method could be modified to apply to other species of plants. Such modification may include the addition of new steps, the deletion of any of the steps described, and/or substitution of reagents.
Direct Agrobacterium Transformation:Freeze-Thaw Method.
An Agrobacterium strain containing the appropriate helper Ti plasmid is grown in 5 mL of YEP medium overnight at 28° C. 2 ml of the overnight culture is added to 50 ml YEP medium in a 259 mL flask and shaken vigorously (250 rpm) at 28° C. until the culture grows to an OD 600 of 0.5 to 1.0. The culture is chilled on ice. The cell suspension is centrifuged at 3000 g for 5 min at 4° C.
The supernatant solution is discarded. The cells are resuspend in 1 mL of 20 mM CaCl 2 solution (ice-cold). 0.1 mL aliquots are dispensed into prechilled Eppendorf tubes. About 1 ug of plasmid DNA is added to the cells.
The cells are frozen in liquid nitrogen. The cells are thawed by incubating the test tube in a 37° C. water bath for 5 min. 1 mL of YEP medium is added to the tube and the tube incubated at 28° C. for 2-4 hr with gentle rocking. This period allows the bacteria to express the antibiotic resistance genes. The tubes are centrifuged for 30 s in an Eppendorf centrifuge. The supernatant solution is discarded and the cells resuspended in 0.1 mL YEP medium
The cells are spread onto a YEP agar plate containing 3-5 ug/mL tetracycline and 10-25 ug/mL kanamycin. The plate is incubated at 28° C. Transformed colonies should appear in 2-3 days.
The skilled artisan would appreciate that the above transformation method could be modified. Such modification may include the addition of new steps, the deletion of any of the steps described, and/or substitution of reagents.
Example 17
Functional Determination of Proteins Using Metabolite Profiling
The present invention encompasses the application of metabolite profiling to the identification of gene function for the polypeptides of the present invention. In one example, transgenic plants could be produced which are either incapable of expressing a protein of the present invention, or that have decreased expression levels of a protein of the present invention. Such transgenic plants could be produced by creating knockout constructs to inactivate or delete the endogenous gene, for example, using methods known in the art. Alternatively, the transgenic plants could be produced by inserting into the plant a construct that expresses antagonists of a protein of the present invention (e.g., antisense oligonucleotides, antisense genes, antibodies, etc.). Other examples of methods of producing transgenic plants, including specific strategies, are known in the art, some of which are described elsewhere herein.
Once a protein of the present invention is inactivated, or its expression inhibited, the resulting metabolite profile of the plant can be ascertained, and the function of the protein assigned. Some of the anticipated metabolic profiles of inhibiting or inactivating the expression of a protein of the present invention in a plant may resemble known nutritional deficiencies, pathogenic diseases, biotic stresses, or abiotic stresses, for example, many of which are disclosed elsewhere herein. In addition, the metabolic profile of a transgenic plant of the present invention may be useful in identifying the specific pathways the polypeptide of the present invention is a member of, in addition, to identifying the potential downstream and/or upstream effectors or affectors, respectively. In addition, it may be possible to identify the mode of action of a polypeptide of the present invention.
A number of methods are known in the art for identifying the metabolic profile of a plant. A non-limiting example is provided by Sauter, H., et al., in “Metabolic Profiling of Plants: A New Diagnostic Technique”, Synthesis and Chemistry of Agrochemicals II, Baker, D. R., Fenyes, J. G., and Moberg, W. K., eds, ACS Symposium Series, 433, Chapter 24, pp. 288-299, (1991). Briefly, transgenic plants of the invention, or plants in which the expression of a polypeptide of the present invention is inhibited or inactivated, are grown in growth chambers. The shoots are harvested and immediately deep frozen until further treatment. The frozen plant samples are weighed and a threefold amount (W:W) of ethanol is added. The mixture is then macerated in a mixer and the resulting suspension is left for 2 hours for extraction. The next steps are filtration, evaporation, and silylation with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). Internal standard alkanes are also added, thus allowing for the calculation of retention coefficients, as well as, quantification. The crude mixture is then subjected to gas chromotography on a methyl silicon gum fused silica capillary column (30 m DB-1. Injection temperature 230° C. Oven temperature 100°-320° C., 4° C./min; 15 min 320° C.). Retention coefficients are then calculated relative to internal standards (n-C10H22=1000, n-C28H58=2800).
The above protocol can be applied to numerous test plants, in addition, to controls. The data from the resulting profiles are then grouped together (i.e., one group for the test plants, another group for the controls) to arrive at an average profile for each group. In the latter step, the corresponding peaks (i.e., those peaks with equal retention coefficients) are grouped together and the peak heights are subjected to statistical analysis.
The differences in metabolic profiles between the test and control plants are determined by calculating the “difference profile” between the two groups. The difference profile is calculated by dividing the peal heights. This difference profile provides a semiquantitative estimate of the change in magnitude of one metabolic with respect to the other.
Once the above is completed, the peaks are then associated with particular metabolites (i.e., the metabolite identify of each peak is determined). By comparing the metabolite profile of proteins known to modulate specific pathways in a plant, to those of the present invention, clues for and/or identification of the function of a polypeptide of the present invention may be determined. Other methods are known in the art, and any one or more steps, may be equally substituted with such methods.
Example 18
RT-PCR Amplification and Cloning of CaaX Prenyl Proteases
Total RNA was isolated from leaf tissue of Arabidopsis thaliana, Brassica napus and Glycine max , using the Qiagen RNeasy kit and used as template to amplify the CaaX prenyl proteases (CPP) genes by RT-PCR. Reaction conditions were as follows; 1× reaction buffer (10 mM Tris-HCl pH 8.8, 1.5 mM MgCl 2 , 50 mM KCl), dNTP's at 200 μM, 1 pM AtCPP BamFW and AtCPP SmaRV primers, 2.5U. Pfu DNA polymerase, and template plus water to a final volume of 100 μL. Reactions were run at 1 minute 94° C., 1 minute 60° C., 1 minute 72° C., for 30 cycles. Primers used to PCR amplify Arabidopsis and Brassica sequences were those identified by SEQ ID NO: 43 and SEQ ID NO: 44. Primers used to PCR amplify the Glycine sequence were those identified by SEQ ID NO: 70 and SEQ ID NO: 71. PCR products were separated from the RT-PCR reaction mixture using the Qiagen PCR column spin kit and ligated into the prepared cloning vector, pBluescript KS+. The vector had been prepared by digestion with EcoRV and treated with Taq polymerase in the presence of dTTP to produce a 3′ overhand suitable for ligation with the PCR products. The ligation products were transformed into E. coli DH5α cells, positive colonies selected and the resulting inserts sequenced. The above methodology is applicable to obtain homologous sequences and may require alternative primers.
AtCPP BamFW: (SEQ ID NO: 43) 5′-AAAGGATCCATGGCGATTCCTTTCATGG-3′ AtCPP SmaRV: (SEQ ID NO: 44) 5′-AAACCCGGGTTAATCTGTCTTCTTGTCTTCTCCA-3′ GmCPP SmaFW: (SEQ ID NO: 70) 5′-AAACCCGGGATGGCGTTTCCCTACATGGAAGCC-3′ GmCPP SacRV: (SEQ ID NO: 71) 5′-AAAGAGCTCTTAGTCTTCCTTCTTATCCGGTTCG-3′

Example 19
Vector Construction
Construction of the pBI121-AtCPP construct (SEQ ID NO: 41) was prepared as follows. The pBI121 vector was digested with BamHI and SmaI. The AtCPP, 1.4 kb DNA fragment from RT-PCR (SEQ ID NO: 7) was digested with BamHI and SmaI and ligated into the pBI121 vector. The GUS sequence was then removed by digestion with SmaI and EcolCRI and the vector ligated after purification of the vector from the GUS insert to produce the pBI121-AtCPP vector ( FIG. 2A ). This construct was used to further generate constructs expressing the CPP gene from Brassica and Glycine. To produce the pBI121-BnCPP construct (SEQ ID NO: 63) primer pairs identified by SEQ ID NO: 43 and SEQ ID NO: 44 are used to PCR amplify the appropriate fragment which is ligated into the prepared parent vector. To produce the pBI121-GmCPP construct (SEQ ID NO: 57) primer pairs identified by SEQ ID NO: 70 and SEQ ID NO: 71 are used to PCR amplify the appropriate fragment which is ligated into the prepared parent vector.
Construction of the pBI121-antisense-AtCPP construct (SEQ ID NO: 51). The antisense fragment was produced using PCR amplification with SEQ ID NO: 7 as template and primers identified as SEQ ID NO: 48 and SEQ ID NO: 49, listed in Table 5. This fragment was digested with BamHI and SmaI and used to replace the sense fragment of the pBI121-AtCPP construct (SEQ ID NO: 41), to yield SEQ ID NO: 51 ( FIG. 2B ). This construct, SEQ ID NO: 51, was used to further generate constructs expressing the antisense CPP gene from Brassica and Glycine . To produce the pBI121-antisense-BnCPP construct (SEQ ID NO: 65) primer pairs identified by SEQ ID NO: 72 and SEQ ID NO: 73 are used to PCR amplify the appropriate fragment which is ligated into the prepared parent vector. To produce the pBI121-antisense-GmCPP construct (SEQ ID NO: 59) primer pairs identified by SEQ ID NO: 74 and SEQ ID NO: 75 are used to PCR amplify the appropriate fragment which is ligated into the prepared parent vector.
Construction of the pBI121-HP-AtCPP construct (SEQ ID NO: 42). The cloning strategy involved truncating the GUS gene of pBI121 and flanking the GUS sequence with a AtCPP fragment in the antisense orientation upstream of the GUS and in the sense orientation on the downstream side of GUS. The pBI121 vector was digested with SmaI and SacI, the GUS sequence and the vector fragments were purified from one another. The isolated GUS fragment was digested using EcoRV and the 1079 bp. blunt ended EcoRV/SacI fragment isolated. This was ligated back into the digested parent vector at the SmaI/SacI sites. This intermediate vector was used in the subsequent production of the hair-pin vectors. The AtCPP fragment to be used as the gene specific hair-pin sequence was isolated by PCR. Primers identified as SEQ ID NO: 45 and SEQ ID NO: 46, listed in Table 5, were used to generate a 596 bp fragment. Cloning of the sense orientation fragment was achieved by digesting the PCR AtCPP fragment with SacI and ligation into the SacI site at the 3′ end of GUS. To insert the same fragment upstream of GUS, the BamHI site was opened and the ends blunted with Klenow. The PCR amplified AtCPP fragment was digested with EcolCRI, which is an isoschizomer of SacI but leaves blunt ends, and ligated into the blunted BamHI site of the vector to yield the final construct ( FIG. 2C ). The intermediate construct used to produce SEQ ID NO: 42 above contained only the truncated GUS gene and no CPP sequences this intermediate vector was used to further generate constructs expressing hair-pin CPP gene constructs from Brassica and Glycine . To produce the pBI121-HP-BnCPP construct (SEQ ID NO: 64) primer pairs identified by SEQ ID NO: 74 and SEQ ID NO: 75 are used to PCR amplify the sense fragment and primer pairs identified by SEQ ID NO: 76 and SEQ ID NO: 77 are used to PCR amplify the antisense fragment. These fragments are cloned into the prepared intermediate vector described above. To produce the pBI121-HP-GmCPP construct (SEQ ID NO: 58) primer pairs identified by SEQ ID NO: 78 and SEQ ID NO: 79 are used to PCR amplify the sense fragment and primer pairs identified by SEQ ID NO: 80 and SEQ ID NO: 81 are used to PCR amplify the antisense fragment. These fragments are cloned into the prepared intermediate vector described above.
The above vector constructs were modified to place the genes under the control of alternative promoters, such as, but not limited to, the RD29A or MuA. This was accomplished by excising the 35S promoter sequence and replacing it with an appropriate promoter sequence. In this way SEQ ID NOs: 55 and 56 were generated and SEQ ID NOs: 54, 57-69 can be constructed.
TABLE 5 AtCPP-HP-SacFW 5′-CTGGAGCTCTTTTACCGAGGTTGGGCCTTGATCC-3′ (SEQ ID NO: 45) AtCPP-HP-SacRV 5′-ATTGAGCTCCCAATGTCCAAGCTCGTGTGCAATA-3′ (SEQ ID NO: 46) AtCPP-anti-SmaFW 5′-AAACCCGGGATGGCGATTCCTTTCATGG-3′ (SEQ ID NO: 48) AtCPP-anti-BamRV 5′-AAAGGATCCTTAATCTGTCTTCTTGTCTTCTCCA-3′ (SEQ ID NO: 49) BnCPP-anti-SmaFW 5′-AAACCCGGGATGGCGATTCCTTTCATGG-3′ (SEQ ID NO: 72) BnCPP-anti-BamRV 5′-AAAGGATCCTTAATCTGTCTTCTTGTCTTCTCC-3′ (SEQ ID NO: 73) BnCPP-HP-Sac-FW 5′-AAAGAGCTCTTCTACCAATGGTGGGACTCG-3′ (SEQ ID NO: 74) BnCPP-HP-Sac-RV 5′-AAAGAGCTCCCAGTGTCCCAGCTCGTGTG-3′ (SEQ ID NO: 75) BnCPP-HP-BamFW 5′-AAAGGATCCTTCTACCAATGGTGGGACTCG-3′ (SEQ ID NO: 76) BnCPP-HP-XbaRV 5′-AAATCTAGACCAGTGTCCCAGCTCGTGTG-3′ (SEQ ID NO: 77) GmCPP-HP-Sac-FW 5′-GATGAGCTCACAAGATCAAGTCACAGCAATGCCT-3′ (SEQ ID NO: 78) GmCPP-HP-Sac-RV 5′-AAAGAGCTCCCGGTTCGTCCAGCGCGGCC-3′ (SEQ ID NO: 79) GmCPP-HP-BamFW 5′-GATGGATCCACAAGATCAAGTCACAGCAATGCCT-3′ (SEQ ID NO: 80) GmCPP-HP-XbaRV 5′-CCTTCTAGACCGGTTCGTCCAGCGCGGCC-3′ (SEQ ID NO: 81)

Example 20
Sequence Analysis
Arabidopsis thaliana PrPase (AtCPP)
A disclosed nucleic acid of 1275 nucleotides referred to as AtCPP is shown in SEQ ID NO: 7.
A disclosed PrPase polypeptide encoded by SEQ ID NO: 7 has 424 amino acid residues and is presented as SEQ ID NO: 8.
The present invention also includes a nucleic acid sequence complementary to the Arabidopsis thaliana CaaX prenyl protease of SEQ ID NO: 7. The disclosed complementary sequence is shown as SEQ ID NO: 9.
Due to the nature of the cloning strategy the sequence presented is not full length but is missing the 5′ and 3′ non-translated regions. The percent identities of the Arabidopsis thaliana nucleotide sequence and its encoded amino acid sequence to that of other PrPase sequences as determined by EMBOSS Pairwise Alignment analysis are shown in FIGS. 6 and 7 .
Using the sequences disclosed herein as hybridization probes, one is able to screen and isolate full length sequences from cDNA or genomic libraries or use the rapid amplification of cDNA ends (RACE) technology or other such PCR techniques.
Brassica napus PrPase (BnCPP)
A disclosed nucleic acid of 1275 nucleotides referred to as BnCPP is shown in SEQ ID NO: 11. A disclosed PrPase polypeptide encoded by SEQ ID NO: 11 has 424 amino acid residues and is presented in SEQ ID NO: 12.
The present invention also includes a nucleic acid sequence complementary to the Brassica napus CaaX prenyl protease of SEQ ID NO: 11. The disclosed complementary sequence is shown as SEQ ID NO: 13.
Due to the nature of the cloning strategy the sequence presented is not full length but is missing the 5′ and 3′ non-translated regions. The percent identities of the Brassica napus nucleotide sequence and its encoded amino acid sequence to that of other PrPase sequences as determined by EMBOSS Pairwise Alignment analysis are shown in FIGS. 6 and 7 .
Using the sequences disclosed herein as hybridization probes, one is able to screen and isolate full length sequences from cDNA or genomic libraries or use the rapid amplification of cDNA ends (RACE) technology or other such PCR techniques.
Glycine max PrPase (GmCPP)
A disclosed nucleic acid of 1275 nucleotides referred to as GmCPP is shown in SEQ ID NO: 18.
A disclosed PrPase polypeptide encoded by SEQ ID NO: 18 has 424 amino acid residues and is presented in SEQ ID NO: 19.
The present invention also includes a nucleic acid sequence complementary to the Glycine max CaaX prenyl protease of SEQ ID NO: 18. The disclosed complementary sequence is shown as SEQ ID NO: 20.
Due to the nature of the cloning strategy the sequence presented is not full length but is missing the 5′ and 3′ non-translated regions. The percent identities of the Glycine max nucleotide sequence and its encoded amino acid sequence to that of other PrPase sequences as determined by EMBOSS Pairwise Alignment analysis are shown in FIGS. 6 and 7 .
Using the sequences disclosed herein as hybridization probes, one is able to screen and isolate full length sequences from cDNA or genomic libraries or use the rapid amplification of cDNA ends (RACE) technology or other such PCR techniques.
The PrPase nucleic acids and amino acids disclosed above have homology to other disclosed PrPase sequences (GenBank ID NOs: AL161491 (AT4g01320), AF007269 and AF353722; WO 02/16625 A2). The nucleic acid and amino acid homology between these and other sequences is shown in the EMBOSS Pairwise Alignment analysis shown in FIGS. 4 and 5 .
Example 21
Plant Transformation
Arabidopsis transgenic plants were made by the method of dipping flowering plants into an Agrobacterium culture, based on the method of Andrew Bent in, Clough S J and Bent A F, 1998. Floral dipping: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana . Wild type plants were grown under standard conditions until the plant has both developing flowers and open flowers. The plant was inverted for 2 minutes into a solution of Agrobacterium culture carrying the appropriate gene construct. Plants were then left horizontal in a tray and kept covered for two days to maintain humidity and then righted and bagged to continue growth and seed development. Mature seed was bulk harvested.
Transformed T1 plants were selected by germination and growth on MS plates containing 50 μg/ml kanamycin. Green, kanamycin resistant (Kan R ) seedlings were identified after 2 weeks growth and transplanted to soil. Plants were bagged to ensure self fertilization and the T2 seed of each plant harvested separately. During growth of T1 plants leaf samples were harvested, DNA extracted and Southern blot and PCR analysis performed.
T2 seeds were analysed for Kan R segregation. From those lines that showed a 3:1 resistant phenotype, surviving T2 plants were grown, bagged during seed set, and T3 seed harvested from each line. T3 seed was again used for Kan R segregation analysis and those lines showing 100% Kan R phenotype were selected as homozygous lines. Further molecular and physiological analysis was done using T3 seedlings.
Transgenic Brassica napus, Glycine max and Zea mays plants were produced using Agrobacterium mediated transformation of cotyledon petiole tissue. Seeds were sterilized as follows. Seeds were wetted with 95% ethanol for a short period of time such as 15 seconds. Approximately 30 ml of sterilizing solution I was added (70% Javex, 100 μl Tween20) and left for approximately 15 minutes. Solution I was removed and replaced with 30 ml of solution II (0.25% mecuric chloride, 100 μl Tween20) and incubated for about 10 minutes. Seeds were rinsed with at least 500 ml double distilled sterile water and stored in a sterile dish. Seeds were germinated on plates of ½ MS medium, pH 5.8, supplemented with 1% sucrose and 0.7% agar. Fully expanded cotyledons were harvested and placed on Medium I (Murashige minimal organics (MMO), 3% sucrose, 4.5 mg/L benzyl adenine (BA), 0.7% phytoagar, pH5.8). An Agrobacterium culture containing the nucleic acid construct of interest was grown for 2 days in AB Minimal media. The cotyledon explants were dipped such that only the cut portion of the petiole is contacted by the Agrobacterium solution. The explants were then embedded in Medium I and maintained for 5 days at 24° C., with 16, 8 hr light dark cycles.
Explants were transferred to Medium II (Medium 1,300 mg/L timentin,) for a further 7 days and then to Medium III (Medium II, 20 mg/L kanamycin). Any root or shoot tissue which had developed at this time was dissected away. Transfer explants to fresh plates of Medium III after 14-21 days. When regenerated shoot tissue developed the regenerated tissue was transferred to Medium IV (MMO, 3% sucrose, 1.0% phytoagar, 300 mg/L timentin, 20 mg/L 20 mg/L kanamycin). Once healthy shoot tissue developed shoot tissue dissected from any callus tissue was dipped in 10×IBA and transferred to Medium V (Murashige and Skooge (MS), 3% sucrose, 0.2 mg/L indole butyric acid (IBA), 0.7% agar, 300 mg/L timentin, 20 mg/L 20 mg/L kanamycin) for rooting. Healthy plantlets were transferred to soil. The above method, with or without modifications, is suitable for the transformation of numerous plant species including Glycine max, Zea mays and cotton.
Transgenic Glycine max, Zea mays and cotton can be produced using Agrobacterium -based methods which are known to one of skill in the art. Alternatively one can use a particle or non-particle biolistic bombardment transformation method. An example of non-particle biolistic transformation is given in U.S. Patent Application 20010026941. This method has been used to produce transgenic Glycine max and Zea mays plants. Viable plants are propagated and homozygous lines are generated. Plants are tested for the presence of drought tolerance, physiological and biochemical phenotypes as described elsewhere.
The following table identifies the constructs and the species which they have been transformed.
TABLE 6 Transformation List SEQ ID NO: Construct Species Transformed 41 pBII121-AtCPP A. thaliana , B. napus 42 pBII121-HP-AtCPP A. thaliana 52 pRD29A-AtCPP A. thaliana , B. napus 53 pRD29A-HP-AtCPP A. thaliana 55 MuA-AtCPP Glycine max , Zea mays

Non-limiting examples of vector constructs suitable for plant transformation are given in SEQ ID NOs: 41, 42, 51-69. The right and left border repeats correspond to the first 24 nucleotide positions and the last 25 nucleotide positions of each construct.
SEQ ID NO: 41 is the nucleic acid sequence of pBI121-AtCPP. The 35S promoter is at positions 2515-3318 and the AtCPP sense sequence is at positions 3334-4608 of SEQ ID NO: 41.
SEQ ID NO: 42 is the nucleic acid sequence of pBI121-HP-AtCPP. The 35S promoter is at positions 2515-3318, the AtCPP anti-sense sequence at positions 3336-3925, the truncated GUS fragment at positions 3936-4958; and the AtCPP sense sequence at positions 4959-5548 of SEQ ID NO: 42.
SEQ ID NO: 51 is the nucleic acid sequence of pBI121-antisense-AtCPP. The 35S promoter is at positions 2515-3318 and the AtCPP anti-sense sequence at positions 3334-4608 of SEQ ID NO: 51.
SEQ ID NO: 52 is the nucleic acid sequence of RD29A-AtCPP. The RD29A promoter is at positions 2515-2865 and the AtCPP sense sequence at positions 3458-4732 of SEQ ID NO: 52.
SEQ ID NO: 53 is the nucleic acid sequence of RD29A-HP-AtCPP. The RD29A promoter is at positions 2515-3442, the AtCPP anti-sense sequence at positions 3460-4069, the truncated GUS fragment at positions 4060-5082, and the A. thaliana CaaX prenyl protease sense fragment at positions 5083-5675 of SEQ ID NO: 53.
SEQ ID NO: 54 is the nucleic acid sequence of RD29A-antisense-AtCPP. The RD29A promoter is at positions 2515-3442 and the AtCPP anti-sense sequence is at positions 3458-4732 of SEQ ID NO: 54.
SEQ ID NO: 55 is the nucleic acid sequence of MuA-AtCPP. The MuA promoter is at positions 2502-2863 and the A. thaliana CaaX prenyl protease sense sequence at positions 2864-4138 of SEQ ID NO: 55.
SEQ ID NO: 56 is the nucleic acid sequence of MuA-GmCPP. The MuA promoter is at positions 2502-2865 and the G. max CaaX prenyl protease sense sequence at positions 2866-4140 of SEQ ID NO: 56.
SEQ ID NO: 57 is the nucleic acid sequence of pBI121-GmCPP. The 35S promoter is at positions 2515-3318 and the G. max CaaX prenyl protease sense sequence at positions 3339-4613 of SEQ ID NO: 57.
SEQ ID NO: 58 is the nucleic acid sequence of pBI121-HP-GmCPP. The 35S promoter is at positions 2515-3318, the antisense prenyl protease fragment of G. max at positions 3328-3856, the G. max sense prenyl protease fragment is at positions 4888-5416, and the truncated GUS fragment at positions 3865-4827 of SEQ ID NO: 58.
SEQ ID NO: 59 is the nucleic acid sequence of pBI121-antisense-GmCPP. The 35S promoter is at positions 2515-3318 is the GmCPP anti-sense sequence at positions 3339-4613 of SEQ ID NO: 59.
SEQ ID NO: 60 is the nucleic acid sequence of pRD29A-GmCPP. The RD29A promoter is at positions 2515-3442 and the GmCPP sense sequence at positions 3463-4737 of SEQ ID NO: 60.
SEQ ID NO: 61 is the nucleic acid sequence of pRD29A-HP-GmCPP. The RD29A promoter is at positions 2515-3442, the GmCPP antisense sequence at positions 3452-3980, and the GmCPP sense sequence at positions 5012-5540 of SEQ ID NO: 61.
SEQ ID NO: 62 is the nucleic acid sequence of pRD29A-antisense-GmCPP. The RD29A promoter is at positions 2515-3442 and the GmCPP antisense sequence at positions 3463-4737 of SEQ ID NO: 62.
SEQ ID NO: 63 is the nucleic acid sequence of pBI121-BnCPP. The 35S promoter is at positions 2515-3318 and the BnCPP antisense sequence at positions 3334-4608 of SEQ ID NO: 63.
SEQ ID NO: 64 is the nucleic acid sequence of pBI121-HP-BnCPP. The 35S promoter is at positions 2515-3318, the BnCPP antisense sequence at positions 3328-3917, the BnCPP sense fragment at positions 4949-5538, and the truncated GUS fragment at positions 3926-4888 of SEQ ID NO: 64.
SEQ ID NO: 65 is the nucleic acid sequence of pBI121-antisense-BnCPP. The 35S promoter is at positions 2515-3318 and the BnCPP antisense sequence at positions 3334-4608 of SEQ ID NO: 65.
SEQ ID NO: 66 is the nucleic acid sequence of pRD29A-BnCPP. The RD29A promoter is at positions 2515-3420 and the BnCPP sense sequence at positions 3458-4732 of SEQ ID NO: 66.
SEQ ID NO: 67 is the nucleic acid sequence of pRD29A-HP-BnCPP. The RD29A promoter is at positions 2515-3442, the BnCPP antisense sequence at positions 3452-4041, the BnCPP sense fragment at positions 5073-5640, and the truncated GUS fragment at positions 4050-5072 of SEQ ID NO: 67.
SEQ ID NO: 68 is the nucleic acid sequence of pRD29A-antisense-BnCPP. The RD29A promoter is at positions 2515-3420 and the BnCPP antisense sequence at positions 3458-4732 of SEQ ID NO: 68.
SEQ ID NO: 69 is the nucleic acid sequence of MuA-BnCPP. The MuA promoter is at positions 2502-2820 and the BnCPP sense sequence at positions 2863-4138 of SEQ ID NO: 69.
Example 22
Southern Analysis
Genomic Southern blot analysis of transgenic Arabidopsis was performed using standard techniques known to one skilled in the art. Typically, 10 μg of DNA was electrophoresed in a 0.8% agarose gel and transferred to an appropriate membrane such as Hybond N+ (Amersham Pharmacia Biotech). Pre-hybridization and hybridization conditions were as suggested by the membrane manufacturer, typically at 65° C. The final stringency wash was typically at 1×SSC and 0.1% SDS at 65° C. The NPTII coding region was typically used as the radiolabeled probe in Southern blot analysis.
Thirty-seven Arabidopsis lines were selected as homozygous pBI121-AtCPP over-expression lines for further examination. The Southern blot depicted a representative blot confirming the presence of the pBI121-AtCPP transgene. Lines were confirmed to be transgenic by PCR analysis using transgene specific primers in the PCR assays.
Thirty-three Arabidopsis lines were selected as homozygous pBI121-HP-AtCPP hair-pin down-regulation lines for further examination. The Southern blot depicted a representative blot confirming the presence of the pBI121-HP-AtCPP hair-pin construct. All lines were confirmed to be transgenic by PCR analysis using transgene specific primers in the PCR assays.
Arabidopsis lines were selected as homozygous pRD29A-AtCPP over-expression lines for further examination. The Southern blot depicted a representative blot confirming the presence of the pRD29A-AtCPP transgene. Lines were confirmed to be transgenic by PCR analysis using transgene specific primers in the PCR assays.
Arabidopsis lines were selected as homozygous pRD29A-HP-AtCPP lines for further examination. The Southern blot depicted a representative blot confirming the presence of the pRD29A-HP-AtCPP transgene. Lines were confirmed to be transgenic by PCR analysis using transgene specific primers in the PCR assays.
Example 23
PCR Analysis of Transgenic Plants
PCR was used as a method to confirm the presence of the transgene in all transgenic lines and every construct. Typical PCR mixtures contained: 1× reaction buffer (10 mM Tris-HCl pH 8.8, 1.5 mM MgCl 2 , 50 mM KCl), dNTP's at 200 μM, 1 pM forward and reverse primer, 2.5U. Taq DNA polymerase, and template plus water to a final volume of 50 μL. Reactions were run at 1 minute 94° C., 1 minute 60° C., 1 minute 72° C., for 30 cycles. Primers used in the analysis of pBI121-AtCPP and pBI121-HP-AtCPP transgenic plants were as shown in Table 7. Primers used in the analysis of pRD29A-AtCPP were RD29AP1 (SEQ ID NO: 82) and SEQ ID NO: 44. Primers used in the analysis of pRD29A-HP-AtCPP transgenic plants were those identified as RD29AP1 (SEQ ID NO: 82), SEQ ID NO: 44 and SEQ ID NO: 45, Nosterm-RV (SEQ ID NO: 83).
TABLE 7 pBI121-AtCPP BamFW: 5′-GCCGACAGTGGTCCCAAAGATGG-3′ (SEQ ID NO: 47) p35S-AtCPP SmaRV: 5′-AAACCCGGGTTAATCTGTCTTCTTGTCTTCTCCA-3′ (SEQ ID NO: 44) p35S-HP-AtCPP BamFW: 5′-CTGGAGCTCTTTTACCGAGGTTGGGCCTTGATCC-3′ (SEQ ID NO: 45) p35S-HP-AtCPP SmaRV: 5′-GCAAGACCGGCAACAGGA-3′ (SEQ ID NO: 50) pRD29AP1: 5′-TTTAAGCTTGGAGCCATAGATGCAATTCAA -3′ (SEQ ID NO: 82) Nosterm-RV: 5′-GCAAGACCGGCAACAGGA-3′ (SEQ ID NO: 83)

Example 24
Northern Analysis of Transgenic Plants
Total RNA was isolated from developing leaf tissue of 27 35S-AtCPP Arabidopsis lines (T3 plants). Approximately 10 μg of total RNA was loaded into each lane. The Northern blot was first probed with P 32 labeled, single-stranded antisense transcript of AtCPP which detects sense transcript, then stripped and re-probed with cDNA of β-tubulin that was used as a reference. The hybridizing bands of AtCPP and β-tubulin were scanned and quantified using the UN-Scan-It programme (Silk Scientific, Utah, USA), and the ratio of the two hybridizing bands for each sample was obtained. The ratio of the wild type plants was set to 100%, and was compared with those of the transgenic lines. Twenty-one out of twenty-seven lines showed higher expression of AtCPP transcript as compared to the wild type. Values ranged from 104% to 282% of wild type. The results of five lines ( 35 , 84 , 76 , 136 , and 156 ) of the 21 over-expressing lines is shown in FIG. 8 .
Example 25
Production of Polyclonal Antibodies Against AtCPP
Anti-AtCPP antibodies were generated using AtCPP fusion protein over-expressed in E. coli . The over-expression vector, pMAL-p2, contains 1175 bp malE gene that is located upstream of AtCPP and encodes a 43 KDa maltose-binding protein (MBP). The 1275 bp BamHI/SmaI DNA fragment of AtCPP was inserted into pMAL-p2 at BamHI and SalI sites. The SalI site was converted into blunt end using Klenow fragment. The resulting fusion protein MBP-AtCPP was then over-expressed in DH5α, and purified by one-step affinity for MBP as described by the manufacturer (New England Biolab). The soluble fraction of the crude bacterial extract containing the MBP-AtCPP fusion protein was loaded to a amylose column (1.5 cm×10.0 cm), and the proteins were eluted with 10 mM maltose in column buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 200 mM NaCl). Fractions containing purified MBP-AtCPP fusion protein were pooled, and concentrated with a Centriprep-30 concentrator (Amicon). All purification steps were carried out at 4° C. To generate an antibody, the purified fusion protein was further separated by SDS-PAGE and the Coomassie stained band corresponding to the fusion protein was excised. The identity of the fusion protein was confirmed by Western analysis using anti-MBP antibodies (purchased from New England Biolab). The protein was eluted from the gel slice by electroelution and then emulsified in Ribi adjuvant (Ribi Immunochem) to a final volume of 1 ml. MBP-AtCPP protein was injected into a 3 kg New Zealand rabbit on day 1 and booster injections were given on day 21 and day 35 with 175 μg of the protein each time. High-titer antisera were obtained one week after the final injection.
Example 26
Western Blot Analysis of 35S-AtCPP Transgenic Lines Using Anti-AtCPP Antibodies
Western analysis was performed to examine expression level of AtCPP in the transgenic lines compared with that of wild type plants. Anti-Bip antibody, an ER lumenal protein (Stressgen, Victoria, BC, Canada) was used as a reference. Total proteins were extracted from developing leaf tissue of five ABA S lines and a wild type control. The antigenic protein bands of AtCPP and Bip were scanned and quantified using the UN-Scan-It programme (Silk Scientific, Utah, USA) and the ratio of the two protein bands for each sample was obtained. The ratio of the wild type plants was set to 100%, and was compared with those of the transgenic lines. Data is presented in FIG. 8 indicating that the AtCPP protein level was increased in the transgenic lines compared to the wild type plants.
Example 27
ABA Sensitivity of Transgenic Seedlings
Approximately 100 seeds were assessed per line per 9 cm plate. Seeds were plated on minimal medium (½ MS) supplemented with no ABA or 1.0 μM ABA. Plates were chilled for 3 days at 4° C. in the dark, and incubated for up to 21 days at 22° C. with 24 hour continuous light. Plates were assessed for germination, cotyledon expansion, true leaf development and seedling vigor. Seedlings were assessed for ABA sensitivity over 21 days of growth at which time sensitive seedlings were arrested at the cotyledon stage, lacked true leaves, and showed inhibition of root growth. Wild type control Columbia plants had two to three pairs of true leaves and a well developed root system. Lines were categorized as ABA sensitive (ABA S ) if less than 1% of plants looked like control, moderately ABA sensitive (ABA MS ) if more than 1% but less than 50% of looked like control, or ABA insensitive (ABA Wt ) if greater than 50% looked like control.
For example, if a plate had 20 healthy seedlings and the control plate had 60 healthy seedlings, the line would be 33% of control and categorized as moderately ABA sensitive.
All four vector constructs (pBI121-AtCPP, pBI121Hp-AtCPP, pRD29AHp-AtCPP, pRD29A-ATCPP) have resulted in transgenic lines of Arabidopsis which have increased sensitivity to ABA which is indicative of stress tolerance. The data for all 4 constructs is shown in FIG. 9 . Of the lines transformed with the pBI121-AtCPP construct to over-express the AtCPP gene, 58% (21 out of 36) were classified as sensitive and an added 30% (11 out of 36) were classified as moderately sensitive. These lines were tested again in T4 and T5 generations and their ABA sensitivity was still present indicating that ABA sensitivity is an inheritable trait. Of the lines transformed with the pBI121-HP-AtCPP construct to down-regulate the AtCPP gene by double stranded RNA-inhibition, 15% (7 out of 45) were classified as sensitive and 31% (14 out of 45) were classified as moderately sensitive. To illustrate the increased sensitivity of transgenic lines to ABA, FIG. 10 shows the results of germination and seedling development over a range of ABA concentrations. Wild type and pRD29A-HP-AtCPP are compared. Of the lines transformed with pRD29AHp-AtCPP 70% (12 out of 17) showed high sensitivity and 24% (4 out of 17) showed moderate sensitivity to ABA. Of the lines transformed with pRD29A-AtCPP 29% (5 out of 17) showed high sensitivity and 12% (2 out of 17) moderate sensitivity to ABA. Clearly all 4 transgene constructs are altering ABA sensitivity and ABA signal transduction.
Example 28
Drought Experiments
Arabidopsis plants were grown five plants per 4″ or 3″ pot, in a replicated water-stress experiment. All pots were filled with equal amounts of homogeneous premixed and wetted soil. Plants were grown under 16 hour daylight (150-200 μmol/m 2 /s) at 22° C. and 70% relative humidity. On the day that the first flower opened drought treatment was initiated. First soil water content in each pot was equalized on a weight basis and any further watering of plants was stopped. Daily measurements of soil water content were taken by recording total pot weight. At the end of the drought treatment (6 to 9 days for experiments in 4″ pots and 4-5 days for experiments in 3″ pots) plants were harvested and shoot dry weights determined. Differences in plant growth were factored into the analysis by expressing water loss on a per gram shoot dry weight basis.
28a) pBI121-AtCPP, Drought Stress Screen:
Analysis of pBI121-AtCPP transgenic lines during water-stress treatment experiments of up to an eight day period, shows a strong trend towards increased soil water content and reduced water loss per gram of shoot biomass. After three days of water-stress treatment most lines had increased soil water content relative to the wild type control with four out of twenty-four lines, 146 , 149 , 156 and 97 , showing a statistically significant difference. The amount of water lost per gram of shoot biomass was lower for all lines except one ( 95 ), and thirteen of these lines were significantly different from the wild type Columbia control ( FIG. 11 ). All of the lines showing a statistically significant lower water loss per gram shoot biomass also showed an increased ABA sensitivity. There is also a strong trend, for all but one line (95), which is ABA Wt , towards greater shoot biomass at the end of the drought stress treatment. Seven of those lines 136 , 146 , 23 , 46 , 76 , 84 and 9 , were statistically significant from control at a p=0.05 value.
28b) pBI121-AtCPP, Water Loss Per Gram Shoot Biomass During Water Stress Treatment:
Lines 35 , 76 , 95 and a wild type control were grown and placed under a water-stress treatment as above. Plants were harvested at 2 days, 4 days and 6 days of drought treatment. The ABA S lines, 35 and 76, showed a statistically significant reduction in water-loss relative to shoot dry weight at all three time points (Table 8). Additionally, the two ABA S transgenic lines had increased shoot biomass, due to increased leaf biomass, and maintained higher soil water contents during drought treatment.
TABLE 8 Water loss (g) per Shoot dry weight (g) after 2, 4 and 6 days of drought-stress treatment. Values in bold indicate statistically significant differences from Columbia. 2 days 4 days 6 days Line Mean Std. Error Mean Std. Error Mean Std. Error 35 212.5 3.5 308.0 9.9 297.7 11.2 76 227.2 5.8 321.2 8.5 293.8 5.0 95 287.0 5.1 377.3 14.8 348.5 25.5 Columbia 265.3 11.8 408.2 7.7 345.9 6.7 Wild type

28c) pBI121-AtCPP, Drought Stress and Shoot Recovery:

Water-stress tolerance and determination of post drought-treatment recovery ability was assessed using 20 of the 24 pBI121-AtCPP transgenic lines. Drought treatment was imposed for 6 days after which the plants were watered and allowed to grow for 6 days. Recovered shoot fresh biomass was then determined. Soil water content of these plants was measured daily during the drought treatment and the results confirm previously seen trends. All ABA sensitive (ABA S ) lines that showed a statistically significantly reduction of water loss on a per gram dry weight basis in experiment 34a, continued to show a significant greater soil water content than control plants in this experiment (Table 9). Additionally, Table 9 shows that the recovered shoot fresh biomass after 6 days of drought treatment was significantly greater in all the ABAs lines than Columbia.
TABLE 9 Soil water content on day 3 of drought treatment and recovered shoot fresh weight after 6 days of drought treatment (values in bold were significantly different from Columbia at p = 0.05) soil water content day 3 recovered shoot ABA status Mean (% biomass Line ABA initial) Std Error Mean (g) Std Error 136 ABA S 46.6 1.9 4.5 0.16 14 ABA S 50.25 0.7 4.1 0.12 146 ABA S 45.9 2.5 4.0 0.11 147 ABA S 45.1 1.7 4.0 0.15 149 ABA S 45.3 1.8 3.8 0.17 156 ABA S 47.1 1.9 4.0 0.134 23 ABA S 49 1.4 4.0 0.17 33 ABA S 46.9 1.6 4.3 0.14 35 ABA S 41.7 1.7 4.0 0.11 46 ABA S 44.8 1.7 3.8 0.09 63 ABA S 46.3 1.4 4.0 0.19 76 ABA S 47.8 1.0 3.9 0.17 79 ABA S 45.4 1.1 4.1 0.09 84 ABA S 46.8 1.9 4.1 0.16 85 ABA S 45.3 1.9 4.0 0.12 9 ABA S 45.2 2.1 3.9 0.12 93 ABA Wt 43.5 1.2 2.8 0.07 94 ABA S 46.9 1.5 3.9 0.13 97 ABA S 53 1.2 3.8 0.16 95 ABA Wt 41.9 1.2 2.7 0.06 Columbia ABA Wt 41.3 1.0 2.7 0.04

28d) pBI121-AtCPP, Seed Yield After Drought Stress Treatment:

Seed yield after drought stress during flowering was examined using ten pBI121-AtCPP transgenic lines, eight of which were ABA S . Plants were grown one per 4″ pot and were exposed to 9 days of drought treatment as described above. A second group of plants was grown and maintained under well watered conditions as the optimal group. After 9 days of drought treatment plants were re-watered and allowed to continue growth and seed set to maturity. After drought-treatment conditions all eight ABA S lines had increased yields relative to controls, which ranged from 109% to 126% of the Columbia (Table 10). Drought-treatment resulted in a reduction of yield in all lines, including controls, relative to plants grown under optimal conditions. Expression of the seed yields obtained from drought-treated group relative to the same line under optimal conditions shows that the transgenics preserve a larger percentage of optimal seed yield than do wild type lines.
TABLE 10 Seed Yield following 9 days drought-treatment Seed Yield ABA status (g per plant) % Line ABA Mean (g) Std Error Columbia % Optimal 156 ABA S 0.735 0.044 126.2 83.7 63 ABA S 0.675 0.061 116.0 71.0 146 ABA S 0.666 0.053 114.4 72.9 94 ABA S 0.644 0.052 110.6 68.8 84 ABA S 0.642 0.049 110.4 61.8 76 ABA S 0.631 0.055 108.5 66.6 136 ABA S 0.630 0.051 108.3 74.1 35 ABA S 0.614 0.054 105.6 74.2 93 ABA Wt 0.567 0.041 97.5 60.0 95 ABA Wt 0.388 0.088 66.7 43.4 Columbia ABA Wt 0.582 0.060 100 53.8

28e) pBI121-AtCPP Seed Yield and Growth Under Optimal Water Conditions:

The lines evaluated above and a number of additional lines were examined in a growth and yield experiment under optimal, well-watered conditions. Results indicated that the ABA S lines were shorter at the stage of first open flower, had more rosette leaves, however, by maturity there were no differences in plant height of transgenics and Columbia. Moreover, the ABA S transgenics showed similar or higher seed yields ranging from 95% to 121% of the wild type control ( FIG. 12 ).
28f) pRD29A-HP-AtCPP Screen for Drought Tolerant Phenotype:
Analysis of 17 transgenic lines identified 7 candidate drought tolerant lines ( 12 , 22 , 23 , 47 , 82 , 83 , 90 ) on the basis of higher soil water content and lower water loss per g of shoot dry weight (Table 11). All 7 drought tolerant candidate lines showed strong ABA sensitivity and lines that did not show drought tolerance did not show ABA sensitivity.
TABLE 11 Soil water content after 3 days of drought treatment and water lost per g shoot dry weight. Values in bold are statistically different from those of Columbia wild type (p = 0.05) soil water content water lost in day 2 2 days/g shootDW ABA status Mean Mean Line ABA (% initial) Std Error (g/g) Std Error 10 ABA S 33.4 1.6 199.1 4.5 11 ABA S 34.6 3.3 173.1 1.6 12 ABA S 36.2 2.0 179.5 5.0 126 ABA MS 32.5 2.6 199.1 4.1 127 ABA MS 33.5 2.0 195.6 10.6 14 ABA S 32.7 1.2 203 4.9 17 ABA S 29.9 1.8 200.7 7.3 22 ABA S 39.3 2.1 170.0 3.0 23 ABA S 35.7 1.4 174.9 2.6 42 ABA MS 28 0.7 185.4 5.8 47 ABA S 35.9 2.2 181.2 7.7 7 ABA Wt 35 1.3 201.8 5.1 82 ABA S 36.7 2.2 178.3 4.0 83 ABA S 40 1.4 180.7 6.9 9 ABA S 31.4 1.4 173.8 8.7 90 ABA S 38.2 1.3 177.6 6.2 93 ABA Wt 30.7 1.8 175.3 4.6 Columbia ABA Wt 32.1 1.2 196.9 6.2

Example 29
Growth Analysis
The growth analysis of most promising constructs has been set up at 3 stages. Eight plants per line were grown in 3″ pots with one plant per pot at 22 C, 16 hr light (150-200 μmol/m 2 /s) and 70% RH. Plants were harvested at vegetative growth stage (2 week old seedlings), bolting growth stage (at first open flower) and mid-flowering growth stage (5 to 7 days from first open flower). Also, in some growth experiments additional group of plants was grown in 4″ pots (one per pot and 10 plants per line) to maturity for seed yield determinations.
29a) pBI121-AtCPP Growth Under Optimal and Biotic Stress Conditions
The growth and productivity of pBI121-AtCPP transgenic Arabidopsis lines was examined at several stages of development under optimal growth conditions. Although optimal growth conditions were maintained, plants were assessed to be under a degree of stress that was later determined to be a result of the soil properties. Soil analysis found a fungal contaminant that was believed to be responsible for the biotic stress. This stress could be negated by sterilization of the soil prior to use. Eight ABA S lines, two with normal ABA sensitivity (ABA Wt ) and a wild type Columbia control were analyzed.
FIG. 13 presents the results of various growth (from mid-flowering stage) and yield parameters and each trait is expressed as a percentage of the Columbia control. The results strongly support an enhanced growth phenotype. This enhanced growth phenotype is present at all growth stages. At the vegetative stage, all ABA S transgenic plants showed an increase in leaf number relative to that of the wild type with four of the eight lines showing a statistically significant difference. The two ABA Wt lines showed the same or fewer leaves relative to wild type.
At the bolting stage ABA S transgenics showed an increase in leaf number but plants were shorter at this stage (first open flower) than controls. The shoot fresh weight of transgenics was significantly increased relative to that of controls, ranging from 80% to 342% of the wild type. The ABA S transgenics displayed a delay in flowering from one to three days. The ABA Wt transgenics did not show delayed flowering, increased shoot fresh weight or increased height.
At the flowering stage of development the enhanced growth phenotype is maintained (greater leaf number and fresh weight), however, there were no observable differences in plant height indicating that transgenics bolt shorter but reach same final plant height.
Of particular significance is the observation, that under these conditions (biotic stress due to presence of fungi in the soil) yields of the ABA S transgenics were significantly higher, ranging from 120% to 229% of the wild type control. The ABA Wt lines showed similar or slightly reduced yields relative to the Columbia control. This finding indicates that ABA S transgenic lines are affected less by the biotic stress. This observation has been confirmed, where 5 of the drought tolerant lines were grown in contaminated soil to maturity. The seed yields of transgenic lines, even though greatly reduced relative to optimal conditions, were 2.5 to 4.5 fold higher than those of Columbia wild type (Table 12).
TABLE 12 Seed yield of pBI121-AtCPP lines grown in contaminated soil. Values in bold indicate statistical differences at p = 0.05 ABA Seed Yield per plant Line sensitivity (g) % of Columbia 156 ABA S 0.33 ± 0.04 316% 23 ABA S 0.35 ± 0.05 336% 76 ABA S 0.31 ± 0.04 296% 84 ABA S 0.25 ± 0.33 237% 9 ABA S 0.48 ± 0.05 455% Columbia ABA Wt 0.11 ± 0.03

29b) pBI121-AtCPP Early Seedling Growth:

Four ABA S and one ABA Wt line plus Columbia were examined for early seedling growth on agar plates. Twenty seeds were plated in a line on agar plates containing 50% MS with 1% sucrose and vitamins and 6 plates per line were used. Plates were placed on slants, which allowed roots to grow downwards. Root length was measured on 7-day old seedlings and shoot and root biomass determined on 11-day old seedlings. Two of the ABA S transgenic lines had significantly longer roots and all 4 ABA S lines had shoot dry weights 114% to 123% of controls and root dry weights of 116% to 151% of controls. As a result, the shoot biomass to rootbiomass ratios were slightly reduced in transgenics. These results indicate that enhanced growth of these transgenics is evident in the early growth stage, shortly after germination, and the root growth is more enhanced relative to shoot growth. In a different experiment seedlings were pulled out of agar and roots were stained with toluidine blue to show their structure. FIG. 14 shows that transgenic lines had more extensive lateral root system, which would account for greater root biomass.
29c) pRD29A-HP-AtCPP Optimal Growth Characteristics
An optimal growth study has been conducted with 10 lines as described before. Vegetative growth data showed that two of the lines ( 12 and 9 ) had significantly more leaves and seven of the lines ( 12 , 22 , 23 , 47 , 82 , 9 ) had significantly greater shoot biomass. Bolting data showed that eight of the lines ( 12 , 22 , 23 , 47 , 82 , 9 , 90 , 93 ) were significantly delayed in flowering by one to two days, and seven of the lines were significantly shorter than Columbia at first open flower. All of the lines except 42 and 7 had significantly greater number of rosette leaves and shoot FW and this trend is maintained into the mid-flowering harvest ( FIG. 15 ). The plant height, however, by mid-flowering harvest was not significantly different between the transgenic lines and control. All the lines that showed this enhanced growth also showed drought tolerance and ABA sensitivity.
Example 30
Ultrastructure pBI121-AtCPP
Two of the drought tolerant and ABA S lines ( 35 and 76 ) plus Wt Columbia were used to examine stem and root cross-sections for any differences in ultrastructure. Free hand sections of mature stems (plants flowering for 10 days) were obtained from above the first node, stained with toluidine blue and preserved with glycerol. The stems of transgenic plants appeared to have more dense cellular structure and contain one or two more vascular bundles than those of Columbia Wt indicating more enhanced water and nutrient transport system.
Leaf disks were taken and fresh weights determined. Transgenic leaf disks were significantly heavier, 20-24% greater than corresponding wild type controls. This increase is believed to be as a result of a thicker leaf.
Example 31
Cold Stress Experiment pBI121-AtCPP
Four drought tolerant, ABA S lines ( 156 , 23 , 35 , 76 ) and one ABA Wt ( 95 ) line plus wild type Columbia were included in a cold stress study. Plants were grown in 3″ pots one per pot) with 10 replicate pots per line at 22 C for 10 days (7 days on agar plates and 4 in soil). The cold stress group was moved into 7° C. for 5 days while the optimal group was left at 22 C. After 5 days in the cold both cold stress group and the optimal group were harvested for shoot biomass determination. ABA S and drought tolerant lines had significantly greater shoot biomass than Columbia in both optimal (25 to 39% greater shoot fresh weight) and cold stress groups (18 to 44% greater shoot DW) (Table 13). Results of an eight-day cold stress showed that differences between the transgenic lines and Columbia were even more pronounced (53 to 61% greater shoot fresh weight). This result indicates greater plant vigor and better ability of transgenics to cope with cold stress.
TABLE 13 Shoot fresh weight of optimal and cold stressed (5 C. for 5 d) pBI121-AtCPP. Values in bold indicate statistical difference at p = 0.05 Optimal shoot FW Cold stress shoot FW ABA % of % of Line sensitivity mg Columbia mg Columbia 156 ABA S 95.4 ± 3.7 137% 23.1 0.7 118% 23 ABA S 96.3 ± 3.9 139% 28.3 1.5 144% 35 ABA S 87.0 ± 1.7 125% 25.3 1.4 130% 76 ABA S 94.7 ± 2.2 136% 27.3 1.5 140% 95 ABAWt 67 ± 2.4 96% 21.4 1.0 109% Columbia ABAWt 69 ± 1.9 19.6 1.1

Example 32
Drought Stress Under High Temperature pBI121-AtCPP
A drought stress experiment was conducted as described above except that day temperature of 32° C. (16 hr) and night temperature of 22° C. (8 hr) was maintained. These temperatures were achieved daily over a 2 hr ramping period. Four ABA S and one ABA Wt line plus Columbia were included. Plants were monitored daily for water loss and soil water content and after 5 days of drought treatment half of the plants were harvested and the other half was re- and watered and allowed to recover for four days. Shoots were harvested and shoot fresh weight determined. The results (Table 14) of this experiment showed that previously identified drought tolerant lines maintained their drought tolerant phenotype at high temperature and were able to recover well from the drought stress at high temperature
TABLE 14 Soil water content on day 2 and water lost in 2 days/final shoot dry weight plus recovery shoot FW after 5 days of drought stress at 32 C. day and 22 C. night temperatures. Values in bold indicate significant differences from the Columbia control. ABA soil water water lost in recovered shoot line sensitivity content day 2 2 d/shoot DW FW (g) 136 ABA S 50.4 ± 1.1 485.7 ± 18.5 1.30 ± 0.04 146 ABA S 52.1 ± 1.0 504.5 ± 7.9 1.15 ± 0.04 35 ABA S 52.2 ± 0.8 502.8 ± 15.8 1.19 ± 0.02 76 ABA S 52.1 ± 0.6 435.6 ± 10.5 1.11 ± 0.03 95 ABAWt 50.0 ± 0.9 518.2 ± 13.0 0.86 ± 0.03 Columbia ABAWt 48.6 ± 0.6 559.7 ± 19.0 0.84 ± 0.03

Example 33
Heat Stress and Seed Yield pBI121-AtCPP
Two ABA S lines and one ABA Wt line plus Columbia were examined for the effect of heat stress during flowering on the final seed yield. Plants were grown in 4 inch pots (one/pot) as described above and 9 days from first open flower the temperature was ramped from 22 C to 43 C over 2 hours and plants were kept at 43 C for 2 hr. Temperature was then ramped back to 22 C over 2 hours and plants were grown under optimal conditions until maturity. The seed yields from this experiment are shown in Table 15. One of the drought-tolerant lines (35) had significantly greater yield than Columbia.
TABLE 15 Seed yield of pBI121-AtCPP lines after two hour 43 C. heat stress 9 days from first open flower. Values in bold are statistically significant from Columbia. ABA seed yield seed yield line sensitivity (g/plant) (% of col.) 35 ABA S 0.55 ± 0.05 347% 76 ABA S 0.24 ± 0.03 148% 95 ABAWt 0.11 ± 0.02 69% Columbia ABAWt 0.16 ± 0.03

The effect of heat shock on lines of pBI121-AtCPP at the early flowering stage was assessed. Three ABA S lines ( 76 , 136 , 97 ) a ABA Wt line (95) and a Columbia wild type control were seeded in 128 cell flats, one flat per line. At the early flowering stage flats were exposed to a temperature of 46.8° C. for 50 minutes and then returned to normal growth conditions. Lack of continued growth from main meristems was defined as main meristem death and scored for each line. Data is shown in Table 16.
TABLE 16 Meristem death due to heat shock Line Wt 95 76 136 97 % Death 91 97 79 59 18

Example 34
Stomata Density Determinations pBI121AtCPP
Two ABA S lines (76 and 35) plus Columbia were examined for stomata density on the upper and lower leaf surface. Nail polish imprints of the upper and lower epidermis were obtained from a fully expanded leaf #5. These imprints were analyzed under the microscope and the number of stomata per 8.7×10 −8 m 2 were counted. There were no significant differences found between transgenics and Columbia in the stomata of the upper or lower epidermis (Table 17). The increases seen in drought tolerance and reduced water loss is not attributable to a reduced number of leaf stomata.
TABLE 17 Stomata numbers per 8.7 × 10 −8 m 2 of abaxial and adaxial epidermis of fully expanded leaf #5 in pBI121AtCPP. stomata stomata ABA on upper on lower line sensitivity epidermis epidermis 35 ABA S 68 ± 5 103 ± 7 76 ABA S 58 ± 6 120 ± 16 Columbia ABAWt 57 ± 6 116 ± 11

Example 35
PrPase Consensus Sequences
Also included in the invention are the PrPase consensus sequences. The consensus sequences were generated by alignment of the PrPase polypeptide and nucleic acid sequences.
The consensus sequence for the nucleic acid sequence alignment of GmCPP (SEQ ID NO: 18), GmPrPase2 (SEQ ID NO: 16), AtCPP (SEQ ID NO: 7), AtPrPase1 (SEQ ID NO: 3), AtPrPase2 (SEQ ID NO: 5), BnCPP (SEQ ID NO: 11), ZmPrPase2 (SEQ ID NO: 23), PpPrPase1 (SEQ ID NO: 1), and three other disclosed Arabidopsis PrPases sequences (AT4g01320 (SEQ ID NO: 27); AF007269 (SEQ ID NO: 29), and AFC1 (SEQ ID NO: 25)) as shown in FIG. 4 is provided in SEQ ID NO: 84. The consensus sequence for the corresponding amino acid sequence alignment shown in FIG. 5 is provided in SEQ ID NO: 85.
The consensus sequence for the nucleic acid alignment of the PrPase sequences of the invention from dicot plants (GmCPP (SEQ ID NO: 18), GmPrPase2 (SEQ ID NO: 16), AtCPP (SEQ ID NO: 7), AtPrPase1 (SEQ ID NO: 3), AtPrPase2 (SEQ ID NO: 5), BnCPP (SEQ ID NO: 11)) and three other disclosed dicot PrPases sequences (AT4g01320 (SEQ ID NO: 27), AF007269 (SEQ ID NO: 29), and AFC1 (SEQ ID NO: 25)) as shown in FIG. 16 is provided in SEQ ID NO: 86. The consensus sequence for the corresponding amino acid sequence alignment shown in FIG. 17 is provided in SEQ ID NO: 87.
The consensus sequence for the nucleic acid alignment of the Arabidopsis PrPase sequences of the invention (AtCPP (SEQ ID NO: 7), AtPrPase1 (SEQ ID NO: 3), AtPrPase2 (SEQ ID NO: 5)) and three other disclosed Arabidopsis PrPases sequences (AT4g01320 (SEQ ID NO: 27), AF007269 (SEQ ID NO: 29), and AFC1 (SEQ ID NO: 25)) as shown in FIG. 18 is provided in SEQ ID NO: 88. The consensus sequence for the corresponding amino acid sequence alignment shown in FIG. 19 is provided in SEQ ID NO: 89.
The consensus sequence for the nucleic acid alignment of the soybean PrPase sequences of the invention: GmCPP (SEQ ID NO: 18) and GmPrPase2 (SEQ ID NO: 16) as shown in FIG. 20 is provided in SEQ ID NO: 90. The consensus sequence for the corresponding amino acid sequence alignment shown in FIG. 21 is provided in SEQ ID NO: 91.
The “X” in the consensus sequence represents any amino acid and the “N” represents any nucleotide. Preferably “X” is a conservative amino acid substitution and the “N” a conservative nucleotide substitution. More preferably, “X” is the amino acid and “N” the nucleotide most prevalent at a given position.