US20020062495A1

US20020062495A1 - Novel polypeptides and polynucleotides relating to the a- and b-subunits of glutamate dehydrogenases and methods of use

Info

Publication number: US20020062495A1
Application number: US09/070,844
Authority: US
Inventors: Robert R. Schmidt; Philip Miller
Original assignee: Individual
Current assignee: Individual
Priority date: 1998-05-01
Filing date: 1998-05-01
Publication date: 2002-05-23
Also published as: US20010000266A1; US20040128710A1; US7485771B2

Abstract

Amino acid and nucleotide sequences relating to the glutamate dehydrogenase (GDH) enzyme are described. The GDH enzymes described herein were discovered in the alga Chlorella sorokiniana in the form of seven different inducible isoenzymes. These isoenzymes are found in the algae as chloroplast-localized hexamers composed of alpha- and beta-subunits. Plants transformed with nucleotide sequences encoding the alpha- or beta-subunits of the enzyme show improved properties, for example, increased growth and improved stress tolerance. A heterohexamer having both α- and β-subunits can have higher aminating:deaminating activity ratio than α-homohexamers or β-homohexamers.

Description

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a divisional application of co-pending application Ser. No. 08/725,596, filed Oct. 3, 1996, which is a continuation-in-part of co-pending application Ser. No. 08/541,033. filed Oct. 6, 1995.[0001]
[0002] This invention was made with government support under USDA Competitive Grant Number 87-CRCR-1-2476. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Inorganic nitrogen acquired by plants is ultimately converted to ammonium before being assimilated in organic nitrogen metabolism. One enzyme postulated to be involved in the assimilatory process is glutamate dehydrogenase (GDH), a group of ubiquitous enzymes found to be present in almost all organisms from microbes to higher plants and animals (Srivastava, H. S., R. P. Singh [1987] Phytochem. 26:597-610). GDH catalyses the reversible conversion of α-ketoglutarate to glutamate via a reductive amination that utilizes reduced β-nicotinamide adenine dinucleotide (NADH) or reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. The role of plant GDHs in the assimilation of ammonium into amino acids has been questioned since the discovery of the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway that is believed to be the favored pathway for ammonium assimilation in higher plants (Miflin, B. J., P. J. Lea [1976] Phytochem. 15:873-885).

The primary objection to GDH playing a major role in plant nitrogen metabolism is its low affinity for ammonium that would require high intracellular ammonium concentrations to function anabolically. Early evidence indicated that GDH is a catabolic enzyme catalyzing the deamination of glutamate with only a partially anabolic function in synthesizing glutamate (Wallgrove, J. C., N. P. Hall, A. C. Kendall, [1987 ] Plant Physiol. 83:155-158). The physiological role of large amounts of GDH present in various plant tissues and organelles is still unclear, and possible conditions under which GDH may play a significant role in carbon and nitrogen metabolism have not been resolved.

The majority of plant GDHs characterized to date are localized in the mitochondria; however, a GDH species differing in several properties (e.g., cofactor specificity, K _mvalues, organelle localization, thermal stability, among others) has been characterized from the chloroplast of a unicellular green alga Chlorella sorokiniana. C. sorokiniana cells have been shown to possess a constitutive, mitochondrial, tetrameric NAD-specific GDH (hereinafter designated “NAD-GDH”) (Meredith, M. J., R. M. Gronostajski, R. R. Schmidt [1978] Plant Physiol. 61:967-974), and seven ammonium-inducible, chloroplast-localized, homo- and heterohexameric NADP-specific GDH isoenzymes (hereinafter designated “NADP-GDH”) (Prunkard, D. E., N. F. Bascomb, R. W. Robinson, R. R. Schmidt [1986] Plant Physiol. 81:349-355; Bascomb, N. F., R. R. Schmidt [1987] Plant Physiol. 83:75-84). The seven chloroplastic NADP-GDH isoenzymes were shown to have different electrophoretic mobilities during native-PAGE, which can result from the formation of homo- and heterohexamers composed of varying ratios of α- and β-subunits (53.5 and 52.3 kilodaltons, respectively).

Chlorella cells cultured in 1 to 2 mM ammonium medium accumulate only the α-homohexamer (Bascomb and Schmidt, supra). The addition of higher ammonium concentrations (3.4 to 29 mM) to nitrate-cultured cells results in the accumulation of both α- and β-subunits in NADP-GDH holoenzymes (Prunkard et al., supra; Bascomb and Schmidt, supra; Bascomb. N. F., D. E. Prunkard, R. R. Schmidt [1987 ] Plant Physiol. 83:85-91). Prunkard et al. (Prunkard, D. E., N. F. Bascomb, N F W. T. Molin, R. R. Schmidt [1986] Plant Physiol. 81:413-422) demonstrated that the NADP-GDH subunit ratio and isoenzyme pattern is influenced by both the carbon and nitrogen source as well as the light conditions under which cells are cultured.

The α- and β-NADP-GDH homohexamers purified from Chlorella cells have strikingly different ammonium K _mvalues: however, the K_mvalues for their other substrates are very similar. The α-homohexamer (composed of six identical α-subunits) that catalyzes the biosynthesis of glutamate is allosterically regulated by NADPH and possesses an unusually low K_mfor ammonium that ranges from 0.02 to 3.5 mM, depending on the NADPH concentration (Bascomb and Schmidt, supra). The K_mvalue for ammonium of the α-homohexamer is the lowest reported ammonium K_mfor any plant GDH characterized to date. In contrast, the β-homohexamer (catabolic form) is a non-allosteric enzyme with an ammonium K_mof approximately 75 mM. From these studies involving purified enzymes, it had been heretofore postulated that the heterohexamers have varying degrees of affinity for ammonium ranging between the K_mvalues for the α- and β-homohexamers. Surprisingly, however, we have discovered that certain heterohexamers can have aminating:deaminating activity ratio which is greater than either the α- or β-homohexamers.

Although the α- and β-subunits have distinct in vivo turnover rates (Bascomb et al. supra) and the corresponding homohexamers have remarkably different ammonium K _mvalues, the α- and β-subunits are derived from precursor proteins of nearly identical size (ca 58,000 Daltons) and were shown to have very similar peptide maps (Prunkard et al.. supra; Bascomb and Schmidt, supra). Moreover, polyclonal antibodies prepared against the β-homohexamer are capable of immunoprecipitating all of the NADP-GDH isoenzymes (Yeung, A. T., K. J. Turner, N. F. Bascomb, R. R. Schmidt [1981] Anal. Biochem. 10:216-228; Bascomb et al., supra), but do not crossreact with the mitochondrial NAD-GDH. In addition, previous research in this laboratory provided genomic cloning and southern blot evidence that indicated the C. sorokiniana genome possesses a single NADP-GDH structural gene (Cock, J. M., K. D. Kim, P. W. Miller, R. G. Hutson, R. R. Schmidt [1991] Plant Mol. Biol. 17:17-27).

The C. sorokiniana nuclear-encoded chloroplastic NADP-GDH isoenzymes are the only chloroplastic localized GDH sequences isolated and characterized from plants. Although the Chlorella GDH isoenzymes had been previously characterized, it has been discovered in the present invention that the two mature subunits arise via specific processing of two similar precursor proteins encoded by two mRNAs formed by alternative splicing of a pre-mRNA derived from a single nuclear gene. Furthermore, the identification of the cleavage site and amino-terminal peptide sequence of the mature functional GDH subunits had not been accomplished prior to the present invention.

BRIEF SUMMARY OF THE INVENTION

The present invention provides the isolation and characterization of two full-length cDNAs from mRNAs isolated from the unicellular green algae Chlorella sorokiniana. The two cDNAs encode the precursor proteins (α-precursor, 56.35 kD; β-precursor, 57.85 kD) that are processed to yield the mature α- and β-subunits (53.5 kD; 52.3 kD, respectively) that compose the active NADP-GDH hexameric isoenzymes. The present invention concerns a single NADP-GDH gene which is alternatively spliced to yield two mRNAs that encode two different chloroplast precursor proteins. These precursor proteins can then be processed to the mature α- and β-subunits of the NADP-GDH isoenzymes. Also described are useful fragments or mutants of the nucleotide and amino acid sequences which retain the disclosed activity or utility. For example, certain fragments of the amino acid sequences provided herein can be useful as transit peptides, providing the protein with the capability to enter and remain in certain cell compartments. The nucleotide sequences which are described herein, and fragments of those nucleotide sequences, can be useful, for example, as primers in amplification procedures or as probes to hybridize to complementary sequences of interest. The nucleotide and amino acid sequences and fragments thereof as described herein can also be useful as molecular weight markers or in identifying and conforming the relatedness of other nucleotide sequences, polypeptides, or isoenzymes which pertain to NADP-GDH.

The present invention further provides methods in which assimilation of inorganic nitrogen into organic nitrogen metabolism of higher plants can be altered by expressing GDH from C. sorokiniana or GDHs isolated from other organisms. The alteration of nitrogen assimilation can have the effect of increasing nitrogen assimilation which, as is well understood in the art, can affect the composition of the plant through an inverse effect on carbon metabolism, e.g., accumulation of carbohydrates. The subject invention also concerns DNA constructs for use in the described methods. The present invention includes the identification of the amino-terminal sequences of the α- and β-subunits which can assemble to form NADP-GDH isoenzymes, e.g., the native hexameric NADP-GDH found in C. sorokiniana chloroplasts. This precise molecular information can be employed to express NADP-GDH with the unique kinetic properties of the C. sorokiniana chloroplastic α- and β-NADP-GDH homohexamers. The present invention also provides recombinant cells or organisms, e.g., transgenic crops or plants which, by expressing the genes of the described polynucleotide sequences to produce corresponding polypeptides, can have an increased yield, improved ammonia assimilatory properties which can advantageously increase their tolerance of ammonia toxicity, improved osmotic stress tolerance, and improved composition of the crop or plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a pattern of NADP-GDH activities in homogenates of synchronous [0012] C. sorokiniana cells cultured for 240 min in 29 mM ammonium medium in continuous light. Aliquots of clarified homogenates, from cell collected at various time intervals, were analyzed spectrophotometrically for both aminating () and deaminating (◯) NADP-GDH activities.
FIG. 2 shows patterns of accumulation of NADP-GDH antigens in illuminated cells cultured in 29 mM ammonium medium for 240 min. At zero time, ammonium was added to synchronous [0013] C. sorokiniana daughter cells and the culture was illuminated. Autoradiographs of Western blots were analyzed by laser densitometry to determine the relative levels of the NADP-GDH α-subunit () and β-subunit (◯) throughout the 240 min induction period.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 is the cDNA for the precursor-protein of the α-subunit of an NADP-specific glutamate dehydrogenase. [0014]
SEQ ID NO.2 is the deduced amino acid sequence of the polynucleotide of SEQ ID NO. 1. [0015]
SEQ ID NO.3 is the cDNA for the precursor-protein of the β-subunit of an NADP-specific glutamate dehydrogenase. [0016]
SEQ ID NO.4 is the deduced amino acid sequence of the polynucleotide of SEQ ID NO.3. [0017]
SEQ ID NO.5 is the N-terminal sequence for the NADP-GDH α-subunit. [0018]
SEQ ID NO.6 is the N-terminal sequence for the NADP-GDH β-subunit. [0019]
SEQ ID NO.7 is the cDNA sequence in the clone designated pBGDc53. [0020]
SEQ ID NO.8 is a primer which hybridizes to the conserved region of NADP-GDH mRNAs. [0021]
SEQ ID NO.9 is a poly(dT) polynucleotide used as an adaptor primer according to the subject invention. [0022]
SEQ ID NO.10 is a polynucleotide used as a primer according to the subject invention. [0023]
SEQ ID NO.11 is a polynucleotide used as a primer according to the subject invention. [0024]
SEQ ID NO.12 is a polynucleotide used as an adaptor primer according to the subject invention. [0025]
SEQ ID NO.13 is the polynucleotide insert in the clone designated [0026] pRGDc 60.
SEQ ID NO.14 is the polynucleotide insert in the clone designated pRGDc 61. [0027]
SEQ ID NO.15 is the polynucleotide used as a primer according to the subject invention. [0028]
SEQ ID NO.16 is the polynucleotide insert in a clone designated pGDc 63. [0029]
SEQ ID NO.17 is the polynucleotide insert of a clone designated pGDc 64. [0030]
SEQ ID NO.18 is the polynucleotide resulting from ligation of purified fragments of the inserts in the clones designated pBGDc 53 and pGDc 63, according to the subject invention. [0031]
SEQ ID NO.19 is the polynucleotide resulting from ligation of purified inserts of the clones designated pGDc 64 and pBGDc 53. [0032]
SEQ ID NO.20 is a polynucleotide used as a primer according to the subject invention. [0033]
SEQ ID NO.21 is a polynucleotide used as a primer hybridizing to the 3′ terminus of the template DNA according to the subject invention. [0034]
SEQ ID NO.22 is a polynucleotide used as a primer according to the subject invention. [0035]
SEQ ID NO.23 is the polynucleotide sequence (cDNA) of the processed, mature NADP-GDH α-subunit. [0036]
SEQ ID NO.24 is the amino acid sequence of the processed, mature NADP-GDH α-subunit. [0037]
SEQ ID NO.25 is the polynucleotide (cDNA) sequence of the processed, mature NADP-GDH β-subunit. [0038]
SEQ ID NO.26 is the amino acid sequence of the processed, mature NADP-GDH β-subunit. [0039]

DETAILED DISCLOSURE OF THE INVENTION

The present invention provides heretofore undescribed polynucleotide sequences, for example, cDNAs for precursor-proteins of α- and β-subunits of an ammonium inducible, chloroplast-localized NADP-specific glutamate dehydrogenase (hereinafter NADP-GDH) from Chlorella sorokiniana. The nucleotide sequences for the precursor proteins of the α- and β-subunits that form NADP-GDH are shown in SEQ ID NOS. 1 and 3, respectively. The deduced amino acid sequences for the precursor-proteins of the α- and β-subunits of the NADP-GDH enzyme from [0040] Chlorella sorokiniana are shown in SEQ ID NOS. 2 and 4, respectively.

E. coli hosts comprising the subject cDNA inserts were deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 USA. The cultures were assigned the following accession numbers by the repository:



Culture	Accession number	Deposit date

E. coli DH5α	ATCC 69925	October 6, 1995
α-NADP-GDH
SEQ No. 1 (+42 bp)
E. coli DH5α	ATCC 69926	October 6, 1995
β-NADP-GDH
SEQ No. 1 (−42 bp)

The subject cultures have been deposited under conditions that assure that access to the culture(s) will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action. [0042]
Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of a deposit(s), and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The depositor acknowledges the duty to replace the deposit(s) should the depository be unable to furnish a sample when requested, due to the condition of the deposit(s). All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them. [0043]
Automated amino acid sequence analysis identifies 20 and 10 amino-terminal amino acid residues of the α- and β-subunits, respectively. Alignment of the α- and β-subunit peptide sequences reveals that the two subunits are identical with the exception of an 11- amino acid extension present in the larger α-subunit. Monoclonal antibodies raised against the α-subunit were shown to recognize the β-subunit providing further evidence that the two subunits are nearly identical. The identification of the unique α- and β-subunit processing sites within the precursor proteins provides the molecular mechanism to explain the different kinetic properties of the α- and β-NADP-GDH homohexameric isoenzymes. [0044]
The aforementioned data provide information applicable to genetically engineer plants with a specific GDH having favorable kinetic properties which can influence both carbon and nitrogen metabolism. Based on the high guanine/cytosine content the cDNAs are highly amenable for heterologous expression in higher plants. The introduction of either or both subunits with their chloroplast targeting sequences or with other organellar targeting sequences in heterologous plant systems can improve nitrogen assimilation and influence the carbon/nitrogen balance. [0045]
It has been discovered that chloroplast localization is related to, and can be dependent on, the N-terminus of the α- or β-precursor protein. Cleavage of the N-terminus of the precursors yields the mature proteins. Accordingly, the chloroplast transit peptide comprises a peptide which forms, or is an active fragment of, the N-terminus cleaved from the precursor protein. Peptides having similar or equivalent amino acid sequences, or that have a tertiary structure or conformation similar to these cleaved peptides can also function as transit peptides. The chloroplast-transit peptide comprises the active fragment of the N-terminal peptide cleaved from the α-precursor (a 40-mer) or the β-precursor (a 37-mer). The polynucleotide sequences encoding the chloroplast-transit peptides can be used by persons of ordinary skill in the art to produce chloroplast-transitpeptides employed with the peptides described herein, or others known in the art. [0046]
Adding, removing, or replacing the chloroplast-transit peptide associated with a protein, e.g., the GDH enzyme, can be employed to localize the protein according to need, by means well known in the art. For example, localization of the enzyme in a chloroplast of a cell can be achieved by the insertion of a chloroplast-transit peptide onto an amino acid sequence lacking such a transit peptide. Species-specific chloroplast-transit peptides can be added or can replace those present to optimize insertion into the chloroplast of a desired species. In addition, localization inside the chloroplast of a protein expressed within the chloroplast can be achieved by direct transformation of the plastid with the polynucleotide sequences encoding an expressed protein. Similarly, removal of a chloroplast-transitpeptide or production of a recombinant protein lacking the peptide can be utilized to sequester the protein in a cellular compartment other than the chloroplast. [0047]
Transformed plants expressing the α-homohexamer can be more tolerant to ammonia toxicity, assimilate ammonium more efficiently, and respond more rapidly to osmotic stress encountered in transiently saline soils by providing glutamate the precursor to the osmoprotectant proline. Expression of, for example, the β-homohexamer or GDH heterohexamers can be used to alter the rate of nitrogen assimilation, favoring accumulation of carbohydrates in fruits and other storage organs. [0048]
Unexpectedly, it was discovered that a hexamer comprising at least one α-subunit and at least one β-subunit, i.e., a heterohexamer, can have advantageous activity. Specifically, the aminating:deaminating activity ratio (i.e.. biosynthetic capacity for synthesis of glutamate) of a chloroplastic NADP-GDH isozyme can be increased by incorporating both α- and β-subunits into the hexameric protein rather than using a homohexamer comprising only the α- or only the β-subunits. In one embodiment of the invention, it can be advantageous to co-express cDNAs encoding both types of subunits in the same plant at different rates/levels such that a particular ratio of α- and β-subunits is obtained in the heterohexamer. For example, we have discovered that an NADP-GDH heterohexamer having at least one of the subunits in the β-form is preferred for increasing aminating:deaminating activity ratio. A more preferred heterohexamer has 2-5 β-subunits. This differential rate of expression of the two cDNAs can be accomplished by placing them under the control of plant promoters with different strengths or under the same promoter that has been modified to generate different levels of expression. The use of this algal NADP-GDH isozyme system in plant biotechnology has advantages over NADP-GDHs from organisms, such as bacteria, that contain only a single form of the enzyme (i.e., no isozymes). [0049]
It is recognized that expression levels of certain recombinant proteins in transgenic plants can be improved via increased expression of stabilized mRNA transcripts; and that, conversely, detection of these stabilized RNA transcripts may be utilized to measure expression of translational product (protein). Low expression of protein RNA in plants and, therefore, of low protein expression, can be resolved through the use of an improved synthetic gene specifying the desired protein from the gene source organism. [0050]
Thus, in one embodiment of the subject invention, bacteria and plants can be genetically engineered to attain desired expression levels of novel proteins having agricultural or otherwise commercial value. To provide genes having enhanced expression in plants, the DNA sequence of the gene can be modified to comprise codons preferred by highly expressed plant genes, to attain an A+T content in nucleotide base composition substantially that found in plants, and also preferably to form a plant initiation sequence, and to eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation and termination of RNA and to avoid sequences that constitute secondary structure hairpins and RNA splice sites. For example, in synthetic genes, the codons used to specify a given amino acid can be selected with regard to the distribution frequency of codon usage employed in highly expressed plant genes to specify that amino acid. As is appreciated by those skilled in the art, the distribution frequency of codon usage utilized in the synthetic gene is a determinant of the level of expression. [0051]
For purposes of the subject invention, “frequency of preferred codon usage” refers to the preference exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. To determine the frequency of usage of a particular codon in a gene, the number of occurrences of that codon in the gene is divided by the total number of occurrences of all codons specifying the same amino acid in the gene. Similarly, the frequency of preferred codon usage exhibited by a host cell can be calculated by averaging frequency of preferred codon usage in a large number of genes expressed by the host cell. It is preferable that this analysis be limited to genes that are highly expressed by the host cell. [0052]
When synthesizing a gene for improved expression in a host cell it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell. [0053]
The percent deviation of the frequency of preferred codon usage for a synthetic gene from that employed by a host cell is calculated first by determining the percent deviation of the frequency of usage of a single codon from that of the host cell followed by obtaining the average deviation over all codons. As defined herein this calculation includes unique codons (i.e., ATG and TGG). In general terms the overall average deviation of the codon usage of a synthetic gene from that of a host cell is calculated using the equation [0054] $A = \sum_{n = 1}^{Z} \frac{\frac{X_{n} - Y_{n}}{X_{n}} \times 100}{Z}$
where X[0055] _n=frequency of usage for codon n in the host cell; Y_n=frequency of usage for codon n in the synthetic gene. Where n represents an individual codon that specifies an amino acid, the total number of codons is Z. The overall deviation of the frequency of codon usage, A, for all amino acids should preferably be less than about 25%, and more preferably less than about 10%. Hence, a gene can be designed such that its distribution frequency of codon usage deviates, preferably, no more than 25% from that of highly expressed plant genes and, more preferably, no more than about 10%. In addition, consideration is given to the percentage G+C content of the degenerate third base (monocotyledons appear to favor G+C in this position, whereas dicotyledons do not). It is also recognized that the XCG (where X is A, T, C or G) nucleotide is the least preferred codon in dicots whereas the XTA codon is avoided in both monocots and dicots. Synthetic genes of this invention also preferably have CG and TA doublet avoidance indices closely approximating those of the chosen host plant. More preferably these indices deviate from that of the host by no more than about 10-15%.
Assembly of the NADP-GDH gene of this invention can be performed using standard technology known in the art. A structural gene designed for enhanced expression in plants of the specific embodiment can be enzymatically assembled within a DNA vector from chemically synthesized oligonucleotide duplex segments. The gene can then be introduced into a plant host cell and expressed by means known to the art. Preferably, the protein produced upon expression of the synthetic gene in plants is functionally equivalent to a native protein in having comparable or improved aminating/deaminating activity. According to the subject invention, functionally equivalent refers to identity or near identity of function. A synthetic gene product which has at least one property relating to its activity or function, which is the same or similar to a natural protein is considered functionally equivalent thereto. [0056]
Modifications in nucleotide sequence of the coding region can be made to alter the A+T content in DNA base composition of a synthetic gene to reflect that normally found in genes for highly expressed proteins native to the host cell. Preferably the A+T content of the synthetic gene is substantially equal to that of said genes for highly expressed proteins. In genes encoding highly expressed plant proteins, the A+T content is approximately 55%. It is preferred that the synthetic gene have an A+T content near this value, and not sufficiently high as to cause destabilization of RNA and, therefore, lower the protein expression levels. More preferably, the A+T content is no more than about 60% and most preferably is about 55%. Also, for ultimate expression in plants, the synthetic gene nucleotide sequence preferably can be modified to form a plant initiation sequence at the 5′ end of the coding region. In addition, particular attention is preferably given to assure that unique restriction sites are placed in strategic positions to allow efficient assembly of oligonucleotide segments during construction of the synthetic gene and to facilitate subsequent nucleotide modification. As a result of these modifications in coding region of the native gene, the preferred synthetic gene is expressed in plants at an enhanced level when compared to that observed with natural structural genes. [0057]
It is known that the relative use of synonymous codons differs between the monocots and the dicots. In general, the most important factor in discriminating between monocot and dicot patterns of codon usage is the percentage G+C content of the degenerate third base. In monocots, 16 of 18 amino acids favor G+C in this position, while dicots only favor G+C in 7 of 18 amino acids. [0058]
For soybean and maize, the maize codon usage pattern resembles that of monocots in general, whereas the soybean codon usage pattern is almost identical to the general dicot pattern. [0059]
In designing a synthetic gene for expression in plants, it is preferred to eliminate sequences which interfere with the efficacy of gene expression. [0060]
A synthetic gene may be synthesized for other purposes in addition to that of achieving enhanced levels of expression. For example, in accordance with the subject invention, one of the nucleotide sequences encoding the α-subunit or the β-subunit of NADP-GDH can be modified such that the products are differentially expressed, favoring expression of one of the subunits. A result of such differential expression is a heterohexamer comprising more of one subunit than the other. Modification may encompass substitution of one or more, but not all, of the oligonucleotide segments used to construct the synthetic gene by a corresponding region of natural sequence. Preferably, differential expression of the nucleotide sequences encoding the α- and β-subunits of the NADP-GDH polypeptides can be employed to produce a heterohexamer having at least one β-subunit, more preferably two to five β-subunits, and most preferably three β-subunits. [0061]
The recombinant DNA molecule comprising a nucleotide sequence of the subject invention can be introduced into plant tissue by any means known to those skilled in the art. The technique used for a given plant species or specific type of plant tissue depends on the known successful techniques. As novel means are developed for the stable insertion of foreign genes into plant cells and for manipulating the modified cells, skilled artisans will be able to select from known means to achieve a desired result. Means for introducing recombinant DNA into plant tissue include, but are not limited to, direct DNA uptake (Paszkowski, J. et al. (1984) EMBO J. 3:2717), electroporation (Fromm, M. et al. (1985) Proc. Natl. Acad. Sci. USA 82:5824), microinjection (Crossway, A. et al. (1986) Mol. Gen. Genet. 202:179), or T-DNA mediated transfer from [0062] Agrobacterium tumefaciens to the plant tissue. There appears to be no fundamental limitation of T-DNA transformation to the natural host range of Agrobacterium. Successful T-DNA-mediated transformation of monocots (Hooykaas-Van Slogteren. G. et al. (1984) Nature 311:763), gymnosperms (Dandekar. A. et al. (1987) Biotechnology 5:587) and algae (Ausich, R., EPO application 108,580) has been reported. Representative T-DNA vector systems are described in the following references: An, G. et al. (1985) EMBO J. 4:277; Herrera-Estrella, L. et al. (1983) Nature 303:209; Herrera-Estrella, L. et al. (1983) EMBO J. 2:987; Herrera-Estrella, L. et al. (1985) in Plant Genetic Engineering, New York: Cambridge University Press, p. 63. Once introduced into the plant tissue the expression of the structural gene may be assayed by any means known to the art, and expression may be measured as mRNA transcribed or as protein synthesized. Techniques are known for the in vitro culture of plant tissue, and in a number of cases, for regeneration in to whole plants. Procedures for transferring the introduced expression complex to commercially useful cultivars are known to those skilled in the art.
In one of its preferred embodiments the invention disclosed herein comprises expression in plant cells of an NADP-GDH gene under control of a plant expressible promoter, that is to say, by inserting the gene into T-DNA under control of a plant expressible promoter and introducing the T-DNA containing the insert into a plant cell using known means. Once plant cells expressing the gene under control of a plant expressible promoter are obtained, plant tissues and whole plants can be regenerated therefrom using methods and techniques well-known in the art. The regenerated plants are then reproduced by conventional means and the introduced genes can be transferred to other strains and cultivars by conventional plant breeding techniques. [0063]
The introduction and expression of the NADP-GDH gene can be used to improve. e.g., increase, yields in a crop. Other uses of the invention, exploiting the properties of the genes introduced into plant species will be readily apparent to those skilled in the art. [0064]
Differences also exist between codon choice in plant nuclear genes and in cholorplasts. Chloroplasts differ from higher plants in that they encode only 30 tRNA species. Since chloroplasts have restricted their tRNA genes, the use of preferred codons by chloroplast-encoded proteins appears more extreme. However, a positive correlation has been reported between the level of iso accepting tRNA for a given amino acid and the frequency with which this codon is used in the chloroplast genome (Pfitzinger et al. (1987) Nucl. Acids Res. 15:1377-l386. In general, the chloroplast codon profile more closely resembles that of unicellular organisms, with a strong bias towards the use of A+T in the degenerate third base. [0065]
Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. [0066]

EXAMPLES

Example 1

Kinetics of C. sorokiniana Chloroplast Glutamate Dehydrogenases

The chloroplastic glutamate dehydrogenase α- and β-isoenzymes used in the following experiments are naturally produced by an organism characterized as [0067] Chlorella sorokiniana.
[0068] C. sorokiniana culture conditions. For kinetic characterization in both the aminating and deaminating directions, the α- and β-holoenzymes were purified from cells that were accumulating only one form of homohexameric GDH isoenzyme.
The [0069] C. sorokiniana cells (UTEX-1230, University of Texas algal culture collection: 3B2NA, Robert R. Schmidt, University of Florida, Microbiology Cell Science Department) were cultured autotrophically as previously described by Prunkard et al., supra in a modified basal salts medium. The modified medium contained in mM concentration: CaCl₂, 0.34; K₂SO₄6.0; KH₂PO₄, 18.4; MgCl₂, 1.5; in μM concentration CoCl₂, 0.189; CuCl₂, 0.352; EDTA, 72; FeCl₃, 71.6; H₃BO₃, 38.8; MnCl₂, 10.1; NH₄VO₄, 0.20; (NH₄)₆MO₇O₂₄, 4.19; NiCl₂, 0.19; SnCl₂, 0.19; ZnCl₂, 0.734. The medium was supplemented with 1 mM NH₄Cl, 29 mM NH₄Cl, or 29 mM KNO₃as a nitrogen source depending on the experimental conditions. The medium containing NH₄Cl was adjusted to pH 7.4. and medium containing KNO₃was adjusted to pH 6.8 with KOH after autoclaving. Cells were supplied with a 2% (v/v) CO₂-air mixture and light intensity sufficient to allow cell division into four progeny.
Purification of the NADP-GDH isoenzymes. For purification of the glutamate dehydrogenase α-isoenzyme, [0070] C. sorokiniana cells were cultured with continuous light in 29 mM ammonium medium in a 30 L Plexiglas chamber as previously described (Baker, A. L., R. R. Schmidt [1963] Biochim. Biophys. Acta 74:75-83). Cells were harvested at 4.0 OD₆₄₀by centrifugation at 30,000 rpm through a Sharples centrifuge and washed two times in 10 mM Tris (pH 8.5 at 4° C.). Pelleted cells (130 g) were stored at −20° C. in 250 mL centrifuge bottles until use. Purification of NADP-GDH was accomplished using a modified procedure of Yeung et al., supra. Procedural modifications involved the substitution of Sephadex G-200 gel (Pharmacia) for G-150 gel in the gel-filtration column, and the addition of NADP⁺ as a stabilizer to a final concentration of 0.1 mM to the gel-filtration buffer and all subsequent storage buffers. As a final modification, the NADP⁻ affinity resin step was omitted and a preparative nondenaturing-PAGE step was substituted (Miller, P. W., W. D. Dunn, R. R. Schmidt [1994] BioRad US/EG Bulletin 1897).
The GDH deaminating enzyme assay solution was composed of 44 mM Tris, 20.4 mM glutamate, and 1.02 mM NADP[0071] ⁻, pH 8.8. The aminating assay solution was composed of 50 mM Tris, 25 mM α-ketoglutarate, 0.357 mM NADPH, and 0.356M (NH₄)₂SO₄, pH 7.4. One unit of enzyme activity was the amount of NADP-GDH required to reduce or to oxidize 1.0 μmol of NADP⁺ or NADPH per minute at 38.5° C.
Sephadex G-200 column fractions possessing NADP-GDH activity were pooled and concentrated via Diaflow filtration. The soluble enzyme (68 mg) was protected from oxidation by the addition of DTT to a final concentration of 10 mM, and dialyzed for 30 minutes against 28.8 mM Tris, 192 mM glycine, 2 mM DTT (pH 8.4). The dialysate was clarified by centrifugation at 20,000 g for 10 minutes at 4° C. and was combined with 3 mL of 40% (w/v) sucrose and 1 mL of 0.02% bromophenol blue. [0072]
For preparative nondenaturing PAGE, a 3 cm tall 7% acrylamide (w/v, 28 acrylamide: 0.735 bis-acrylamide, pH 8.8) resolving gel, and a 2 cm tall 2% acrylamide (w/v, 1.6 acrylamide: 0.4 bis-acrylamide, pH 6.6) stacking gel were cast in the 28 mm ID gel tube of the Model 491 Prep Cell. All acrylamide stocks were pretreated with AG501-X8 mixed bed resin to remove any contaminating acrylic acid residue to prevent in vitro N-acylation of proteins during electrophoresis. The protein sample was electrophoresed at 15 mA constant power for 20 minutes and then for 3.5 hours at a constant power of 30 mA. Six milliliter fractions were collected and assayed for NADP-GDH deaminating activity and GDH containing fractions were pooled. The enzyme in the pooled fractions in 10 mM KPO[0073] ₄(pH 6.2), 0.1 mM NADP⁺ was concentrated by Diaflow ultrafiltration to 1 mg/mL as determined by the method of Bradford, using BSA as a standard. The concentrated enzyme preparation was stored at −20° C. The purity of the preparation was determined by silver-staining to visualize proteins resolved by 10% (w/v) Tris-Tricine SDS-PAGE (Schagger, H., G. von Jagow [1987] Anal. Biochem. 166:368-379).
The NADP-GDH β-isoenzyme was purified from a mixture of cells cultured for 240 minutes in 1 mM ammonium medium (14 g), 90 minutes in 1 mM ammonium medium (6 g), and for 20, 40, 60, and 80 minutes in 29 mM ammonium medium (1 g/time point) according to Bascomb and Schmidt, supra. The NADP-GDH β-isoenzyme was partially purified using a scaled down modified procedure of Yeung et al., supra. The DEAE sephacel ion exchange columns (pH 7.4, and pH 6) were scaled down to a 40 mL bed volume and a 400 mL linear KCl gradient (0 to 0.4M) was used to elute the proteins in 3 mL fractions. The pH 6 DEAE ion-exchange column fractions containing NADP-GDH were combined into two pools; corresponding to the leading and trailing halves of the NADP-GDH activity peak. The separate pooled fractions were dialyzed against 10 mM KPO[0074] ₄(pH 6.2), 2 mM DTT for 16 hours, and affinity purified using Type 3 NADP⁺ affinity gel (Pharmacia) as previously described (Bascomb and Schmidt, supra). The NADP-GDH in the pooled fractions was concentrated via Diaflow ultrafiltration to 2 mg/ml protein, as determined by the method of Bradford (Bradford, M. M. [1976] Anal. Biochem. 72:248-254), and stored at 4° C. until further use. After resolution of the proteins by 8% (w/v) Tris-Tricine SDS-PAGE, the purity of the preparation was determined by silver staining.
Table 1 summarizes the K[0075] _mvalues determined for both the α- and β-homohexameric isoenzyme aminating reaction.

TABLE 1

GDH Isoform Substrate K_mValue (mM)

α-homohexamer NADPH 0.14

NH₄ ⁺ 0.02-3.5

α-ketoglutarate 0.35*

β-homohexamer NADPH 0.14

NH₄ ⁺ 77

α-ketoglutarate 12
[0076]

TABLE 2

GDH Isoform Substrate K_mValue (mM)

α-homohexamer NADP 0.04

Glutamate 38.2

β-homohexamer NADP+ 0.04

Glutamate 32.3
Activity of the α-, β-heterohexamer. The aminating and deaminating activities of the mixture of native NADP-GDH isoenzymes (heterohexamers composed of varying ratios of the α- and β-subunits) were also measured with saturating levels of substrates throughout the 240 minute induction period (FIG. 1). The aminating and deaminating activities showed initial induction lags of 20 to 40 min, respectively. The aminating activity increased rapidly during the first 100 min, decreased sharply between 100 min and 140 min, and increased sharply once again between 140 min and 240 min. In contrast, the deaminating activity increased in almost a linear manner throughout the induction after the initial induction-lag. [0077]
During the 240 min induction period in 29 mM ammonium medium, the patterns of accumulation of the [0078] Chlorella sorokiniana NADP-GDH α- and β-subunits in isoenzymes were also examined by use of a western blot immunodetection procedure following SDS polyacrylamide-gel electrophoresis (see FIG. 2). The NADP-GDH β-subunit was detected at T₀and increased for the first 40 min followed by a gradual decrease through the remainder of the induction period. The α-subunit was first detected at 20 min. This subunit accumulated at a low rate for the first 80 min. showed a marked increase between 80 min and 100 min, and thereafter accumulated in a linear manner at a lower rate for the remainder of the induction period. The transition from the β-subunit being the prominent species to the α-subunit being prominent occurred between 60 and 80 min.
The aminating:deaminating activity ratio and the α:β subunit ratio were calculated to determine if changes in the subunit ratio in the mixture of NADP-GDH isoenzymes correlated with the predicted aminating:deaminating activity ratio during the time-course of the induction period (Table 3). Surprisingly, the highest aminating:deaminating ratio was observed at 60 min when the subunit ratio showed the β-subunit to be the prominent NADP-GDH antigen, whereas the α-subunit was the prominent form when the aminating:deaminating activity ratio was the lowest. This latter result was not predictable in advance. [0079]
Prior to this discovery, substrate kinetic studies of purified α- and β-homohexamers, the cc-homohexamer, with its very high affinity for ammonium (relative to the β-homohexamer), was assumed to be the isoenzyme-form with the highest aminating activity (i.e., biosynthetic capacity for glutamate synthesis). The results suggested that the individual subunits would act independently with respect to their kinetic properties in homo- and heterohexamers. [0080]
Comparison of the aminating:deaminating activity ratio with the α:β subunit ratio throughout the 240 min induction in 29 mM ammonium medium revealed an unexpected correlation between the maxima in these ratios (Table 3). [0081]

Table 3. NADP-GDH aminating:deaminating activity and α-subunit:β-subunit ratios during ammonium induction period in C. sorokiniana cells.

TABLE 3


Time (min)	Am:Deam Activity	α:β Subunit

0	2.87	0.28
20	2.96	0.58
40	3.81	0.49
60	4.51	0.80
80	3.49	1.57
100	2.73	8.74
140	1.61	11.23
240	1.13	34.79

The peak in aminating:deaminating ratio occurred at 60 min at which time the β-subunit was the prominent but not exclusive antigen, whereas the α-subunit was prominent when the aminating:deaminating ratio was lowest. Interestingly, the aminating activity was highest when both subunits were present, suggesting that heterohexamer(s), formed by combination(s) of the α- and β-subunits, can have a higher aminating activity than a homohexamer. Based on the much lower K[0083] _mof the purified α-homohexamer that the β-homohexamer for ammonium, it had been predicted earlier that the α-homohexamer would have a higher aminating activity than any heterohexamer composed of the two subunits (Bascomb and Schmidt, 1987).

Example 2

Sequencing of Polypeptides and Polynucleotides

Amino-terminal sequencing of the mature subunits. An aliquot of a preparation of purified NADP-GDH α-subunit (120 pmol) and a partially purified preparation of NADP-GDH α-subunit (80 pmol) and β-subunit (50 pmol) were resolved by 8% (w/v) Tris-Tricine SDS-PAGE and electroblotted to a PVDF membrane (Immobilon-P[0084] ^SQ, Millipore) as described by Plough et al. (Plough, M., A. L. Jensen, V. Barkholt [1989] Anal. Biochem. 181:33-39). To prevent in vitro acylation of the protein amino-terminal residues, all polyacrylamide solutions used in PAGE were treated with AG501-X8 mixed bed resin to remove contaminating acrylic acid. An Applied Biosystems, Inc. model 470A gas phase sequencer was utilized for automated Edman degradation amino sequence analysis. The PTH-aa derivatives were identified by RP-HPLC. Protein sequence analysis of the electroblotted proteins was provided by the Interdisciplinary Center for Biotechnology Research Protein Chemistry Core facility at the University of Florida.
The following N-terminal sequence was determined for the α-subunit:AVSLEEQISAMDATTGDFTA(SEQ ID NO. 5). The following N-terminal sequence was determined for the β-subunit: DATTGDFTAL (SEQ ID NO. 6). These sequences are identical to the ORF identified in the two NADP-GDH cDNAs and indicate the positions of the internal cleavage sites utilized to remove the chloroplast targeting peptide sequences. The chloroplast targeting peptide sequences (or chloroplast-transit peptides) can be useful for cell compartment localization with these and other amino acid sequences. The polynucleotides encoding the chloroplast-transit peptides can be used with other polynucleotide sequences to encode chloroplast-transit peptides. [0085]
cDNA isolation and sequencing. A pellet of [0086] C. sorokiniana cells stored at −70° C. was resuspended 1 to 10 (w/v) in RNA breakage buffer: 0.1M Tris (pH8.5), 0.4M LiCl, 10 mM EGTA, 5 mM EDTA, 100 units/mL sodium heparin (Sigma, 100 units/mg), and 1 mM aurintricarboxylic acid (Sigma). The cell suspension was centrifuged at 7000 g for 5 minutes at 4° C. and the supernatant was discarded. The cell pellet was resuspended 1 to 10 (w/v) in RNA breakage buffer and ruptured by passage through a French pressure cell at 20,000 p.s.i. The cell homogenate was collected in a disposable 50 mL conical tube containing 0.05 times volume 20% (w/v) SDS, 0.05 times volume 0.5M EDTA (pH 8), 200 pg/mL proteinase K, and allowed to incubate at room temperature for 15 minutes. One-half volume of TE buffer (Tris 10 mM:EDTA 1 mM, pH 8.0) equilibrated phenol was added to the homogenate and after a 3 minutes incubation a one-half volume of chloroform:isoamylalcohol(24:1, v/v) was added and mixed for 10 minutes on a wrist action shaker. The extracted homogenate was transferred to a 30 mL siliconized corex tube and centrifuged at 1000 g for 10 minutes at 4° C. The upper aqueous phase was removed and repeatedly extracted with an equal volume of chloroform: isoamyl-alcohol (24:1, v/v), as described above, until the aqueous interface was clear. After the final extraction, the aqueous phase was combined with an equal volume of 2×LiCl-Urea buffer (4M LiCl, 4M urea, 2 mM EDTA, 1 mM aurintricarboxylic acid; Sigma) and the RNA was precipitated on ice for 16 hours at 4° C. The RNA precipitate was centrifuged at 4000 g for 20 minutes at 4° C. and the resulting pellet was rinsed once with 1×LiCl-Urea buffer and centrifuged again to pellet the RNA. The RNA pellet was solubilized in TE (pH 7.5) and an aliquot was quantified spectrophotometrically at 260 nm. After quantitation, the mRNA fraction was isolated from total cellular RNA using an oligo(dT) spin column kit. Poly(A)⁺ RNA (50 μg) from each preparation was combined and utilized for the commercial production of a custom λUni-ZAP XR C. sorokiniana cDNA library (Stratagene Cloning Systems, Palo Alto, Calif.).
The amplified λZAP library, containing 2×10[0087] ¹⁰pfu/mL, was plated on twenty 150 mm petri plates at 50,000 pfu per plate for a total of 1×10⁶pfu screened. The phage plaques were absorbed to duplicate Hybond-N 132 mm circular membranes and treated according to the plaque blotting protocol of Amersham (1985, Amersham International plc. Arlington Heights, Ill.). Membranes were prehybridized in a common container in 200 mL of 2×PIPES (0.8M NaCl, 20 mM PIPES, pH 6.5), 50% (w/v) formamide, 0.5% (w/v) SDS, 100 μg/mL denatured sheared salmon sperm DNA at 40° C. Blocked membranes were hybridized at 42° C. in ten heat-sealable bags (four membranes/bag) in prehybridization buffer containing 1×10⁶cpm/membrane of a ³²P-labeled NADP-GDH 242 bp HCR cDNA probe on a lab rocker. The membranes were washed three times in 200 mL of 0.1×SSC, 0.1% (w/v) SDS for 20 minutes per wash at 50° C. Duplicate membranes were wrapped in plastic wrap and exposed to Kodak X-Omat AR film at −70 ° C. for 28 hours. Putative NADP-GDH cDNA plaques, detected on duplicate membranes, were cored from the plate and plaque purified by secondary and tertiary screenings with the 242 bp conserved region probe. Putative NADP-GDH cDNA phage clones, selected in the primary screening, were combined and screened a second time with a ³²P-labeled 130 bp Eco RI/Bgl II cDNA fragment isolated from the 5′ terminus of the most complete 5′ end NADP-GDH cDNA clone. Ten plaque pure NADP-GDH clones were subcloned in pBluescript KS⁻ (Stratagene) and transformed into E. coli DH5αF′ (Bethesda Research Laboratories, BRL) via an in vivo excision protocol provided by Stratagene. All plasmid isolations were performed as described by Kraft et al. (Kraft, R., J. Tardiff, K. S. Krauter, L. A. Leinwand [1988] Biotechniques 6:544-547). Sequence analysis revealed all ten clones were identical at their 3′-termini and differed by varying degrees of truncation at their 5′-termini. The longest cDNA clone with a complete 3′-terminus designated pBGDc53 (SEQ ID NO. 7) was not long enough to encode either subunit; therefore, the 5′-terminal sequences were determined by RACE PCR.
The 5′-terminal NADP-GDH cDNA sequences were cloned using a modified anchored PCR procedure for the rapid amplification of cDNA ends (Frohman, M. A. [1990] In D. H. Gelford, J. J. Snincky, T. J. White, eds, [0088] PCR Protocols, Academic Press, San Diego, Calif., pp 28-38; Jain, R., R. H. Gorner, J. J. Murtagh [1992] Biotechniques 12:58-59). A mixture of poly(A)⁺ RNA, used in the synthesis of the λZAP library, was utilized to clone the 5′ end of the NADP-GDH mRNA. One hundred nanograms of the mRNA mixture were combined with 10 ng of a gene-specific primer (5′-CTCAAAGGCAAGGAACTTCATG-3′. SEQ ID NO.8), designed to hybridize to the conserved region of NADP-GDH mRNAs, heated for 5 minutes, and chilled on ice. First strand DNA synthesis was performed using Superscript reverse transcriptase (BRL) according to the supplier's protocol. The terminated reverse transcription reaction was treated with one unit of ribonuclease H for 20 minutes at 37° C., 5 minutes at 95° C., and extracted once with chloroform:isoamyl alcohol (24:1, v/v). Excess primers and dNTPs were removed by centrifugation at 2000 rpm through an Ultrafree-MC filterfuge tube (30,000 MW cutoff, Millipore) and the retentate was concentrated to 10 μl on a Savant Speedvac. The first-strand synthesis products were combined with 10 μL of tailing mix (1×tailing buffer [Promega Corp.], 0.4 mM dATP, 10 units terminal deoxytransferase)and incubated at 37° C. for 10 minutes. The reaction mixture was heated to 95° C. for 5 minutes, diluted to 0.5 mL with TE (pH 8), and utilized as a cDNA pool. A mixture of 5 μL of the cDNA pool, 5 μL of Vent™ polymerase 10×buffer (New England Biolabs), 200 μM of each dNTP, 25 pmol of a gene specific primer (SEQ ID NO. 8), 5 pmol of the poly(dT) adaptor primer (5′-GGGTCGACATTCTAGACAGAATTCGTGGATCC(T)₁₈-3′; SEQ ID NO. 9), 0.2 units Perfectmatch™ DNA polymerase enhancer (Stratagene), and 1 unit of Vent™ polymerase (NEB) in 50 μL was amplified according to Jain et al., supra. The PCR products were purified away from the excess primers by centrifugation at 2,000 rpm through an Ultrafree-MC unit. The retentate was collected and subjected to two more rounds of amplification using a new nested gene specific primer at each step (5′-GGACGAGTACTGCACGC-3′, SEQ ID NO. 10; 5′-GATCTCGGTCAGCAGCTG-3′, SEQ ID NO. 11, respectively) and an adaptor primer (5′-GGGTCGACATTCTAGACAGAA-3′; SEQ ID NO. 12). PCR amplifications were performed in a Model 480 thermocycler (Perkin-Elmer Cetus), and all custom oligonucleotides were synthesized by the ICBR DNA synthesis facility, University of Florida. The standard PCR reaction mixture consisted of 10 μL of 10×Vent™ polymerase buffer, 100 μM of each dNTP, 0.4 units of Perfectmatch™, 50 pmol of each primer, 1 unit Vent™ DNA polymerase in a 100 μl reaction volume. The 5′ RACE-PCR products were gel purified, subcloned into the SmaI site of pUC 18. and transformed into E. coli DH5α for further characterization. RACE PCR identified two 5′ cDNA clones, which overlapped with the previously identified pBGDc 53 clone, that differed by a 42 nt insert identified in one clone designated pRGDc 60 (SEQ ID NO. 13) and lacking in the second cDNA designated pRGDc 61 (SEQ ID NO. 14).
Two additional cDNA clones lacking the RACE PCR polylinker, but possessing the complete 5′-termini corresponding to [0089] pRGDc 60 and 61 were constructed by RT-PCR amplification from mRNA using reaction conditions as described above and the gene specific primer pair (5′-CTTTCTGCTCGCCCTCTC-3′, SEQ ID NO.15, and SEQ ID NO. 11, above). The two PCR products were cloned into the SmaI site of pBluescript SK+ (Stratagene) and transformed into E. coli DH5α for further characterization. The cDNA clone that possessed the 42 nt insert was designated pGDc 63 (SEQ ID NO. 16) whereas the cDNA lacking the insert was designated pGDc 64 (SEQ ID NO. 17).
Full-length NADP-GDH cDNAs were constructed by restriction endonuclease treating pGDc 63 and 64 with EcoRi/ApaLI and gel purifying the resultant (264 bp; 222 bp, respectively) fragments. The gel purified fragments were ligated to a purified ApaLI/XhoI restriction fragment of pBGDc 53 and the full length ligation products (SEQ ID NO. 18; SEQ ID NO. 19) were gel agarose gel purified and utilized in subsequent PCR reactions. [0090]
Expression of α- and β-homohexamers in [0091] E. coli. Using the gel purified product (SEQ ID NO. 18), PCR mutagenesis was performed to remove the chloroplast targeting signal from the full-length cDNA and yield cDNAs encoding specifically the mature α- and β-subunits. Two sets of primer pairs were designed to synthesize α- and β-GDH subunit genes.
The following primer was designed to add a methionine to the amino terminus of the processed mature α-NADP-GDH subunit (alanine-41) to allow translation initiation and to generate a 5′ VdeI site for subcloning purposes: 5 ′-CATATGGCCGTCTCGCTGGAGGAG-3′ (SEQ ID NO. 20). The following second primer was designed to hybridize to the 3′ terminus of the template DNA at a position 20 nt 3′ of the endogenous TAA termination codon: 5′-GTTGGATTGCCGGTGAGCC-3′ (SEQ ID NO. 21). [0092]
The following primer was designed to add a methionine to the amino terminus of the processed mature β-subunit (aspartate-38) to allow translation initiation and to generate a 5′ NdeI site for subcloning purposes: 5′-CATATGGACGCCACCACCGGC-3′ (SEQ ID NO. 22). The second 3′ primer used in the PCR amplification was the 3 ′-terminus primer (SEQ ID NO. 21) described for the α-subunit amplification. [0093]
PCR cycling conditions were as follows: 95° C., 50 seconds; 64° C. 1 minute; 72° C., 1 minute 35 seconds (30 cycles). Primer, dNTP, Vent polymerase, and other reaction component concentrations were as previously described. The 1506 bp α-NADP-GDH subunit gene (SEQ ID NO. 23) and 1473 bp β-GDH subunit gene (SEQ ID NO. 25) PCR products were gel purified and given a 3′ adenine nucleotide overhang by incubating the purified fragment with 100 μM dATP and Taq polymerase for 15 minutes at 72° C. The modified PCR products were cloned into the PCRII T/A cloning vector (Invitrogen) and transformed into competent [0094] E. coli cells. Clones bearing the inserts were selected by blue-white screening, plasmid purified, and digested with NdeI/BamHI to select for the proper orientation in the cloning vector. The selected plasmids were restricted with NdeI and BamHI (BamHI site provided by vector) and directionally cloned under the control of the IPTG inducible T7 polymerase promoter of pET 11a and pET 15b bacterial expression vectors (Novagen) linearized with NdeI/BamHI, and transformed into DH5α. Transformants were screened by NdeI/BamHI restriction analysis and clones possessing the properly oriented α- and β-subunit cDNAs (SEQ ID NO. 23; SEQ ID NO. 25) were selected, plasmid purified, and transformed into E. coli BL21(DE3) for protein expression purposes.
[0095] E. coli BL2 I (DE3) cells transformed with pET 11a-α-cDNA and pET 11a-β-cDNA constructs were induced with 100 mM IPTG for 1 hour. Protein extracts from the induced cells were tested by enzyme analysis for NADP-GDH activity, and the denatured proteins were resolved by SDS gel electrophoresis, and visualized by coomassie staining. The proteins expressed by the mature α-subunit cDNA (SEQ ID NO. 23) and the β-subunit cDNA (SEQ ID NO. 25) have the amino acid sequences shown in SEQ ID NO. 24 (α-subunit) and SEQ ID NO. 26 (β-subunit). The recombinant GDH subunits were verified by cross reactivity with rabbit anti-Chlorella NADP-GDH antibodies.
Under conditions not optimized for maximal induction, the [0096] E. coli cells, possessing the α- and β-GDH cDNAs and induced with IPTG, showed 60- and 7,000-fold increases in NADP-GDH activity relative to uninduced controls, respectively. The recombinant α- and β-NADP-GDHs are currently being analyzed to verify kinetic and biochemical properties.
The over-expression and assembly of the [0097] C. sorokiniana chloroplastic GDHs into active enzymes provides proof that the DNA constructs engineered via PCR are transcribed and translated into authentic proteins. The aforementioned constructs were then utilized for cytosolic expression of the algal GDHs in transgenic plants.
Transformation of plants. A method for producing genetically transformed plants that express increased levels of a specific GDH requires the introduction of a double-stranded recombinant DNA molecule into the nuclear genome of a plant cell. The DNA molecule must (1) contain a structural DNA for the GDH enzyme being introduced into the plant cell; (2) possess a promoter which functions in plants to regulate the production of an RNA sequence in a constitutive or tissue-specific manner by RNA polymerase enzyme; and (3) have a 3 ′-untranslated region which functions to cause transcriptional termination and the addition of polyadenylated nucleotides to the 3′ end of the RNA. The resulting primary RNA molecule is subsequently processed in the nucleus, a process which involves the removal of intronic sequences and the addition of polyadenylate nucleotides to the 3′ end of the mRNA. [0098]
Promoters which are useful in the present invention are those that can initiate transcription in a constitutive manner or in a tissue-specific manner where glutamate production or catabolism is desired. An example of a useful constitutive promoter is the CaMV enhanced 35S promoter that directs the synthesis of RNA in a tissue independent manner. Promoters which cause production of GDH specifically in seeds, stems, roots, leaves, or specific cell types in these tissues are useful in the present invention. For example, the seed-specific Phaseolin promoter is one such tissue-specific promoter. Thus native promoters for maize, wheat, barley, and rice may be obtained and used in the present invention as well as heterologous promoters from other organisms shown to function in a constitutive/tissue-specific manner. [0099]
Introns. Generally, optimal expression in monocotyledonousplants is obtained when an intron sequence is inserted between the promoter sequence and the structural gene sequence. An example of such an intron sequence is the HSP 70 intron described in WO 93/19189. [0100]
Polyadenylation signal. The DNA constructs of the present invention can possess a 3′ untranslated region which functions in plants to direct the addition of polyadenylate nucleotides to the 3′ end of the RNA. An example of a suitable 3′ untranslated region is the polyadenylation signal of the Agrobacterium tumor inducing plasmid, i.e., nopaline synthatase (NOS) gene. [0101]
Plastid targeting sequence. The DNA constructs of the present invention can optionally contain a plastid targeting sequence. The plastid targeting sequence directs the import of the protein into the plastid, and is removed during importation. The plastid targeting sequence can be, but is not limited to, the native chloroplast targeting peptide (CTP) identified in the [0102] C. sorokiniana NADP-GDH full-length cDNAs which encode the precursor proteins. A fusion of a selected plastid targeting sequence and the mature α- and β-NADP-GDH subunit sequences can be made by standard procedures and used in the present invention. GDH subunits lacking these targeting sequences are typically found in the cytoplasm of the cell. Such a cytosolic localized enzyme can be useful in capturing ammonium or glutamate compartmentalized in the cytosol of the cell.
GDH gene sources. The GDH gene used in the DNA constructs of the present invention can be any GDH gene. It is not limited to the [0103] C. sorokiniana GDH genes described above, although they are preferred. For example a GDH gene from bacteria or fungi can be used. The examples provided use the α- and β-GDH genes of C. sorokiniana, but should not be interpreted in any way to limit the scope of the present invention. Individuals skilled in the art will recognize that various other genes as well as alterations can be made to genes and methods described herein while not departing from the spirit and scope of the present invention. For example, mutagenesis and routine screening can be implemented by techniques well known in the art to produce mutant variants that lack regulation by the cofactor NADPH.
Transient expression in maize protoplasts. In order to test the expression of the [0104] C. sorokiniana GDH subunits and their assembly into active enzymes in Zea mays cells, vectors were constructed to contain the CaMV E35S promoter, the coding sequence for the mature α-subunit (pMON21904) or β- subunit (pMON21905), the NOS 3′-untranslated polyadenylation region, and kanamycin resistance for selection in E. coli. The α- and β-subunit genes were isolated as a XbaI-EcoRI fragment from pET 11a-α-cDNA and pET 11a-β-cDNA, respectively. The GDH genes were ligated into the XbaI-EcoRI E35S promoter, NOS 3′, kanamycin resistance bearing region of pMON22072 to give pMON21904, and pMON21905. The DNA constructs were electroporated into maize and wheat protoplast according to the method of Sheen et al. (The Plant Cell Vol. 3, 225-245).
Analysis of transformed maize protoplasts. Pelleted protoplast samples transformed with pMON21904 (α-subunit), pMON21905 (β-subunit), pMON21709 (kanamycin negative control DNA), and no DNA were thawed in 0.2 mL of GDH cell breakage buffer (Yeung et al. supra) on ice. The cells in each suspension were homogenized twice for 30 seconds, chilled on ice, and clarified at 14,000 rpm for 10 minutes. Cell extracts were assayed in the deaminating direction at 38.5° C. according to Yeung et al., supra. Total protein content of the cell extracts was determined using the BioRad microprotein assay according to the manufacturer's protocol. Activities were normalized against total protein content for comparisons among different preparations. One unit of GDH activity is defined as the amount of enzyme necessary to reduce 1 μmol of NADP per minute at 38.5° C. [0105]
Protoplasts transformed with the control vector pMON2 1709 (n=3) or protoplasts not transformed (n=3) had no detectable NADP-GDH activity. Protoplasts transformed with pMON21904 (n=3) expressed 3.31 Units mg[0106] ⁻¹protein of GDH activity, whereas pMON21905 transformed protoplasts (n=3) 1.96 Units mg⁻¹protein.
The high level of activity observed for the protoplasts transformed with the cytoplasmic expressed [0107] C. sorokiniana α- and β-NADP-GDH genes provides evidence that the GDH subunits are expressed in heterologous plant systems. Additionally, expression levels demonstrate that the subunits are assembled into active enzymes. Generally, it would be readily apparent to persons of ordinary skill in the art that superfluous sequences added to the described sequences, or fragments of the nucleotide or amino acid sequences described herein, which result in polynucleotides or amino acid sequences that function similarly or equivalently to the sequences expressly described herein, should also be considered part of this invention. They can easily and routinely be produced by techniques well known in the art, for example, by time-controlled Bal31 exonuclease digestion of the full-length DNA, followed by expression of the resulting fragments and routine screening of the expression products as described in the foregoing example. In addition, it would be readily accepted by ordinarily skilled artisans that the function, property, or utility of the described sequences can be negatived by inserting mutations into the sequences by standard techniques and procedures. These mutations which, by implication, effectively serve to remove the property or function inherent in the sequences as described are hereby expressly included as part of the invention. For example, a clear distinction between the α- and β-subunits of the C. sorokiniana is the 11-amino acid polypeptide sequence at the N-terminus of the α-subunit, but absent in the β-subunit. This sequence can affect the affinity, specificity, and modulation of ammonium compounds by the enzyme. Therefore, it would be apparent that inserting (if absent) or removing (if present) the appropriate sequence, or its functional equivalent, to effect a difference in certain characteristics of other GDH genes, or their products, would be easily carried out by those persons.
It should also be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. [0108]
1 26 2140 base pairs nucleic acid double linear cDNA CDS 33..1610 1 CTCCTTTCTG CTCGCCCTCT CTCCGTCCCG CC ATG CAG ACC GCC CTC GTC GCC 53 Met Gln Thr Ala Leu Val Ala 1 5 AAG CCT ATC GTG GCC GCC CCG CTG GCG GCA CGC CCG CGC TGC CTC GCG 101 Lys Pro Ile Val Ala Ala Pro Leu Ala Ala Arg Pro Arg Cys Leu Ala 10 15 20 CCG TGG CCG TGC GCG TGG GTC CGC TCC GCC AAG CGC GAT GTC CGC GCC 149 Pro Trp Pro Cys Ala Trp Val Arg Ser Ala Lys Arg Asp Val Arg Ala 25 30 35 AAG GCC GTC TCG CTG GAG GAG CAG ATC TCC GCG ATG GAC GCC ACC ACC 197 Lys Ala Val Ser Leu Glu Glu Gln Ile Ser Ala Met Asp Ala Thr Thr 40 45 50 55 GGC GAC TTC ACG GCG CTG CAG AAG GCG GTG AAG CAG ATG GCC ACC AAG 245 Gly Asp Phe Thr Ala Leu Gln Lys Ala Val Lys Gln Met Ala Thr Lys 60 65 70 GCG GGC ACT GAG GGC CTG GTG CAC GGC ATC AAG AAC CCC GAC GTG CGC 293 Ala Gly Thr Glu Gly Leu Val His Gly Ile Lys Asn Pro Asp Val Arg 75 80 85 CAG CTG CTG ACC GAG ATC TTC ATG AAG GAC CCG GAG CAG CAG GAG TTC 341 Gln Leu Leu Thr Glu Ile Phe Met Lys Asp Pro Glu Gln Gln Glu Phe 90 95 100 ATG CAG GCG GTG CGC GAG GTG GCC GTC TCC CTG CAG CCC GTG TTC GAG 389 Met Gln Ala Val Arg Glu Val Ala Val Ser Leu Gln Pro Val Phe Glu 105 110 115 AAG CGC CCC GAG CTG CTG CCC ATC TTC AAG CAG ATC GTT GAG CCT GAG 437 Lys Arg Pro Glu Leu Leu Pro Ile Phe Lys Gln Ile Val Glu Pro Glu 120 125 130 135 CGC GTG ATC ACC TTC CGC GTG TCC TGG CTG GAC GAC GCC GGC AAC CTG 485 Arg Val Ile Thr Phe Arg Val Ser Trp Leu Asp Asp Ala Gly Asn Leu 140 145 150 CAG GTC AAC CGC GGC TTC CGC GTG CAG TAC TCG TCC GCC ATC GGC CCC 533 Gln Val Asn Arg Gly Phe Arg Val Gln Tyr Ser Ser Ala Ile Gly Pro 155 160 165 TAC AAG GGC GGC CTG CGC TTC CAC CCC TCC GTG AAC CTG TCC ATC ATG 581 Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Leu Ser Ile Met 170 175 180 AAG TTC CTT GCC TTT GAG CAG ATC TTC AAG AAC AGC CTG ACC ACC CTG 629 Lys Phe Leu Ala Phe Glu Gln Ile Phe Lys Asn Ser Leu Thr Thr Leu 185 190 195 CCC ATG GGC GGC GGC AAG GGC GGC TCC GAC TTC GAC CCC AAG GGC AAG 677 Pro Met Gly Gly Gly Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys 200 205 210 215 AGC GAC GCG GAG GTG ATG CGC TTC TGC CAG TCC TTC ATG ACC GAG CTG 725 Ser Asp Ala Glu Val Met Arg Phe Cys Gln Ser Phe Met Thr Glu Leu 220 225 230 CAG CGC CAC ATC AGC TAC GTG CAG GAC GTG CCC GCC GGC GAC ATC GGC 773 Gln Arg His Ile Ser Tyr Val Gln Asp Val Pro Ala Gly Asp Ile Gly 235 240 245 GTG GGC GCG CGC GAG ATT GGC TAC CTT TTC GGC CAG TAC AAG CGC ATC 821 Val Gly Ala Arg Glu Ile Gly Tyr Leu Phe Gly Gln Tyr Lys Arg Ile 250 255 260 ACC AAG AAC TAC ACC GGC GTG CTG ACC CCG AAG GGC CAG GAG TAT GGC 869 Thr Lys Asn Tyr Thr Gly Val Leu Thr Pro Lys Gly Gln Glu Tyr Gly 265 270 275 GGC TCC GAG ATC CGC CCC GAG GCC ACC GGC TAC GGC GCC GTG CTG TTT 917 Gly Ser Glu Ile Arg Pro Glu Ala Thr Gly Tyr Gly Ala Val Leu Phe 280 285 290 295 GTG GAG AAC GTG CTG AAG GAC AAG GGC GAG AGC CTC AAG GGC AAG CGC 965 Val Glu Asn Val Leu Lys Asp Lys Gly Glu Ser Leu Lys Gly Lys Arg 300 305 310 TGC CTG GTG TCT GGC GCG GGC AAC GTG GCC CAG TAC TGC GCG GAG CTG 1013 Cys Leu Val Ser Gly Ala Gly Asn Val Ala Gln Tyr Cys Ala Glu Leu 315 320 325 CTG CTG GAG AAG GGC GCC ATC GTG CTG TCG CTG TCC GAC TCC CAG GGC 1061 Leu Leu Glu Lys Gly Ala Ile Val Leu Ser Leu Ser Asp Ser Gln Gly 330 335 340 TAC GTG TAC GAG CCC AAC GGC TTC ACG CGC GAG CAG CTG CAG GCG GTG 1109 Tyr Val Tyr Glu Pro Asn Gly Phe Thr Arg Glu Gln Leu Gln Ala Val 345 350 355 CAG GAC ATG AAG AAG AAG AAC AAC AGC GCC CGC ATC TCC GAG TAC AAG 1157 Gln Asp Met Lys Lys Lys Asn Asn Ser Ala Arg Ile Ser Glu Tyr Lys 360 365 370 375 AGC GAC ACC GCC GTG TAT GTG GGC GAC CGC CGC AAG CCT TGG GAG CTG 1205 Ser Asp Thr Ala Val Tyr Val Gly Asp Arg Arg Lys Pro Trp Glu Leu 380 385 390 GAC TGC CAG GTG GAC ATC GCC TTC CCC TGC GCC ACC CAG AAC GAG ATC 1253 Asp Cys Gln Val Asp Ile Ala Phe Pro Cys Ala Thr Gln Asn Glu Ile 395 400 405 GAT GAG CAC GAC GCC GAG CTG CTG ATC AAG CAC GGC TGC CAG TAC GTG 1301 Asp Glu His Asp Ala Glu Leu Leu Ile Lys His Gly Cys Gln Tyr Val 410 415 420 GTG GAG GGC GCC AAC ATG CCC TCC ACC AAC GAG GCC ATC CAC AAG TAC 1349 Val Glu Gly Ala Asn Met Pro Ser Thr Asn Glu Ala Ile His Lys Tyr 425 430 435 AAC AAG GCC GGC ATC ATC TAC TGC CCC GGC AAG GCG GCC AAC GCC GGC 1397 Asn Lys Ala Gly Ile Ile Tyr Cys Pro Gly Lys Ala Ala Asn Ala Gly 440 445 450 455 GGC GTG GCG GTC AGC GGC CTG GAG ATG ACC CAG AAC CGC ATG AGC CTG 1445 Gly Val Ala Val Ser Gly Leu Glu Met Thr Gln Asn Arg Met Ser Leu 460 465 470 AAC TGG ACT CGC GAG GAG GTT CGC GAC AAG CTG GAG CGC ATC ATG AAG 1493 Asn Trp Thr Arg Glu Glu Val Arg Asp Lys Leu Glu Arg Ile Met Lys 475 480 485 GAC ATC TAC GAC TCC GCC ATG GGG CCG TCC CGC AGA TAC AAT GTT GAC 1541 Asp Ile Tyr Asp Ser Ala Met Gly Pro Ser Arg Arg Tyr Asn Val Asp 490 495 500 CTG GCT GCG GGC GCC AAC ATC GCG GGC TTC ACC AAG GTG GCT GAT GCC 1589 Leu Ala Ala Gly Ala Asn Ile Ala Gly Phe Thr Lys Val Ala Asp Ala 505 510 515 GTC AAG GCC CAG GGC GCT GTT TAAGCTGCCC AGGCCCAAGC CACGGCTCAC 1640 Val Lys Ala Gln Gly Ala Val 520 525 CGGCAATCCA ACCCAACCAA CTCAACGGCC AGGACCTTTT CGGAAGCGGC GCCTTTTTCC 1700 CAGCCAGGGC CCTCACCTGC CCTTTCATAA CCCTGCTATT GCCGCCGTGC CCCTGCAATT 1760 CCACCCCAAG AAGAACTAGC GGCACTTGAC TGCATCAGGA CGGCTATTTT TTTCGCGACG 1820 CGCGCTCACC CCGAGAGCCT CTCTCCCCCG AGCCCTAAGC GCTGACGTCC GCCCGACTTT 1880 GCCTCGCACA TCGCTCGGTT TTGACCCCCT CCAGTCTACC CACCCTGTTG TGAAGCCTAC 1940 CAGCTCAATT GCCTTTTAGT GTATGTGCGC CCCCTCCTGC CCCCGAATTT TCCTGCCATG 2000 AGACGTGCGG TTCCTAGCCT GGTGACCCCA AGTAGCAGTT AGTGTGCGTG CCTTGCCCTG 2060 CGCTGCCCGG GATGCGATAC TGTGACCTGA GAGTGCTTGT GTAAACACGA CGAGTCAAAA 2120 AAAAAAAAAA AAAAAAAAAA 2140 526 amino acids amino acid linear protein 2 Met Gln Thr Ala Leu Val Ala Lys Pro Ile Val Ala Ala Pro Leu Ala 1 5 10 15 Ala Arg Pro Arg Cys Leu Ala Pro Trp Pro Cys Ala Trp Val Arg Ser 20 25 30 Ala Lys Arg Asp Val Arg Ala Lys Ala Val Ser Leu Glu Glu Gln Ile 35 40 45 Ser Ala Met Asp Ala Thr Thr Gly Asp Phe Thr Ala Leu Gln Lys Ala 50 55 60 Val Lys Gln Met Ala Thr Lys Ala Gly Thr Glu Gly Leu Val His Gly 65 70 75 80 Ile Lys Asn Pro Asp Val Arg Gln Leu Leu Thr Glu Ile Phe Met Lys 85 90 95 Asp Pro Glu Gln Gln Glu Phe Met Gln Ala Val Arg Glu Val Ala Val 100 105 110 Ser Leu Gln Pro Val Phe Glu Lys Arg Pro Glu Leu Leu Pro Ile Phe 115 120 125 Lys Gln Ile Val Glu Pro Glu Arg Val Ile Thr Phe Arg Val Ser Trp 130 135 140 Leu Asp Asp Ala Gly Asn Leu Gln Val Asn Arg Gly Phe Arg Val Gln 145 150 155 160 Tyr Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro 165 170 175 Ser Val Asn Leu Ser Ile Met Lys Phe Leu Ala Phe Glu Gln Ile Phe 180 185 190 Lys Asn Ser Leu Thr Thr Leu Pro Met Gly Gly Gly Lys Gly Gly Ser 195 200 205 Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe Cys 210 215 220 Gln Ser Phe Met Thr Glu Leu Gln Arg His Ile Ser Tyr Val Gln Asp 225 230 235 240 Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Tyr Leu 245 250 255 Phe Gly Gln Tyr Lys Arg Ile Thr Lys Asn Tyr Thr Gly Val Leu Thr 260 265 270 Pro Lys Gly Gln Glu Tyr Gly Gly Ser Glu Ile Arg Pro Glu Ala Thr 275 280 285 Gly Tyr Gly Ala Val Leu Phe Val Glu Asn Val Leu Lys Asp Lys Gly 290 295 300 Glu Ser Leu Lys Gly Lys Arg Cys Leu Val Ser Gly Ala Gly Asn Val 305 310 315 320 Ala Gln Tyr Cys Ala Glu Leu Leu Leu Glu Lys Gly Ala Ile Val Leu 325 330 335 Ser Leu Ser Asp Ser Gln Gly Tyr Val Tyr Glu Pro Asn Gly Phe Thr 340 345 350 Arg Glu Gln Leu Gln Ala Val Gln Asp Met Lys Lys Lys Asn Asn Ser 355 360 365 Ala Arg Ile Ser Glu Tyr Lys Ser Asp Thr Ala Val Tyr Val Gly Asp 370 375 380 Arg Arg Lys Pro Trp Glu Leu Asp Cys Gln Val Asp Ile Ala Phe Pro 385 390 395 400 Cys Ala Thr Gln Asn Glu Ile Asp Glu His Asp Ala Glu Leu Leu Ile 405 410 415 Lys His Gly Cys Gln Tyr Val Val Glu Gly Ala Asn Met Pro Ser Thr 420 425 430 Asn Glu Ala Ile His Lys Tyr Asn Lys Ala Gly Ile Ile Tyr Cys Pro 435 440 445 Gly Lys Ala Ala Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met 450 455 460 Thr Gln Asn Arg Met Ser Leu Asn Trp Thr Arg Glu Glu Val Arg Asp 465 470 475 480 Lys Leu Glu Arg Ile Met Lys Asp Ile Tyr Asp Ser Ala Met Gly Pro 485 490 495 Ser Arg Arg Tyr Asn Val Asp Leu Ala Ala Gly Ala Asn Ile Ala Gly 500 505 510 Phe Thr Lys Val Ala Asp Ala Val Lys Ala Gln Gly Ala Val 515 520 525 2099 base pairs nucleic acid double linear cDNA CDS 33..1568 3 CTCCTTTCTG CTCGCCCTCT CTCCGTCCCG CC ATG CAG ACC GCC CTC GTC GCC 53 Met Gln Thr Ala Leu Val Ala 1 5 AAG CCT ATC GTG GCC TGC GCG TGG GTC CGC TCC GCC AAG CGC GAT GTC 101 Lys Pro Ile Val Ala Cys Ala Trp Val Arg Ser Ala Lys Arg Asp Val 10 15 20 CGC GCC AAG GCC GTC TCG CTG GAG GAG CAG ATC TCC GCG ATG GAC GCC 149 Arg Ala Lys Ala Val Ser Leu Glu Glu Gln Ile Ser Ala Met Asp Ala 25 30 35 ACC ACC GGC GAC TTC ACG GCG CTG CAG AAG GCG GTG AAG CAG ATG GCC 197 Thr Thr Gly Asp Phe Thr Ala Leu Gln Lys Ala Val Lys Gln Met Ala 40 45 50 55 ACC AAG GCG GGC ACT GAG GGC CTG GTG CAC GGC ATC AAG AAC CCC GAC 245 Thr Lys Ala Gly Thr Glu Gly Leu Val His Gly Ile Lys Asn Pro Asp 60 65 70 GTG CGC CAG CTG CTG ACC GAG ATC TTC ATG AAG GAC CCG GAG CAG CAG 293 Val Arg Gln Leu Leu Thr Glu Ile Phe Met Lys Asp Pro Glu Gln Gln 75 80 85 GAG TTC ATG CAG GCG GTG CGC GAG GTG GCC GTC TCC CTG CAG CCC GTG 341 Glu Phe Met Gln Ala Val Arg Glu Val Ala Val Ser Leu Gln Pro Val 90 95 100 TTC GAG AAG CGC CCC GAG CTG CTG CCC ATC TTC AAG CAG ATC GTT GAG 389 Phe Glu Lys Arg Pro Glu Leu Leu Pro Ile Phe Lys Gln Ile Val Glu 105 110 115 CCT GAG CGC GTG ATC ACC TTC CGC GTG TCC TGG CTG GAC GAC GCC GGC 437 Pro Glu Arg Val Ile Thr Phe Arg Val Ser Trp Leu Asp Asp Ala Gly 120 125 130 135 AAC CTG CAG GTC AAC CGC GGC TTC CGC GTG CAG TAC TCG TCC GCC ATC 485 Asn Leu Gln Val Asn Arg Gly Phe Arg Val Gln Tyr Ser Ser Ala Ile 140 145 150 GGC CCC TAC AAG GGC GGC CTG CGC TTC CAC CCC TCC GTG AAC CTG TCC 533 Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Leu Ser 155 160 165 ATC ATG AAG TTC CTT GCC TTT GAG CAG ATC TTC AAG AAC AGC CTG ACC 581 Ile Met Lys Phe Leu Ala Phe Glu Gln Ile Phe Lys Asn Ser Leu Thr 170 175 180 ACC CTG CCC ATG GGC GGC GGC AAG GGC GGC TCC GAC TTC GAC CCC AAG 629 Thr Leu Pro Met Gly Gly Gly Lys Gly Gly Ser Asp Phe Asp Pro Lys 185 190 195 GGC AAG AGC GAC GCG GAG GTG ATG CGC TTC TGC CAG TCC TTC ATG ACC 677 Gly Lys Ser Asp Ala Glu Val Met Arg Phe Cys Gln Ser Phe Met Thr 200 205 210 215 GAG CTG CAG CGC CAC ATC AGC TAC GTG CAG GAC GTG CCC GCC GGC GAC 725 Glu Leu Gln Arg His Ile Ser Tyr Val Gln Asp Val Pro Ala Gly Asp 220 225 230 ATC GGC GTG GGC GCG CGC GAG ATT GGC TAC CTT TTC GGC CAG TAC AAG 773 Ile Gly Val Gly Ala Arg Glu Ile Gly Tyr Leu Phe Gly Gln Tyr Lys 235 240 245 CGC ATC ACC AAG AAC TAC ACC GGC GTG CTG ACC CCG AAG GGC CAG GAG 821 Arg Ile Thr Lys Asn Tyr Thr Gly Val Leu Thr Pro Lys Gly Gln Glu 250 255 260 TAT GGC GGC TCC GAG ATC CGC CCC GAG GCC ACC GGC TAC GGC GCC GTG 869 Tyr Gly Gly Ser Glu Ile Arg Pro Glu Ala Thr Gly Tyr Gly Ala Val 265 270 275 CTG TTT GTG GAG AAC GTG CTG AAG GAC AAG GGC GAG AGC CTC AAG GGC 917 Leu Phe Val Glu Asn Val Leu Lys Asp Lys Gly Glu Ser Leu Lys Gly 280 285 290 295 AAG CGC TGC CTG GTG TCT GGC GCG GGC AAC GTG GCC CAG TAC TGC GCG 965 Lys Arg Cys Leu Val Ser Gly Ala Gly Asn Val Ala Gln Tyr Cys Ala 300 305 310 GAG CTG CTG CTG GAG AAG GGC GCC ATC GTG CTG TCG CTG TCC GAC TCC 1013 Glu Leu Leu Leu Glu Lys Gly Ala Ile Val Leu Ser Leu Ser Asp Ser 315 320 325 CAG GGC TAC GTG TAC GAG CCC AAC GGC TTC ACG CGC GAG CAG CTG CAG 1061 Gln Gly Tyr Val Tyr Glu Pro Asn Gly Phe Thr Arg Glu Gln Leu Gln 330 335 340 GCG GTG CAG GAC ATG AAG AAG AAG AAC AAC AGC GCC CGC ATC TCC GAG 1109 Ala Val Gln Asp Met Lys Lys Lys Asn Asn Ser Ala Arg Ile Ser Glu 345 350 355 TAC AAG AGC GAC ACC GCC GTG TAT GTG GGC GAC CGC CGC AAG CCT TGG 1157 Tyr Lys Ser Asp Thr Ala Val Tyr Val Gly Asp Arg Arg Lys Pro Trp 360 365 370 375 GAG CTG GAC TGC CAG GTG GAC ATC GCC TTC CCC TGC GCC ACC CAG AAC 1205 Glu Leu Asp Cys Gln Val Asp Ile Ala Phe Pro Cys Ala Thr Gln Asn 380 385 390 GAG ATC GAT GAG CAC GAC GCC GAG CTG CTG ATC AAG CAC GGC TGC CAG 1253 Glu Ile Asp Glu His Asp Ala Glu Leu Leu Ile Lys His Gly Cys Gln 395 400 405 TAC GTG GTG GAG GGC GCC AAC ATG CCC TCC ACC AAC GAG GCC ATC CAC 1301 Tyr Val Val Glu Gly Ala Asn Met Pro Ser Thr Asn Glu Ala Ile His 410 415 420 AAG TAC AAC AAG GCC GGC ATC ATC TAC TGC CCC GGC AAG GCG GCC AAC 1349 Lys Tyr Asn Lys Ala Gly Ile Ile Tyr Cys Pro Gly Lys Ala Ala Asn 425 430 435 GCC GGC GGC GTG GCG GTC AGC GGC CTG GAG ATG ACC CAG AAC CGC ATG 1397 Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Thr Gln Asn Arg Met 440 445 450 455 AGC CTG AAC TGG ACT CGC GAG GAG GTT CGC GAC AAG CTG GAG CGC ATC 1445 Ser Leu Asn Trp Thr Arg Glu Glu Val Arg Asp Lys Leu Glu Arg Ile 460 465 470 ATG AAG GAC ATC TAC GAC TCC GCC ATG GGG CCG TCC CGC AGA TAC AAT 1493 Met Lys Asp Ile Tyr Asp Ser Ala Met Gly Pro Ser Arg Arg Tyr Asn 475 480 485 GTT GAC CTG GCT GCG GGC GCC AAC ATC GCG GGC TTC ACC AAG GTG GCT 1541 Val Asp Leu Ala Ala Gly Ala Asn Ile Ala Gly Phe Thr Lys Val Ala 490 495 500 GAT GCC GTC AAG GCC CAG GGC GCT GTT TAAGCTGCCC AGGCCCAAGC 1588 Asp Ala Val Lys Ala Gln Gly Ala Val 505 510 CACGGCTCAC CGGCAATCCA ACCCAACCAA CTCAACGGCC AGGACCTTTT CGGAAGCGGC 1648 GCCTTTTTCC CAGCCAGGGC CCTCACCTGC CCTTTCATAA CCCTGCTATT GCCGCCGTGC 1708 CCCTGCAATT CCACCCCAAG AAGAACTAGC GGCACTTGAC TGCATCAGGA CGGCTATTTT 1768 TTTCGCGACG CGCGCTCACC CCGAGAGCCT CTCTCCCCCG AGCCCTAAGC GCTGACGTCC 1828 GCCCGACTTT GCCTCGCACA TCGCTCGGTT TTGACCCCCT CCAGTCTACC CACCCTGTTG 1888 TGAAGCCTAC CAGCTCAATT GCCTTTTAGT GTATGTGCGC CCCCTCCTGC CCCCGAATTT 1948 TCCTGCCATG AGACGTGCGG TTCCTAGCCT GGTGACCCCA AGTAGCAGTT AGTGTGCGTG 2008 CCTTGCCCTG CGCTGCCCGG GATGCGATAC TGTGACCTGA GAGTGCTTGT GTAAACACGA 2068 CGAGTCAAAA AAAAAAAAAA AAAAAAAAAA A 2099 512 amino acids amino acid linear protein 4 Met Gln Thr Ala Leu Val Ala Lys Pro Ile Val Ala Cys Ala Trp Val 1 5 10 15 Arg Ser Ala Lys Arg Asp Val Arg Ala Lys Ala Val Ser Leu Glu Glu 20 25 30 Gln Ile Ser Ala Met Asp Ala Thr Thr Gly Asp Phe Thr Ala Leu Gln 35 40 45 Lys Ala Val Lys Gln Met Ala Thr Lys Ala Gly Thr Glu Gly Leu Val 50 55 60 His Gly Ile Lys Asn Pro Asp Val Arg Gln Leu Leu Thr Glu Ile Phe 65 70 75 80 Met Lys Asp Pro Glu Gln Gln Glu Phe Met Gln Ala Val Arg Glu Val 85 90 95 Ala Val Ser Leu Gln Pro Val Phe Glu Lys Arg Pro Glu Leu Leu Pro 100 105 110 Ile Phe Lys Gln Ile Val Glu Pro Glu Arg Val Ile Thr Phe Arg Val 115 120 125 Ser Trp Leu Asp Asp Ala Gly Asn Leu Gln Val Asn Arg Gly Phe Arg 130 135 140 Val Gln Tyr Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe 145 150 155 160 His Pro Ser Val Asn Leu Ser Ile Met Lys Phe Leu Ala Phe Glu Gln 165 170 175 Ile Phe Lys Asn Ser Leu Thr Thr Leu Pro Met Gly Gly Gly Lys Gly 180 185 190 Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg 195 200 205 Phe Cys Gln Ser Phe Met Thr Glu Leu Gln Arg His Ile Ser Tyr Val 210 215 220 Gln Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly 225 230 235 240 Tyr Leu Phe Gly Gln Tyr Lys Arg Ile Thr Lys Asn Tyr Thr Gly Val 245 250 255 Leu Thr Pro Lys Gly Gln Glu Tyr Gly Gly Ser Glu Ile Arg Pro Glu 260 265 270 Ala Thr Gly Tyr Gly Ala Val Leu Phe Val Glu Asn Val Leu Lys Asp 275 280 285 Lys Gly Glu Ser Leu Lys Gly Lys Arg Cys Leu Val Ser Gly Ala Gly 290 295 300 Asn Val Ala Gln Tyr Cys Ala Glu Leu Leu Leu Glu Lys Gly Ala Ile 305 310 315 320 Val Leu Ser Leu Ser Asp Ser Gln Gly Tyr Val Tyr Glu Pro Asn Gly 325 330 335 Phe Thr Arg Glu Gln Leu Gln Ala Val Gln Asp Met Lys Lys Lys Asn 340 345 350 Asn Ser Ala Arg Ile Ser Glu Tyr Lys Ser Asp Thr Ala Val Tyr Val 355 360 365 Gly Asp Arg Arg Lys Pro Trp Glu Leu Asp Cys Gln Val Asp Ile Ala 370 375 380 Phe Pro Cys Ala Thr Gln Asn Glu Ile Asp Glu His Asp Ala Glu Leu 385 390 395 400 Leu Ile Lys His Gly Cys Gln Tyr Val Val Glu Gly Ala Asn Met Pro 405 410 415 Ser Thr Asn Glu Ala Ile His Lys Tyr Asn Lys Ala Gly Ile Ile Tyr 420 425 430 Cys Pro Gly Lys Ala Ala Asn Ala Gly Gly Val Ala Val Ser Gly Leu 435 440 445 Glu Met Thr Gln Asn Arg Met Ser Leu Asn Trp Thr Arg Glu Glu Val 450 455 460 Arg Asp Lys Leu Glu Arg Ile Met Lys Asp Ile Tyr Asp Ser Ala Met 465 470 475 480 Gly Pro Ser Arg Arg Tyr Asn Val Asp Leu Ala Ala Gly Ala Asn Ile 485 490 495 Ala Gly Phe Thr Lys Val Ala Asp Ala Val Lys Ala Gln Gly Ala Val 500 505 510 20 amino acids amino acid single linear peptide 5 Ala Val Ser Leu Glu Glu Gln Ile Ser Ala Met Asp Ala Thr Thr Gly 1 5 10 15 Asp Phe Thr Ala 20 10 amino acids amino acid single linear peptide 6 Asp Ala Thr Thr Gly Asp Phe Thr Ala Leu 1 5 10 1969 base pairs nucleic acid double linear cDNA 7 CAGATCTCCG CGATGGACGC CACCACCGGC GACTTCACGG CGCTGCAGAA GGCGGTGAAG 60 CAGATGGCCA CCAAGGCGGG CACTGAGGGC CTGGTGCACG GCATCAAGAA CCCCGACGTG 120 CGCCAGCTGC TGACCGAGAT CTTCATGAAG GACCCGGAGC AGCAGGAGTT CATGCAGGCG 180 GTGCGCGAGG TGGCCGTCTC CCTGCAGCCC GTGTTCGAGA AGCGCCCCGA GCTGCTGCCC 240 ATCTTCAAGC AGATCGTTGA GCCTGAGCGC GTGATCACCT TCCGCGTGTC CTGGCTGGAC 300 GACGCCGGCA ACCTGCAGGT CAACCGCGGC TTCCGCGTGC AGTACTCGTC CGCCATCGGC 360 CCCTACAAGG GCGGCCTGCG CTTCCACCCC TCCGTGAACC TGTCCATCAT GAAGTTCCTT 420 GCCTTTGAGC AGATCTTCAA GAACAGCCTG ACCACCCTGC CCATGGGCGG CGGCAAGGGC 480 GGCTCCGACT TCGACCCCAA GGGCAAGAGC GACGCGGAGG TGATGCGCTT CTGCCAGTCC 540 TTCATGACCG AGCTGCAGCG CCACATCAGC TACGTGCAGG ACGTGCCCGC CGGCGACATC 600 GGCGTGGGCG CGCGCGAGAT TGGCTACCTT TTCGGCCAGT ACAAGCGCAT CACCAAGAAC 660 TACACCGGCG TGCTGACCCC GAAGGGCCAG GAGTATGGCG GCTCCGAGAT CCGCCCCGAG 720 GCCACCGGCT ACGGCGCCGT GCTGTTTGTG GAGAACGTGC TGAAGGACAA GGGCGAGAGC 780 CTCAAGGGCA AGCGCTGCCT GGTGTCTGGC GCGGGCAACG TGGCCCAGTA CTGCGCGGAG 840 CTGCTGCTGG AGAAGGGCGC CATCGTGCTG TCGCTGTCCG ACTCCCAGGG CTACGTGTAC 900 GAGCCCAACG GCTTCACGCG CGAGCAGCTG CAGGCGGTGC AGGACATGAA GAAGAAGAAC 960 AACAGCGCCC GCATCTCCGA GTACAAGAGC GACACCGCCG TGTATGTGGG CGACCGCCGC 1020 AAGCCTTGGG AGCTGGACTG CCAGGTGGAC ATCGCCTTCC CCTGCGCCAC CCAGAACGAG 1080 ATCGATGAGC ACGACGCCGA GCTGCTGATC AAGCACGGCT GCCAGTACGT GGTGGAGGGC 1140 GCCAACATGC CCTCCACCAA CGAGGCCATC CACAAGTACA ACAAGGCCGG CATCATCTAC 1200 TGCCCCGGCA AGGCGGCCAA CGCCGGCGGC GTGGCGGTCA GCGGCCTGGA GATGACCCAG 1260 AACCGCATGA GCCTGAACTG GACTCGCGAG GAGGTTCGCG ACAAGCTGGA GCGCATCATG 1320 AAGGACATCT ACGACTCCGC CATGGGGCCG TCCCGCAGAT ACAATGTTGA CCTGGCTGCG 1380 GGCGCCAACA TCGCGGGCTT CACCAAGGTG GCTGATGCCG TCAAGGCCCA GGGCGCTGTT 1440 TAAGCTGCCC AGGCCCAAGC CACGGCTCAC CGGCAATCCA ACCCAACCAA CTCAACGGCC 1500 AGGACCTTTT CGGAAGCGGC GCCTTTTTCC CAGCCAGGGC CCTCACCTGC CCTTTCATAA 1560 CCCTGCTATT GCCGCCGTGC CCCTGCAATT CCACCCCAAG AAGAACTAGC GGCACTTGAC 1620 TGCATCAGGA CGGCTATTTT TTTCGCGACG CGCGCTCACC CCGAGAGCCT CTCTCCCCCG 1680 AGCCCTAAGC GCTGACGTCC GCCCGACTTT GCCTCGCACA TCGCTCGGTT TTGACCCCCT 1740 CCAGTCTACC CACCCTGTTG TGAAGCCTAC CAGCTCAATT GCCTTTTAGT GTATGTGCGC 1800 CCCCTCCTGC CCCCGAATTT TCCTGCCATG AGACGTGCGG TTCCTAGCCT GGTGACCCCA 1860 AGTAGCAGTT AGTGTGCGTG CCTTGCCCTG CGCTGCCCGG GATGCGATAC TGTGACCTGA 1920 GAGTGCTTGT GTAAACACGA CGAGTCAAAA AAAAAAAAAA AAAAAAAAA 1969 22 base pairs nucleic acid double linear cDNA 8 CTCAAAGGCA AGGAACTTCA TG 22 50 base pairs nucleic acid double linear cDNA 9 GGGTCGACAT TCTAGACAGA ATTCGTGGAT CCTTTTTTTT TTTTTTTTTT 50 17 base pairs nucleic acid double linear cDNA 10 GGACGAGTAC TGCACGC 17 18 base pairs nucleic acid double linear CDNA 11 GATCTCGGTC AGCAGCTG 18 21 base pairs nucleic acid double linear CDNA 12 GGGTCGACAT TCTAGACAGA A 21 367 base pairs nucleic acid double linear CDNA 13 GGGTCGACAT TCTAGACAGA ATTCGTGGAT CCTTTTTTTT TTTTTTTTTT TTTTTTCTCC 60 TTTCTGCTCG CCCTCTCTCC GTCCCGCCAT GCAGACCGCC CTCGTCGCCA AGCCTATCGT 120 GGCCGCCCCG CTGGCGGCAC GCCCGCGCTG CCTCGCGCCG TGGCCGTGCG CGTGGGTCCG 180 CTCCGCCAAG CGCGATGTCC GCGCCAAGGC CGTCTCGCTG GAGGAGCAGA TCTCCGCGAT 240 GGACGCCACC ACCGGCGACT TCACGGCGCT GCAGAAGGCG GTGAAGCAGA TGGCCACCAA 300 GGCGGGCACT GAGGGCCTGG TGCACGGCAT CAAGAACCCC GACGTGCGCC AGCTGCTGAC 360 CGAGATC 367 325 base pairs nucleic acid double linear CDNA 14 GGGTCGACAT TCTAGACAGA ATTCGTGGAT CCTTTTTTTT TTTTTTTTTT TTTTTTCTCC 60 TTTCTGCTCG CCCTCTCTCC GTCCCGCCAT GCAGACCGCC CTCGTCGCCA AGCCTATCGT 120 GGCCTGCGCG TGGGTCCGCT CCGCCAAGCG CGATGTCCGC GCCAAGGCCG TCTCGCTGGA 180 GGAGCAGATC TCCGCGATGG ACGCCACCAC CGGCGACTTC ACGGCGCTGC AGAAGGCGGT 240 GAAGCAGATG GCCACCAAGG CGGGCACTGA GGGCCTGGTG CACGGCATCA AGAACCCCGA 300 CGTGCGCCAG CTGCTGACCG AGATC 325 18 base pairs nucleic acid double linear CDNA 15 CTTTCTGCTC GCCCTCTC 18 308 base pairs nucleic acid double linear CDNA 16 CTTTCTGCTC GCCCTCTCTC CGTCCCGCCA TGCAGACCGC CCTCGTCGCC AAGCCTATCG 60 TGGCCGCCCC GCTGGCGGCA CGCCCGCGCT GCCTCGCGCC GTGGCCGTGC GCGTGGGTCC 120 GCTCCGCCAA GCGCGATGTC CGCGCCAAGG CCGTCTCGCT GGAGGAGCAG ATCTCCGCGA 180 TGGACGCCAC CACCGGCGAC TTCACGGCGC TGCAGAAGGC GGTGAAGCAG ATGGCCACCA 240 AGGCGGGCAC TGAGGGCCTG GTGCACGGCA TCAAGAACCC CGACGTGCGC CAGCTGCTGA 300 CCGAGATC 308 266 base pairs nucleic acid double linear CDNA 17 CTTTCTGCTC GCCCTCTCTC CGTCCCGCCA TGCAGACCGC CCTCGTCGCC AAGCCTATCG 60 TGGCCTGCGC GTGGGTCCGC TCCGCCAAGC GCGATGTCCG CGCCAAGGCC GTCTCGCTGG 120 AGGAGCAGAT CTCCGCGATG GACGCCACCA CCGGCGACTT CACGGCGCTG CAGAAGGCGG 180 TGAAGCAGAT GGCCACCAAG GCGGGCACTG AGGGCCTGGT GCACGGCATC AAGAACCCCG 240 ACGTGCGCCA GCTGCTGACC GAGATC 266 2137 base pairs nucleic acid double linear CDNA 18 CTTTCTGCTC GCCCTCTCTC CGTCCCGCCA TGCAGACCGC CCTCGTCGCC AAGCCTATCG 60 TGGCCGCCCC GCTGGCGGCA CGCCCGCGCT GCCTCGCGCC GTGGCCGTGC GCGTGGGTCC 120 GCTCCGCCAA GCGCGATGTC CGCGCCAAGG CCGTCTCGCT GGAGGAGCAG ATCTCCGCGA 180 TGGACGCCAC CACCGGCGAC TTCACGGCGC TGCAGAAGGC GGTGAAGCAG ATGGCCACCA 240 AGGCGGGCAC TGAGGGCCTG GTGCACGGCA TCAAGAACCC CGACGTGCGC CAGCTGCTGA 300 CCGAGATCTT CATGAAGGAC CCGGAGCAGC AGGAGTTCAT GCAGGCGGTG CGCGAGGTGG 360 CCGTCTCCCT GCAGCCCGTG TTCGAGAAGC GCCCCGAGCT GCTGCCCATC TTCAAGCAGA 420 TCGTTGAGCC TGAGCGCGTG ATCACCTTCC GCGTGTCCTG GCTGGACGAC GCCGGCAACC 480 TGCAGGTCAA CCGCGGCTTC CGCGTGCAGT ACTCGTCCGC CATCGGCCCC TACAAGGGCG 540 GCCTGCGCTT CCACCCCTCC GTGAACCTGT CCATCATGAA GTTCCTTGCC TTTGAGCAGA 600 TCTTCAAGAA CAGCCTGACC ACCCTGCCCA TGGGCGGCGG CAAGGGCGGC TCCGACTTCG 660 ACCCCAAGGG CAAGAGCGAC GCGGAGGTGA TGCGCTTCTG CCAGTCCTTC ATGACCGAGC 720 TGCAGCGCCA CATCAGCTAC GTGCAGGACG TGCCCGCCGG CGACATCGGC GTGGGCGCGC 780 GCGAGATTGG CTACCTTTTC GGCCAGTACA AGCGCATCAC CAAGAACTAC ACCGGCGTGC 840 TGACCCCGAA GGGCCAGGAG TATGGCGGCT CCGAGATCCG CCCCGAGGCC ACCGGCTACG 900 GCGCCGTGCT GTTTGTGGAG AACGTGCTGA AGGACAAGGG CGAGAGCCTC AAGGGCAAGC 960 GCTGCCTGGT GTCTGGCGCG GGCAACGTGG CCCAGTACTG CGCGGAGCTG CTGCTGGAGA 1020 AGGGCGCCAT CGTGCTGTCG CTGTCCGACT CCCAGGGCTA CGTGTACGAG CCCAACGGCT 1080 TCACGCGCGA GCAGCTGCAG GCGGTGCAGG ACATGAAGAA GAAGAACAAC AGCGCCCGCA 1140 TCTCCGAGTA CAAGAGCGAC ACCGCCGTGT ATGTGGGCGA CCGCCGCAAG CCTTGGGAGC 1200 TGGACTGCCA GGTGGACATC GCCTTCCCCT GCGCCACCCA GAACGAGATC GATGAGCACG 1260 ACGCCGAGCT GCTGATCAAG CACGGCTGCC AGTACGTGGT GGAGGGCGCC AACATGCCCT 1320 CCACCAACGA GGCCATCCAC AAGTACAACA AGGCCGGCAT CATCTACTGC CCCGGCAAGG 1380 CGGCCAACGC CGGCGGCGTG GCGGTCAGCG GCCTGGAGAT GACCCAGAAC CGCATGAGCC 1440 TGAACTGGAC TCGCGAGGAG GTTCGCGACA AGCTGGAGCG CATCATGAAG GACATCTACG 1500 ACTCCGCCAT GGGGCCGTCC CGCAGATACA ATGTTGACCT GGCTGCGGGC GCCAACATCG 1560 CGGGCTTCAC CAAGGTGGCT GATGCCGTCA AGGCCCAGGG CGCTGTTTAA GCTGCCCAGG 1620 CCCAAGCCAC GGCTCACCGG CAATCCAACC CAACCAACTC AACGGCCAGG ACCTTTTCGG 1680 AAGCGGCGCC TTTTTCCCAG CCAGGGCCCT CACCTGCCCT TTCATAACCC TGCTATTGCC 1740 GCCGTGCCCC TGCAATTCCA CCCCAAGAAG AACTAGCGGC ACTTGACTGC ATCAGGACGG 1800 CTATTTTTTT CGCGACGCGC GCTCACCCCG AGAGCCTCTC TCCCCCGAGC CCTAAGCGCT 1860 GACGTCCGCC CGACTTTGCC TCGCACATCG CTCGGTTTTG ACCCCCTCCA GTCTACCCAC 1920 CCTGTTGTGA AGCCTACCAG CTCAATTGCC TTTTAGTGTA TGTGCGCCCC CTCCTGCCCC 1980 CGAATTTTCC TGCCATGAGA CGTGCGGTTC CTAGCCTGGT GACCCCAAGT AGCAGTTAGT 2040 GTGCGTGCCT TGCCCTGCGC TGCCCGGGAT GCGATACTGT GACCTGAGAG TGCTTGTGTA 2100 AACACGACGA GTCAAAAAAA AAAAAAAAAA AAAAAAA 2137 2096 base pairs nucleic acid double linear CDNA 19 CTTTCTGCTC GCCCTCTCTC CGTCCCGCCA TGCAGACCGC CCTCGTCGCC AAGCCTATCG 60 TGGCCTGCGC GTGGGTCCGC TCCGCCAAGC GCGATGTCCG CGCCAAGGCC GTCTCGCTGG 120 AGGAGCAGAT CTCCGCGATG GACGCCACCA CCGGCGACTT CACGGCGCTG CAGAAGGCGG 180 TGAAGCAGAT GGCCACCAAG GCGGGCACTG AGGGCCTGGT GCACGGCATC AAGAACCCCG 240 ACGTGCGCCA GCTGCTGACC GAGATCTTCA TGAAGGACCC GGAGCAGCAG GAGTTCATGC 300 AGGCGGTGCG CGAGGTGGCC GTCTCCCTGC AGCCCGTGTT CGAGAAGCGC CCCGAGCTGC 360 TGCCCATCTT CAAGCAGATC GTTGAGCCTG AGCGCGTGAT CACCTTCCGC GTGTCCTGGC 420 TGGACGACGC CGGCAACCTG CAGGTCAACC GCGGCTTCCG CGTGCAGTAC TCGTCCGCCA 480 TCGGCCCCTA CAAGGGCGGC CTGCGCTTCC ACCCCTCCGT GAACCTGTCC ATCATGAAGT 540 TCCTTGCCTT TGAGCAGATC TTCAAGAACA GCCTGACCAC CCTGCCCATG GGCGGCGGCA 600 AGGGCGGCTC CGACTTCGAC CCCAAGGGCA AGAGCGACGC GGAGGTGATG CGCTTCTGCC 660 AGTCCTTCAT GACCGAGCTG CAGCGCCACA TCAGCTACGT GCAGGACGTG CCCGCCGGCG 720 ACATCGGCGT GGGCGCGCGC GAGATTGGCT ACCTTTTCGG CCAGTACAAG CGCATCACCA 780 AGAACTACAC CGGCGTGCTG ACCCCGAAGG GCCAGGAGTA TGGCGGCTCC GAGATCCGCC 840 CCGAGGCCAC CGGCTACGGC GCCGTGCTGT TTGTGGAGAA CGTGCTGAAG GACAAGGGCG 900 AGAGCCTCAA GGGCAAGCGC TGCCTGGTGT CTGGCGCGGG CAACGTGGCC CAGTACTGCG 960 CGGAGCTGCT GCTGGAGAAG GGCGCCATCG TGCTGTCGCT GTCCGACTCC CAGGGCTACG 1020 TGTACGAGCC CAACGGCTTC ACGCGCGAGC AGCTGCAGGC GGTGCAGGAC ATGAAGAAGA 1080 AGAACAACAG CGCCCGCATC TCCGAGTACA AGAGCGACAC CGCCGTGTAT GTGGGCGACC 1140 GCCGCAAGCC TTGGGAGCTG GACTGCCAGG TGGACATCGC CTTCCCCTGC GCCACCCAGA 1200 ACGAGATCGA TGAGCACGAC GCCGAGCTGC TGATCAAGCA CGGCTGCCAG TACGTGGTGG 1260 AGGGCGCCAA CATGCCCTCC ACCAACGAGG CCATCCACAA GTACAACAAG GCCGGCATCA 1320 TCTACTGCCC CGGCAAGGCG GCCAACGCCG GCGGCGTGGC GGTCAGCGGC CTGGAGATGA 1380 CCCAGAACCG CATGAGCCTG AACTGGACTC GCGAGGAGGT TCGCGACAAG CTGGAGCGCA 1440 TCATGAAGGA CATCTACGAC TCCGCCATGG GGCCGTCCCG CAGATACAAT GTTGACCTGG 1500 CTGCGGGCGC CAACATCGCG GGCTTCACCA AGGTGGCTGA TGCCGTCAAG GCCCAGGGCG 1560 CTGTTTAAGC TGCCCAGGCC CAAGCCACGG CTCACCGGCA ATCCAACCCA ACCAACTCAA 1620 CGGCCAGGAC CTTTTCGGAA GCGGCGCCTT TTTCCCAGCC AGGGCCCTCA CCTGCCCTTT 1680 CATAACCCTG CTATTGCCGC CGTGCCCCTG CAATTCCACC CCAAGAAGAA CTAGCGGCAC 1740 TTGACTGCAT CAGGACGGCT ATTTTTTTCG CGACGCGCGC TCACCCCGAG AGCCTCTCTC 1800 CCCCGAGCCC TAAGCGCTGA CGTCCGCCCG ACTTTGCCTC GCACATCGCT CGGTTTTGAC 1860 CCCCTCCAGT CTACCCACCC TGTTGTGAAG CCTACCAGCT CAATTGCCTT TTAGTGTATG 1920 TGCGCCCCCT CCTGCCCCCG AATTTTCCTG CCATGAGACG TGCGGTTCCT AGCCTGGTGA 1980 CCCCAAGTAG CAGTTAGTGT GCGTGCCTTG CCCTGCGCTG CCCGGGATGC GATACTGTGA 2040 CCTGAGAGTG CTTGTGTAAA CACGACGAGT CAAAAAAAAA AAAAAAAAAA AAAAAA 2096 25 base pairs nucleic acid double linear CDNA 20 CATATGGCCG TCTCGCTGGG AGGAG 25 19 base pairs nucleic acid double linear CDNA 21 GTTGGATTGC CGGTGAGCC 19 21 base pairs nucleic acid double linear CDNA 22 CATATGGACG CCACCACCGG C 21 1506 base pairs nucleic acid double linear CDNA CDS 4..1464 23 CAT ATG GCC GTC TCG CTG GAG GAG CAG ATC TCC GCG ATG GAC GCC ACC 48 Met Ala Val Ser Leu Glu Glu Gln Ile Ser Ala Met Asp Ala Thr 515 520 525 ACC GGC GAC TTC ACG GCG CTG CAG AAG GCG GTG AAG CAG ATG GCC ACC 96 Thr Gly Asp Phe Thr Ala Leu Gln Lys Ala Val Lys Gln Met Ala Thr 530 535 540 AAG GCG GGC ACT GAG GGC CTG GTG CAC GGC ATC AAG AAC CCC GAC GTG 144 Lys Ala Gly Thr Glu Gly Leu Val His Gly Ile Lys Asn Pro Asp Val 545 550 555 CGC CAG CTG CTG ACC GAG ATC TTC ATG AAG GAC CCG GAG CAG CAG GAG 192 Arg Gln Leu Leu Thr Glu Ile Phe Met Lys Asp Pro Glu Gln Gln Glu 560 565 570 575 TTC ATG CAG GCG GTG CGC GAG GTG GCC GTC TCC CTG CAG CCC GTG TTC 240 Phe Met Gln Ala Val Arg Glu Val Ala Val Ser Leu Gln Pro Val Phe 580 585 590 GAG AAG CGC CCC GAG CTG CTG CCC ATC TTC AAG CAG ATC GTT GAG CCT 288 Glu Lys Arg Pro Glu Leu Leu Pro Ile Phe Lys Gln Ile Val Glu Pro 595 600 605 GAG CGC GTG ATC ACC TTC CGC GTG TCC TGG CTG GAC GAC GCC GGC AAC 336 Glu Arg Val Ile Thr Phe Arg Val Ser Trp Leu Asp Asp Ala Gly Asn 610 615 620 CTG CAG GTC AAC CGC GGC TTC CGC GTG CAG TAC TCG TCC GCC ATC GGC 384 Leu Gln Val Asn Arg Gly Phe Arg Val Gln Tyr Ser Ser Ala Ile Gly 625 630 635 CCC TAC AAG GGC GGC CTG CGC TTC CAC CCC TCC GTG AAC CTG TCC ATC 432 Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Leu Ser Ile 640 645 650 655 ATG AAG TTC CTT GCC TTT GAG CAG ATC TTC AAG AAC AGC CTG ACC ACC 480 Met Lys Phe Leu Ala Phe Glu Gln Ile Phe Lys Asn Ser Leu Thr Thr 660 665 670 CTG CCC ATG GGC GGC GGC AAG GGC GGC TCC GAC TTC GAC CCC AAG GGC 528 Leu Pro Met Gly Gly Gly Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly 675 680 685 AAG AGC GAC GCG GAG GTG ATG CGC TTC TGC CAG TCC TTC ATG ACC GAG 576 Lys Ser Asp Ala Glu Val Met Arg Phe Cys Gln Ser Phe Met Thr Glu 690 695 700 CTG CAG CGC CAC ATC AGC TAC GTG CAG GAC GTG CCC GCC GGC GAC ATC 624 Leu Gln Arg His Ile Ser Tyr Val Gln Asp Val Pro Ala Gly Asp Ile 705 710 715 GGC GTG GGC GCG CGC GAG ATT GGC TAC CTT TTC GGC CAG TAC AAG CGC 672 Gly Val Gly Ala Arg Glu Ile Gly Tyr Leu Phe Gly Gln Tyr Lys Arg 720 725 730 735 ATC ACC AAG AAC TAC ACC GGC GTG CTG ACC CCG AAG GGC CAG GAG TAT 720 Ile Thr Lys Asn Tyr Thr Gly Val Leu Thr Pro Lys Gly Gln Glu Tyr 740 745 750 GGC GGC TCC GAG ATC CGC CCC GAG GCC ACC GGC TAC GGC GCC GTG CTG 768 Gly Gly Ser Glu Ile Arg Pro Glu Ala Thr Gly Tyr Gly Ala Val Leu 755 760 765 TTT GTG GAG AAC GTG CTG AAG GAC AAG GGC GAG AGC CTC AAG GGC AAG 816 Phe Val Glu Asn Val Leu Lys Asp Lys Gly Glu Ser Leu Lys Gly Lys 770 775 780 CGC TGC CTG GTG TCT GGC GCG GGC AAC GTG GCC CAG TAC TGC GCG GAG 864 Arg Cys Leu Val Ser Gly Ala Gly Asn Val Ala Gln Tyr Cys Ala Glu 785 790 795 CTG CTG CTG GAG AAG GGC GCC ATC GTG CTG TCG CTG TCC GAC TCC CAG 912 Leu Leu Leu Glu Lys Gly Ala Ile Val Leu Ser Leu Ser Asp Ser Gln 800 805 810 815 GGC TAC GTG TAC GAG CCC AAC GGC TTC ACG CGC GAG CAG CTG CAG GCG 960 Gly Tyr Val Tyr Glu Pro Asn Gly Phe Thr Arg Glu Gln Leu Gln Ala 820 825 830 GTG CAG GAC ATG AAG AAG AAG AAC AAC AGC GCC CGC ATC TCC GAG TAC 1008 Val Gln Asp Met Lys Lys Lys Asn Asn Ser Ala Arg Ile Ser Glu Tyr 835 840 845 AAG AGC GAC ACC GCC GTG TAT GTG GGC GAC CGC CGC AAG CCT TGG GAG 1056 Lys Ser Asp Thr Ala Val Tyr Val Gly Asp Arg Arg Lys Pro Trp Glu 850 855 860 CTG GAC TGC CAG GTG GAC ATC GCC TTC CCC TGC GCC ACC CAG AAC GAG 1104 Leu Asp Cys Gln Val Asp Ile Ala Phe Pro Cys Ala Thr Gln Asn Glu 865 870 875 ATC GAT GAG CAC GAC GCC GAG CTG CTG ATC AAG CAC GGC TGC CAG TAC 1152 Ile Asp Glu His Asp Ala Glu Leu Leu Ile Lys His Gly Cys Gln Tyr 880 885 890 895 GTG GTG GAG GGC GCC AAC ATG CCC TCC ACC AAC GAG GCC ATC CAC AAG 1200 Val Val Glu Gly Ala Asn Met Pro Ser Thr Asn Glu Ala Ile His Lys 900 905 910 TAC AAC AAG GCC GGC ATC ATC TAC TGC CCC GGC AAG GCG GCC AAC GCC 1248 Tyr Asn Lys Ala Gly Ile Ile Tyr Cys Pro Gly Lys Ala Ala Asn Ala 915 920 925 GGC GGC GTG GCG GTC AGC GGC CTG GAG ATG ACC CAG AAC CGC ATG AGC 1296 Gly Gly Val Ala Val Ser Gly Leu Glu Met Thr Gln Asn Arg Met Ser 930 935 940 CTG AAC TGG ACT CGC GAG GAG GTT CGC GAC AAG CTG GAG CGC ATC ATG 1344 Leu Asn Trp Thr Arg Glu Glu Val Arg Asp Lys Leu Glu Arg Ile Met 945 950 955 AAG GAC ATC TAC GAC TCC GCC ATG GGG CCG TCC CGC AGA TAC AAT GTT 1392 Lys Asp Ile Tyr Asp Ser Ala Met Gly Pro Ser Arg Arg Tyr Asn Val 960 965 970 975 GAC CTG GCT GCG GGC GCC AAC ATC GCG GGC TTC ACC AAG GTG GCT GAT 1440 Asp Leu Ala Ala Gly Ala Asn Ile Ala Gly Phe Thr Lys Val Ala Asp 980 985 990 GCC GTC AAG GCC CAG GGC GCT GTT TAAGCTGCCC AGGCCCAAGC CACGGCTCAC 1494 Ala Val Lys Ala Gln Gly Ala Val 995 CGGCAATCCA AC 1506 487 amino acids amino acid linear protein 24 Met Ala Val Ser Leu Glu Glu Gln Ile Ser Ala Met Asp Ala Thr Thr 1 5 10 15 Gly Asp Phe Thr Ala Leu Gln Lys Ala Val Lys Gln Met Ala Thr Lys 20 25 30 Ala Gly Thr Glu Gly Leu Val His Gly Ile Lys Asn Pro Asp Val Arg 35 40 45 Gln Leu Leu Thr Glu Ile Phe Met Lys Asp Pro Glu Gln Gln Glu Phe 50 55 60 Met Gln Ala Val Arg Glu Val Ala Val Ser Leu Gln Pro Val Phe Glu 65 70 75 80 Lys Arg Pro Glu Leu Leu Pro Ile Phe Lys Gln Ile Val Glu Pro Glu 85 90 95 Arg Val Ile Thr Phe Arg Val Ser Trp Leu Asp Asp Ala Gly Asn Leu 100 105 110 Gln Val Asn Arg Gly Phe Arg Val Gln Tyr Ser Ser Ala Ile Gly Pro 115 120 125 Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Leu Ser Ile Met 130 135 140 Lys Phe Leu Ala Phe Glu Gln Ile Phe Lys Asn Ser Leu Thr Thr Leu 145 150 155 160 Pro Met Gly Gly Gly Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys 165 170 175 Ser Asp Ala Glu Val Met Arg Phe Cys Gln Ser Phe Met Thr Glu Leu 180 185 190 Gln Arg His Ile Ser Tyr Val Gln Asp Val Pro Ala Gly Asp Ile Gly 195 200 205 Val Gly Ala Arg Glu Ile Gly Tyr Leu Phe Gly Gln Tyr Lys Arg Ile 210 215 220 Thr Lys Asn Tyr Thr Gly Val Leu Thr Pro Lys Gly Gln Glu Tyr Gly 225 230 235 240 Gly Ser Glu Ile Arg Pro Glu Ala Thr Gly Tyr Gly Ala Val Leu Phe 245 250 255 Val Glu Asn Val Leu Lys Asp Lys Gly Glu Ser Leu Lys Gly Lys Arg 260 265 270 Cys Leu Val Ser Gly Ala Gly Asn Val Ala Gln Tyr Cys Ala Glu Leu 275 280 285 Leu Leu Glu Lys Gly Ala Ile Val Leu Ser Leu Ser Asp Ser Gln Gly 290 295 300 Tyr Val Tyr Glu Pro Asn Gly Phe Thr Arg Glu Gln Leu Gln Ala Val 305 310 315 320 Gln Asp Met Lys Lys Lys Asn Asn Ser Ala Arg Ile Ser Glu Tyr Lys 325 330 335 Ser Asp Thr Ala Val Tyr Val Gly Asp Arg Arg Lys Pro Trp Glu Leu 340 345 350 Asp Cys Gln Val Asp Ile Ala Phe Pro Cys Ala Thr Gln Asn Glu Ile 355 360 365 Asp Glu His Asp Ala Glu Leu Leu Ile Lys His Gly Cys Gln Tyr Val 370 375 380 Val Glu Gly Ala Asn Met Pro Ser Thr Asn Glu Ala Ile His Lys Tyr 385 390 395 400 Asn Lys Ala Gly Ile Ile Tyr Cys Pro Gly Lys Ala Ala Asn Ala Gly 405 410 415 Gly Val Ala Val Ser Gly Leu Glu Met Thr Gln Asn Arg Met Ser Leu 420 425 430 Asn Trp Thr Arg Glu Glu Val Arg Asp Lys Leu Glu Arg Ile Met Lys 435 440 445 Asp Ile Tyr Asp Ser Ala Met Gly Pro Ser Arg Arg Tyr Asn Val Asp 450 455 460 Leu Ala Ala Gly Ala Asn Ile Ala Gly Phe Thr Lys Val Ala Asp Ala 465 470 475 480 Val Lys Ala Gln Gly Ala Val 485 1473 base pairs nucleic acid double linear CDNA CDS 4..1431 25 CAT ATG GAC GCC ACC ACC GGC GAC TTC ACG GCG CTG CAG AAG GCG GTG 48 Met Asp Ala Thr Thr Gly Asp Phe Thr Ala Leu Gln Lys Ala Val 490 495 500 AAG CAG ATG GCC ACC AAG GCG GGC ACT GAG GGC CTG GTG CAC GGC ATC 96 Lys Gln Met Ala Thr Lys Ala Gly Thr Glu Gly Leu Val His Gly Ile 505 510 515 AAG AAC CCC GAC GTG CGC CAG CTG CTG ACC GAG ATC TTC ATG AAG GAC 144 Lys Asn Pro Asp Val Arg Gln Leu Leu Thr Glu Ile Phe Met Lys Asp 520 525 530 CCG GAG CAG CAG GAG TTC ATG CAG GCG GTG CGC GAG GTG GCC GTC TCC 192 Pro Glu Gln Gln Glu Phe Met Gln Ala Val Arg Glu Val Ala Val Ser 535 540 545 550 CTG CAG CCC GTG TTC GAG AAG CGC CCC GAG CTG CTG CCC ATC TTC AAG 240 Leu Gln Pro Val Phe Glu Lys Arg Pro Glu Leu Leu Pro Ile Phe Lys 555 560 565 CAG ATC GTT GAG CCT GAG CGC GTG ATC ACC TTC CGC GTG TCC TGG CTG 288 Gln Ile Val Glu Pro Glu Arg Val Ile Thr Phe Arg Val Ser Trp Leu 570 575 580 GAC GAC GCC GGC AAC CTG CAG GTC AAC CGC GGC TTC CGC GTG CAG TAC 336 Asp Asp Ala Gly Asn Leu Gln Val Asn Arg Gly Phe Arg Val Gln Tyr 585 590 595 TCG TCC GCC ATC GGC CCC TAC AAG GGC GGC CTG CGC TTC CAC CCC TCC 384 Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser 600 605 610 GTG AAC CTG TCC ATC ATG AAG TTC CTT GCC TTT GAG CAG ATC TTC AAG 432 Val Asn Leu Ser Ile Met Lys Phe Leu Ala Phe Glu Gln Ile Phe Lys 615 620 625 630 AAC AGC CTG ACC ACC CTG CCC ATG GGC GGC GGC AAG GGC GGC TCC GAC 480 Asn Ser Leu Thr Thr Leu Pro Met Gly Gly Gly Lys Gly Gly Ser Asp 635 640 645 TTC GAC CCC AAG GGC AAG AGC GAC GCG GAG GTG ATG CGC TTC TGC CAG 528 Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe Cys Gln 650 655 660 TCC TTC ATG ACC GAG CTG CAG CGC CAC ATC AGC TAC GTG CAG GAC GTG 576 Ser Phe Met Thr Glu Leu Gln Arg His Ile Ser Tyr Val Gln Asp Val 665 670 675 CCC GCC GGC GAC ATC GGC GTG GGC GCG CGC GAG ATT GGC TAC CTT TTC 624 Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Tyr Leu Phe 680 685 690 GGC CAG TAC AAG CGC ATC ACC AAG AAC TAC ACC GGC GTG CTG ACC CCG 672 Gly Gln Tyr Lys Arg Ile Thr Lys Asn Tyr Thr Gly Val Leu Thr Pro 695 700 705 710 AAG GGC CAG GAG TAT GGC GGC TCC GAG ATC CGC CCC GAG GCC ACC GGC 720 Lys Gly Gln Glu Tyr Gly Gly Ser Glu Ile Arg Pro Glu Ala Thr Gly 715 720 725 TAC GGC GCC GTG CTG TTT GTG GAG AAC GTG CTG AAG GAC AAG GGC GAG 768 Tyr Gly Ala Val Leu Phe Val Glu Asn Val Leu Lys Asp Lys Gly Glu 730 735 740 AGC CTC AAG GGC AAG CGC TGC CTG GTG TCT GGC GCG GGC AAC GTG GCC 816 Ser Leu Lys Gly Lys Arg Cys Leu Val Ser Gly Ala Gly Asn Val Ala 745 750 755 CAG TAC TGC GCG GAG CTG CTG CTG GAG AAG GGC GCC ATC GTG CTG TCG 864 Gln Tyr Cys Ala Glu Leu Leu Leu Glu Lys Gly Ala Ile Val Leu Ser 760 765 770 CTG TCC GAC TCC CAG GGC TAC GTG TAC GAG CCC AAC GGC TTC ACG CGC 912 Leu Ser Asp Ser Gln Gly Tyr Val Tyr Glu Pro Asn Gly Phe Thr Arg 775 780 785 790 GAG CAG CTG CAG GCG GTG CAG GAC ATG AAG AAG AAG AAC AAC AGC GCC 960 Glu Gln Leu Gln Ala Val Gln Asp Met Lys Lys Lys Asn Asn Ser Ala 795 800 805 CGC ATC TCC GAG TAC AAG AGC GAC ACC GCC GTG TAT GTG GGC GAC CGC 1008 Arg Ile Ser Glu Tyr Lys Ser Asp Thr Ala Val Tyr Val Gly Asp Arg 810 815 820 CGC AAG CCT TGG GAG CTG GAC TGC CAG GTG GAC ATC GCC TTC CCC TGC 1056 Arg Lys Pro Trp Glu Leu Asp Cys Gln Val Asp Ile Ala Phe Pro Cys 825 830 835 GCC ACC CAG AAC GAG ATC GAT GAG CAC GAC GCC GAG CTG CTG ATC AAG 1104 Ala Thr Gln Asn Glu Ile Asp Glu His Asp Ala Glu Leu Leu Ile Lys 840 845 850 CAC GGC TGC CAG TAC GTG GTG GAG GGC GCC AAC ATG CCC TCC ACC AAC 1152 His Gly Cys Gln Tyr Val Val Glu Gly Ala Asn Met Pro Ser Thr Asn 855 860 865 870 GAG GCC ATC CAC AAG TAC AAC AAG GCC GGC ATC ATC TAC TGC CCC GGC 1200 Glu Ala Ile His Lys Tyr Asn Lys Ala Gly Ile Ile Tyr Cys Pro Gly 875 880 885 AAG GCG GCC AAC GCC GGC GGC GTG GCG GTC AGC GGC CTG GAG ATG ACC 1248 Lys Ala Ala Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Thr 890 895 900 CAG AAC CGC ATG AGC CTG AAC TGG ACT CGC GAG GAG GTT CGC GAC AAG 1296 Gln Asn Arg Met Ser Leu Asn Trp Thr Arg Glu Glu Val Arg Asp Lys 905 910 915 CTG GAG CGC ATC ATG AAG GAC ATC TAC GAC TCC GCC ATG GGG CCG TCC 1344 Leu Glu Arg Ile Met Lys Asp Ile Tyr Asp Ser Ala Met Gly Pro Ser 920 925 930 CGC AGA TAC AAT GTT GAC CTG GCT GCG GGC GCC AAC ATC GCG GGC TTC 1392 Arg Arg Tyr Asn Val Asp Leu Ala Ala Gly Ala Asn Ile Ala Gly Phe 935 940 945 950 ACC AAG GTG GCT GAT GCC GTC AAG GCC CAG GGC GCT GTT TAAGCTGCCC 1441 Thr Lys Val Ala Asp Ala Val Lys Ala Gln Gly Ala Val 955 960 AGGCCCAAGC CACGGCTCAC CGGCAATCCA AC 1473 476 amino acids amino acid linear protein 26 Met Asp Ala Thr Thr Gly Asp Phe Thr Ala Leu Gln Lys Ala Val Lys 1 5 10 15 Gln Met Ala Thr Lys Ala Gly Thr Glu Gly Leu Val His Gly Ile Lys 20 25 30 Asn Pro Asp Val Arg Gln Leu Leu Thr Glu Ile Phe Met Lys Asp Pro 35 40 45 Glu Gln Gln Glu Phe Met Gln Ala Val Arg Glu Val Ala Val Ser Leu 50 55 60 Gln Pro Val Phe Glu Lys Arg Pro Glu Leu Leu Pro Ile Phe Lys Gln 65 70 75 80 Ile Val Glu Pro Glu Arg Val Ile Thr Phe Arg Val Ser Trp Leu Asp 85 90 95 Asp Ala Gly Asn Leu Gln Val Asn Arg Gly Phe Arg Val Gln Tyr Ser 100 105 110 Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val 115 120 125 Asn Leu Ser Ile Met Lys Phe Leu Ala Phe Glu Gln Ile Phe Lys Asn 130 135 140 Ser Leu Thr Thr Leu Pro Met Gly Gly Gly Lys Gly Gly Ser Asp Phe 145 150 155 160 Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe Cys Gln Ser 165 170 175 Phe Met Thr Glu Leu Gln Arg His Ile Ser Tyr Val Gln Asp Val Pro 180 185 190 Ala Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Tyr Leu Phe Gly 195 200 205 Gln Tyr Lys Arg Ile Thr Lys Asn Tyr Thr Gly Val Leu Thr Pro Lys 210 215 220 Gly Gln Glu Tyr Gly Gly Ser Glu Ile Arg Pro Glu Ala Thr Gly Tyr 225 230 235 240 Gly Ala Val Leu Phe Val Glu Asn Val Leu Lys Asp Lys Gly Glu Ser 245 250 255 Leu Lys Gly Lys Arg Cys Leu Val Ser Gly Ala Gly Asn Val Ala Gln 260 265 270 Tyr Cys Ala Glu Leu Leu Leu Glu Lys Gly Ala Ile Val Leu Ser Leu 275 280 285 Ser Asp Ser Gln Gly Tyr Val Tyr Glu Pro Asn Gly Phe Thr Arg Glu 290 295 300 Gln Leu Gln Ala Val Gln Asp Met Lys Lys Lys Asn Asn Ser Ala Arg 305 310 315 320 Ile Ser Glu Tyr Lys Ser Asp Thr Ala Val Tyr Val Gly Asp Arg Arg 325 330 335 Lys Pro Trp Glu Leu Asp Cys Gln Val Asp Ile Ala Phe Pro Cys Ala 340 345 350 Thr Gln Asn Glu Ile Asp Glu His Asp Ala Glu Leu Leu Ile Lys His 355 360 365 Gly Cys Gln Tyr Val Val Glu Gly Ala Asn Met Pro Ser Thr Asn Glu 370 375 380 Ala Ile His Lys Tyr Asn Lys Ala Gly Ile Ile Tyr Cys Pro Gly Lys 385 390 395 400 Ala Ala Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Thr Gln 405 410 415 Asn Arg Met Ser Leu Asn Trp Thr Arg Glu Glu Val Arg Asp Lys Leu 420 425 430 Glu Arg Ile Met Lys Asp Ile Tyr Asp Ser Ala Met Gly Pro Ser Arg 435 440 445 Arg Tyr Asn Val Asp Leu Ala Ala Gly Ala Asn Ile Ala Gly Phe Thr 450 455 460 Lys Val Ala Asp Ala Val Lys Ala Gln Gly Ala Val 465 470 475

Claims

1. A method for modulating nitrogen metabolism in plant cells, said method comprising the steps of transforming a plant cell with a polynucleotide encoding a polypeptide having glutamate dehydrogenase activity, and culturing said cell under conditions whereby descendant cells are produced which comprise said polynucleotide and wherein said polynucleotide is expressed, whereby nitrogen metabolism is modulated.

2. The method of claim 1, further comprising the step of regenerating said descendant cells to form a plant.

3. The method of claim 1, wherein said nitrogen metabolism comprises increasing the assimilation of inorganic nitrogen into organic nitrogen.

4. The method of claim 1, wherein said polypeptide is selected from the group consisting of an alpha subunit of glutamate dehydrogenase, a beta subunit of glutamate dehydrogenase, and fragments or mutants thereof which exhibit glutamate dehydrogenase activity.

5. The method of claim 1, wherein said polypeptide is operably linked to a chloroplast transit peptide.

6. The method of claim 5, wherein the chloroplast transit peptide comprises the amino terminus of SEQ ID NO. 2 or SEQ ID NO. 4, or a fragment or mutant thereof sufficient to exhibit chloroplast transit activity.

7. The method of claim 1 wherein said polynucleotide is selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 24, SEQ ID NO. 26, and fragments or mutants thereof.

8. The method of claim 1, wherein said polynucleotide is operably linked to a plant polyadenylation sequence.

9. The method of claim 1, further comprising engineering said polynucleotide to maximize expression in said plant cell, said engineering comprising determining favored codon usage in said plant cell and altering said polynucleotide by increasing the frequency of favored codons.

10. A method of modulating the properties of a plant comprising culturing a plant comprising cells that comprise a polynucleotide encoding a polypeptide having glutamate dehydrogenase activity under conditions where said polynucleotide is expressed in said cells, whereby properties of said plant are modulated.

11. The method of claim 10, wherein at least one property modulated by said method is selected from the group consisting of increased crop yield, altered ammonium assimilation, altered osmotic stress tolerance, and altered composition of said plant.

12. The method of claim 12, wherein said polynucleotide is selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 24, SEQ ID NO. 26, and fragments or mutants thereof sufficient to encode a polypeptide having glutamate dehydrogenase activity.

13. A plant produced by the method of claim 2, comprising cells which express a polynucleotide encoding a polypeptide exhibiting glutamate dehydrogenase activity, whereby nitrogen metabolism in said cells is modulated.

14. A descendant of the plant of claim 13, said descendant comprising cells expressing said polypeptide and wherein nitrogen metabolism of said cells of said descendant is thereby modulated.