AU6259198A - Glutrnagln amidotransferase - a novel essential translational component - Google Patents

Description

GlutRNA^Gln Amidotransferase - A Novel Essential Translational Component CROSS REFERENCE TO RELATED APPLICATIONS

This application is related to U.S. Provisional Application Serial No. 60/037,275; filed February 3, 1997, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention is in the field of inhibitors of protein translation,

particularly translation of proteins within microorganisms and organelles. This

invention relates to newly identified polynucleotides and polypeptides, and their

production and uses, as well as their variants, agonists and antagonists, and their uses.

In particular, in these and in other regards, the invention relates to novel

polynucleotides and polypeptides ofthe Glu-tRNA^Gln Amidotransferase family,

hereinafter referred to as "Glu-tRN A^Gln AdT" or "AdT". The present invention further

provides methods and compositions for use in identifying and using protein

translation inhibitors as antibacterial, antifungal or herbicidal agents.

BACKGROUND OF THE INVENTION

Prior to their incorporation into protein, amino acids are chemically linked to

small RNA molecules called transfer RNA (tRNA). For each ofthe 20 different

amino acids, a specific enzyme catalyzes its linkage to the 3' end of its specific tRNA

molecule. While the general mechanism of protein biosynthesis (the translation process) is conserved throughout the living kingdom there exist two different

pathways for the formation of GlntRNA^Gln. While the two pathways for GlntRNA^G!n

formation are evolutionarily conserved, the reason for existence ofthe different

pathways is as yet not known. In gram-negative eubacteria and in the cytoplasm of

eukaryotic cells the enzyme glutaminyl-tRNA synthetase (GlnRS) acylates glutamine

directly to the cognate tRNA to provide GlntRNA^Gln. Interestingly, GlnRS is not

detectable in several biological systems. In certain organisms and organelles

including the archae, gram-positive eubacteria, mitochondria and chloroplasts a

different pathway of GlntRNA^Gln formation, a transamidation pathway is operative

(Curnow et al. (1996) Nature 382: 589-590; Curnow et al (1997) Proc. Natl. Acad.

Sci. USA 94(22): 11819-11826; Schon et a/. (1988) 5toc/ztmte 70(3):391-394; Wilcox

& Nirenberg (1968) Proc. Natl Acad. Sci. USA 61(l):229-236; Schδn et al. (1988)

Nature 331:187-190. This pathway (depicted in Figure 1) is initiated by misacylation

of tRNA^Gln by glutamyl-tRNA synthetase (GluRS) forming GlutRNA^Gln. The

incorrectly charged tRNA is then converted to GlntRNA^Gln by GlutRNA^Gln

amidotransferase (AdT). AdT catalyzes the amidation of glutamate to glutamine only

when the glutamate is covalently attached to tRNAGln. It has been shown that the

partially purified GlutRNA^Gln amidotransferase activity from Bacillus megaterium in

the presence of ATP, Mg++, and an amide-nitrogen donor (glutamine) will carry out

the amidation of GlutRNA^Gln to GlntRNA^Gln (Wilcox & Nirenberg, 1968). Subsequent work demonstrated, in vitro, that the amidation proceeds through the

activated intermediate (phospho-GlutRNA^Gln) (Wilcox (1969) Cold Spring Harb.

Symp. Quant. Biol 34:521-528; Wilcox (1969) Eur J. Biochem 11(3):405-412). Since

the initial aminoacylation product, GlutRNA^Gln, would be toxic to the cell due to the

fact that it would result in faulty protein translation, it must be converted to the

correctly charged tRNA.

It appears that this pathway is the primary source of GlntRNA^Gln within these

cells and may act as a regulatory mechanism for glutamine metabolism.

Evolutionarily, it has been suggested that glutamine was the last amino acid formed.

Therefore it may be postulated that cells which employ the transamidation pathway

utilized the gene encoding GluRS to generate the AdT. Likewise, in the cells in

which the direct glutaminylation pathway operates, the enzyme GlnRS may have

evolved from a GluRS gene duplication (Rogers & Soil (1995) J. Mol Evol. 40 (5)

p476-81). This is reasonable since both enzymes are required to specifically

recognize and bind tRNAGln and free glutamine. However, database searches and, in

particular, a detailed analysis ofthe Mycoplasma genome (Fraser et al. (1995) Science

270(5235):397-403), the only gram-positive organism sequenced and published to

date, have shown no significant homologies to GluRS and GlnRS in the currently

available sequence information. Thus, the amidotransferase may not have significant

homology to the aminoacyl-tRNA synthetases. Despite the unquestioned evolutionary and biochemical significance in understanding this system, there have

been very few investigations of this enzyme to date (Wilcox & Nirenberg, 1968;

Wilcox, 1969; Strauch et al. (1988) J. 5acteriol. 170:916-920; and Jahn (1990) J. Biol.

Chem. 265(14):8059-64).

SUMMARY OF THE INVENTION

The present invention is based, in part, on the isolation and characterization of

a heterotrimeric protein designated AdT that is involved in generating GlntRNA^Gln

from GlutRNA^Gln. This invention further provides polypeptides that have been

identified as novel AdT polypeptides by homology between the amino acid sequence

of GlutRNA^Gln AdT and a known amino acid sequence.

This invention further provides polynucleotides that encode AdT

polypeptides. In particular, this invention provides the polynucleotide sequence

encoding GlutRNA^Gln AdT comprising the sequence set out in Figure 3 (SEQ ID

NO:l), or a variant thereof, such as naturally occurring allelic variants of AdT and

polypeptides encoded thereby. Thus, this invention provides polynucleotides that

hybridize to AdT polynucleotide sequences, particularly under stringent conditions.

This invention provides GlutRNA^Gln AdT protein from B. subtilis comprising

the amino acid sequences encoded by the nucleotide sequence of Figure 3 (SEQ ID

NOS:l, 3, 5 and 7), as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.

Particularly preferred variants include AdT polypeptides encoded by naturally

occurring alleles ofthe AdT gene. Methods for producing the aforementioned AdT

polypeptides are also provided by this invention.

The invention also provides isolated nucleic acid molecules encoding AdT,

particularly B. subtilis AdT, including niRNAs, cDNAs, and genomic DNAs,

including biologically, diagnostically, prophylactically, clinically or therapeutically

useful variants thereof, and compositions comprising the same.

In accordance with yet another aspect ofthe invention, there are provided

inhibitors to such AdT polypeptides, useful as antibacterial agents, antifungal agents

and herbicides. Thus, the present invention provides compositions and methods for

use in identifying agonists and antagonists ofthe AdT protein.

This invention provides compositions and methods for (i) assessing AdT

expression, (ii) treating disease, for example, diseases associated with excessive or

deficient amounts of available AdT, (iii) assaying genetic variation, and (iv) and

administering an AdT polypeptide or polynucleotide to a cell or to a multicellular

organism to raise an immunological response. In certain preferred embodiments of

this aspect ofthe invention there are provided antibodies against AdT polypeptides.

This invention also provides compositions and methods for protecting plants,

especially crop plants. For example, this invention provides antagonists of AdT which are useful as herbicides, as well as the herbicidal compositions which include

such inhibitors of AdT. This invention also provides non-inhibited mutants of AdT

and functional derivatives thereof which are resistant to inhibition from certain

herbicides, especially herbicides containing inhibitors of AdT. The polynucleotides

coding for the non-inhibited AdT can be placed in plants by various transformation

methods so as to render the plants tolerant or resistant to certain herbicides containing

inhibitors of AdT. Therefore, methods of treating weeds utilizing the application of

AdT inhibitors to transgenic plants containing the non-inhibited mutants of AdT are

also encompassed by this invention.

Various changes and modifications within the spirit and scope ofthe disclosed

invention will become readily apparent to those skilled in the art from reading the

following description and from reading the other parts ofthe present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the transamidation pathway for the formation of GlntRNA^Gln.

Figure 2 shows the gene arrangement ofthe AdT gene.

Figure 3 shows the nucleic acid sequence ofthe AdT protein from B. subtilis.

DESCRIPTION OF THE INVENTION

I. General Description The present invention is based, in part, on the identification and

characterization of a heterotrimeric protein that is responsible for generating GlntRNA

within a cell, herein after the AdT protein. The present invention specifically

provides the amino acid sequences of each ofthe three subunits of an AdT protein

isolated from B. subtilis, as well as nucleotide sequences that encode the AdT protein.

The AdT protein and nucleic acid molecules can serve as targets in methods for

identifying agents for use in inhibiting protein synthesis, particularly antimicrobial,

antifungal and herbicide agents.

II. Specific Embodiments

A. AdT Protein

Prior to the present invention the art had taught that there was an enzyme

involved in converting GlutRNA^Gln to GlntRNA⁰¹" . However the isolation and

characterization ofthe protein responsible for generating GlntRNA remained

unknown. The present invention provides, in part, the amino acid sequences ofthe

three subunits ofthe B. subtilis AdT protein. Quite unexpectedly, this AdT protein

was found to be a heterotrimeric protein.

In one embodiment, the present invention provides the ability to produce a

previously unknown protein using the cloned nucleic acid molecules herein described

or by synthesizing a protein having the amino acid sequence herein disclosed. As used herein, the AdT protein refers to a protein that has the amino acid

sequence B. subtilis AdT encoded by the polynucleotide of Fig. 1, allelic variants

thereof and conservative substitutions thereof that have AdT activity. The AdT

protein is comprised of 3 subunits: the A (SEQ ID NO:4), B (SEQ ID NO:6) and C

(SEQ ID NO: 8) subunits, referred to herein collectively as aAdT, bAdT and CAdT

subunits, respectively. For the sake of convenience, the collective subunits will be

referred to as the AdT protein or the AdT protein ofthe present invention. A skilled

artisan can readily recognize within the context whether a single subunit or the

collective protein is being referred to.

The polypeptides ofthe invention include the polypeptides encoded by SEQ

ID NO:l (Figure 3) as well as polypeptides and fragments, particularly those which

have the biological activity of AdT and also those which have at least 70% sequence

identity to the polypeptides encoded by SEQ ID NO: lor the relevant portion,

preferably at least 80% identity to the polypeptides encoded by SEQ ID NO:l, and

more preferably at least 90% similarity (more preferably at least 90% identity) to the

polypeptides encoded by SEQ LD NO:l and still more preferably at least 95%

similarity (still more preferably at least 95% identity) to the polypeptides encoded by

SEQ LD NO: 1 and also include portions of such polypeptides.

The AdT proteins ofthe present invention include the specifically identified

and characterized variant herein described as well as allelic variants, conservative substitution variants and homologues that can be isolated/generated and characterized

without undue experimentation following the methods outlined below. For the sake

of convenience, all AdT proteins will be collectively referred to as the AdT proteins

or the AdT proteins ofthe present invention.

The term "AdT proteins" includes all naturally occurring allelic variants ofthe

B. subtilis AdT protein that possess normal AdT activity. In general, allelic variants

ofthe AdT protein will have a slightly different amino acid sequence than that

specifically encoded by SEQ ID NO:l but will be able to convert GlutRNA to

GlntRNA. Allelic variants, though possessing a slightly different amino acid

sequence than those recited above, will posses the ability to generate GlntRNA.

Typically, allelic variants ofthe AdT protein will contain conservative amino acid

substitutions from the AdT sequences herein described or will contain a substitution

of an amino acid from a corresponding position in an AdT homologue (an AdT

protein isolated from an organism other than B. subtilis).

The AdT proteins ofthe present invention are preferably in isolated form. As

used herein, a protein is said to be isolated when physical, mechanical or chemical

methods are employed to remove the AdT protein from cellular constituents that are

normally associated with the protein. A skilled artisan can readily employ standard

purification methods to obtain an isolated AdT protein. One purification scheme is outlined in Example 1. The nature and degree of isolation will depend on the

intended use.

The cloning of an AdT encoding nucleic acid molecule makes it possible to

generate defined fragments ofthe AdT proteins ofthe present invention. As discussed

below, fragments ofthe AdT proteins ofthe present invention are particularly useful

in generating subunit specific antibodies, in identifying agents that bind to a AdT

protein and in isolating homologues ofthe B. subtilis AdT protein.

Fragments ofthe AdT proteins can be generated using standard peptide

synthesis technology and the amino acid sequences disclosed herein. Alternatively,

recombinant methods can be used to generate nucleic acid molecules that encode a

fragment ofthe AdT protein.

Fragments ofthe AdT protein subunits that contain particularly interesting

structures can be identified using art-known methods such as immunogenicity,

Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or

Jameson- Wolf analysis. Fragments containing such residues are particularly useful in

generating subunit specific anti-AdT antibodies.

As described below, members ofthe AdT family of proteins can be used for,

but are not limited to: 1) a target to identify agents that block or stimulate AdT

activity, 2) a target or bait to identify and isolate binding partners that bind an AdT protein, 3)identifying agents that block or stimulate the activity of an AdT protein and

4) an assay target to identify AdT mediated activity or disease.

B. Anti-AdT Antibodies

The present invention further provides antibodies that selectively bind one or

more ofthe AdT proteins ofthe present invention, or to a specific subunit of an AdT

protein ofthe present invention. The most preferred antibodies will bind to either an

entire heterotrimeric AdT protein but not to an isolated subunit or will bind to an

isolated subunit but not to the assembled trimeric protein. Anti- AdT antibodies that

are particularly contemplated include monoclonal and polyclonal antibodies as well as

fragments containing the antigen binding domain and/or one or more complement

determining regions of these antibodies.

Antibodies are generally prepared by immunizing a suitable mammalian host

using an AdT protein, or fragment, in isolated or immunoconjugated form (Harlow,

Antibodies, Cold Spring Harbor Press, NY (1989)). Regions ofthe AdT protein that

show immunogenic structure can readily be identified using art-known methods.

Other important regions and domains can readily be identified using protein analytical

and comparative methods known in the art.

Fragments containing these residues are particularly suited in generating

specific classes of anti- AdT antibodies. Methods for preparing a protein for use as an immunogen and for preparing

immunogenic conjugates of a protein with a carrier such as BSA, KLH, or other

carrier proteins are well known in the art. In some circumstances, direct conjugation

using, for example, carbodiimide reagents may be used; in other instances linking

reagents such as those supplied by Pierce Chemical Co., Rockford, IL, may be

effective.

Administration of an AdT immunogen is conducted generally by injection

over a suitable time period and with use of a suitable adjuvant, as is generally

understood in the art. During the immunization schedule, titers of antibodies can be

taken to determine adequacy of antibody formation.

While the polyclonal antisera produced in this way may be satisfactory for

some applications, for pharmaceutical compositions, monoclonal antibody

preparations are preferred. Immortalized cell lines which secrete a desired

monoclonal antibody may be prepared using the standard method of Kohler and

Milstein or modifications which effect immortalization of lymphocytes or spleen

cells, as is generally known. The immortalized cell lines secreting the desired

antibodies are screened by immunoassay in which the antigen is the AdT protein or

peptide fragment. When the appropriate immortalized cell culture secreting the

desired antibody is identified, the cells can be cultured either in vitro or by production

in ascites fluid. The desired monoclonal antibodies are then recovered from the culture

supernatant or from the ascites supernatant. Fragments ofthe monoclonals or the

polyclonal antisera which contain the immunologically significant portion can be used

as antagonists, as well as the intact antibodies. Use of immunologically reactive

fragments, such as the Fab, Fab', of F(ab')2 fragments is often preferable, especially in

a therapeutic context, as these fragments are generally less immunogenic than the

whole immunoglobulin.

The antibodies or fragments may also be produced, using current technology,

by recombinant means. Regions that bind specifically to the desired regions ofthe

transporter can also be produced in the context of chimeric or CDR grafted antibodies

of multiple species origin.

As described below, anti- AdT antibodies are useful as modulators of AdT

activity, are useful in immunoassays for detecting AdT expression/activity and for

purifying homologues ofthe B. subtilis AdT protein.

C. AdT Encoding Nucleic Acid Molecules

As described above, the present invention is based, in part, on isolating nucleic

acid molecules from B. subtilis that encode the three subunits ofthe AdT protein.

Accordingly, the present invention further provides nucleic acid molecules that

encode the AdT protein, as herein defined, preferably in isolated form. For convenience, all AdT encoding nucleic acid molecules will be referred to as AdT

encoding nucleic acid molecules, the AdT genes, or AdT. The nucleotide sequence of

the B. subtilis nucleic acid molecule that encodes each ofthe subunits of AdT is

provided in SEQ ID NO: 1. The start and stop codons for each of subunits A (SEQ

ID NO:4), B (SEQ ID NO:6) and C (SEQ ID NO:8) are designated in the nucleotide

sequence for AdT provided in Figure 3.

Further preferred embodiments ofthe invention are polynucleotides that are at

least 70% sequence identical over their entire length to a polynucleotide encoding

AdT polypeptides having an amino acid sequence encoded by SEQ ID NO:l, and

polynucleotides which are complementary to such polynucleotides. Alternatively,

most highly preferred are polynucleotides that comprise a region that is at least 80%

identical over their entire length to a polynucleotide encoding AdT polypeptide and

polynucleotides complementary thereto. In this regard, polynucleotides at least 90%

identical over their entire length to the same are particularly preferred, and among

these particularly preferred polynucleotides, those with at least 95% are especially

preferred. Furthermore, those with at least 97% are highly preferred among those

with at least 95%, and among these those with at least 98% and at least 99% are

particularly highly preferred, with at least 99% being the more preferred. The invention further relates to variants ofthe herein above described

polynucleotides which encode for variants ofthe polypeptides having the deduced

amino acid sequences of SEQ ID NO:l.

Variants that are fragments ofthe polypeptides ofthe invention may be

employed for producing the corresponding full-length polypeptide by peptide

synthesis; therefore, these variants may be employed as intermediates for producing

the full-length polypeptides. Variants that are fragments ofthe polynucleotides ofthe

invention may be used to synthesize full-length polynucleotides ofthe invention.

Variants that are fragments ofthe polynucleotides ofthe invention may be used to

synthesize full-length polynucleotides ofthe invention. Such methods are widely

available, such as those disclosed in WO 97/26340 and WO 97/38716.

A fragment is a variant polypeptide having an amino acid sequence that

entirely is the same as part but not all ofthe amino acid sequence ofthe

aforementioned polypeptides. AdT polypeptides fragments may be "free-standing,"

or comprised within a larger polypeptide of which they form a part or region, most

preferably as a single continuous region, a single larger polypeptide.

Further particularly preferred embodiments are polynucleotides encoding AdT

variants, which have the amino acid sequence ofthe AdT polypeptides encoded by

SEQ ID NO:l in which several, a few, 10 to 15, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no

amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not

alter the properties and activities of AdT.

As used herein, a "nucleic acid molecule" is defined as an RNA or DNA

molecule that encodes a peptide as defined above, or is complementary to a nucleic

acid sequence encoding such peptides. Particularly preferred nucleic acid molecules

will have a nucleotide sequence identical to or complementary to the B. subtilis DNA

sequences herein disclosed. Specifically contemplated are genomic DNA,

polycistronic mRNA and antisense molecules, as well as nucleic acids based on an

alternative backbone or including alternative bases, whether derived from natural

sources or synthesized. Such nucleic acid molecules, however, are defined further as

being novel and unobvious over any prior art nucleic acid molecules encoding

non- AdT proteins isolated from organisms other than B. subtilis.

As used herein, a nucleic acid molecule is said to be "isolated" when the

nucleic acid molecule is substantially separated from contaminant nucleic acid

molecules that encode polypeptides other than AdT. A skilled artisan can readily

employ nucleic acid isolation procedures to obtain an isolated AdT encoding nucleic

acid molecule.

The present invention further provides fragments ofthe AdT encoding nucleic

acid molecules ofthe present invention. As used herein, a fragment of an AdT

encoding nucleic acid molecule refers to a small portion ofthe entire protein encoding sequence. The size ofthe fragment will be determined by its intended use. For

example, if the fragment is chosen so as to encode an active portion ofthe AdT

protein, such an active domain or effector binding site, then the fragment will need to

be large enough to encode the functional region(s) ofthe AdT protein. If the fragment

is to be used as a nucleic acid probe or PCR primer, then the fragment length^* is

chosen so as to obtain a relatively small number of false positives during

probing/priming. Fragments ofthe B. subtilis AdT nucleic acid molecule that are

particularly useful as selective hybridization probes or PCR can be readily determined

using art-known methods.

Fragments ofthe AdT encoding nucleic acid molecules ofthe present

invention (i.e., synthetic oligonucleotides) that are used as probes or specific primers

for the polymerase chain reaction (PCR), or to synthesize gene sequences encoding

AdT proteins, can easily be synthesized by chemical techniques, for example, the

phosphotriester method of Matteucci, et al., J Am Chem Soc (1981) 103:3185-3191 or

using automated synthesis methods. In addition, larger DNA segments can readily be

prepared by well known methods, such as synthesis of a group of oligonucleotides

that define various modular segments ofthe AdT gene, followed by ligation of

oligonucleotides to build the complete modified AdT gene.

The AdT encoding nucleic acid molecules ofthe present invention may further

be modified so as to contain a detectable label for diagnostic and probe purposes. As described above, such probes can be used to identify nucleic acid molecules encoding

other allelic variants or homologues ofthe AdT proteins and as described below, such

probes can be used to diagnose the presence of a AdT protein as a means for

diagnosing a pathological condition caused by AdT mediated translation. A variety of

such labels are known in the art and can readily be employed with the AdT encoding

molecules herein described. Suitable labels include, but are not limited to, biotin,

radiolabeled nucleotides, biotin, and the like. A skilled artisan can employ any ofthe

art-known labels to obtain a labeled AdT encoding nucleic acid molecule.

D. Isolation of Other AdT Encoding Nucleic Acid Molecules

The identification ofthe AdT protein from B. subtilis and the corresponding

nucleic acid molecules, has made possible the identification of and isolation of AdT

proteins from organisms other than B. subtilis, hereinafter referred to collectively as

AdT homologues. The preferred source ofthe AdT homologues are pathogenic

microorganisms such as bacteria and fungi, as well as plants in which it is desirable to

control growth. The most preferred sources are gram positive bacteria, pathogenic

fungi and plant organelles such as chloroplasts.

Essentially, a skilled artisan can readily use the amino acid sequence ofthe B.

subtilis AdT protein to generate antibody probes to screen expression libraries

prepared from cells. Typically, polyclonal antiserum from mammals such as rabbits immunized with the purified protein (as described below) or monoclonal antibodies

can be used to probe an expression library, prepared from a target organism, to obtain

the appropriate coding sequence for AdT protein homologue. The cloned cDNA

sequence can be expressed as a fusion protein, expressed directly using its own

control sequences, or expressed by constructing an expression cassette using control

sequences appropriate to the particular host used for expression ofthe enzyme.

Alternatively, a portion ofthe AdT encoding sequence herein described can be

synthesized and used as a probe to retrieve DNA encoding a member ofthe AdT

family of proteins from organisms other than B. subtilis. Oligomers containing

approximately 18-20 nucleotides (encoding about a 6-7 amino acid stretch) are

prepared and used to screen genomic DNA or cDNA libraries to obtain hybridization

under stringent conditions or conditions of sufficient stringency to eliminate an undue

level of false positives. This method can be used to identify and isolate altered and

variants ofthe AdT encoding sequences.

Additionally, pairs of oligonucleotide primers can be prepared for use in a

polymerase chain reaction (PCR) to selectively amplify/clone an AdT-encoding

nucleic acid molecule, or fragment thereof. A PCR denature/anneal/extend cycle for

using such PCR primers is well known in the art and can readily be adapted for use in

isolating other AdT encoding nucleic acid molecules. Regions ofthe B. subtilis AdT

gene that are particularly well suited for use as a probe or as primers can be readily identified. In general, the preferred primers will flank one or more ofthe subunit

encoding regions ofthe B. subtilis AdT gene.

Homologues ofthe herein disclosed AdT proteins will share homology. In

general, nucleic acid molecules that encode AdT homologues will hybridize to the

B. subtilis sequences under high stringency. Such sequences will typically contain at

least 70% homology, preferably at least 80%, most preferably at least 90% homology

to the B. subtilis sequences.

E. Recombinant DNA Molecules Containing an AdT Encoding Nucleic Acid

Molecule

The present invention further provides recombinant DNA molecules that

contain one or more ofthe AdT encoding sequences herein described, or a fragment of

the herein-described nucleic acid molecules. As used herein, an recombinant DNA

molecule is a DNA molecule that has been subjected to molecular manipulation in

vitro. Methods for generating recombinant DNA molecules are well known in the art,

for example, see Sambrook et al., Molecular Cloning (1989). In the preferred

recombinant DNA molecules, an AdT encoding DNA sequence that encodes an AdT

protein, or AdT subunit is operably linked to one or more expression control

sequences and/or vector sequences. The recombinant DNA molecule can encode either a single subunit ofthe AdT protein, or can encode an operon that contains all

three ofthe AdT subunits.

The choice of vector and/or expression control sequences to which one ofthe

AdT encoding sequences ofthe present invention is operably linked depends directly,

as is well known in the art, on the functional properties desired, e.g., protein

expression, and the host cell to be transformed. A vector contemplated by the present

invention is at least capable of directing the replication or insertion into the host

chromosome, and preferably also expression, of an AdT encoding sequence included

in the recombinant DNA molecule.

Expression control elements that are used for regulating the expression of an

operably linked protein encoding sequence are known in the art and include, but are

not limited to, inducible promoters, constitutive promoters, secretion signals,

enhancers, transcription terminators and other regulatory elements. Preferably, an

inducible promoter that is readily controlled, such as being responsive to a nutrient in

the host cell's medium, is used.

In one embodiment, the vector containing an AdT encoding nucleic acid

molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability

to direct autonomous replication and maintenance ofthe recombinant DNA molecule

intrachromosomally in a prokaryotic host cell, such as a bacterial host cell,

transformed therewith. Such replicons are well known in the art. In addition, vectors that include a prokaryotic replicon may also include a gene whose expression confers

a detectable marker such as a drug resistance. Typical bacterial drug resistance genes

are those that confer resistance to ampicillin or tetracycline.

Vectors that include a prokaryotic replicon can further include a prokaryotic or

viral promoter capable of directing the expression (transcription and translation) ofthe

AdT encoding sequence in a bacterial host cell, such as E. coli. A promoter is an

expression control element formed by a DNA sequence that permits binding of RNA

polymerase and transcription to occur. Promoter sequences compatible with bacterial

hosts are typically provided in plasmid vectors containing convenient restriction sites

for insertion of a DNA segment ofthe present invention. Typical of such vector

plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories

(Richmond, CA), pPL and pKK223 available from Pharmacia, Piscataway, NJ.

Expression vectors compatible with eukaryotic cells, preferably those

compatible with vertebrate cells, can also be used to variant recombinant DNA

molecules that contain an AdT encoding sequence. Eukaryotic cell expression vectors

are well known in the art and are available from several commercial sources.

Typically, such vectors are provided containing convenient restriction sites for

insertion ofthe desired DNA segment. Typical of such vectors are PSVL and

pKSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector pCDM8 described herein, and the like eukaryotic

expression vectors.

Eukaryotic cell expression vectors used to construct the recombinant DNA

molecules ofthe present invention may further include a selectable marker that is

effective in an eukaryotic cell, preferably a drug resistance selection marker. A

preferred drug resistance marker is the gene whose expression results in neomycin

resistance, i.e., the neomycin phosphotransferase (neo) gene. Southern et al., JMol

Anal Genet (1982) 1 :327-341. Alternatively, the selectable marker can be present on

a separate plasmid, and the two vectors are introduced by cotransfection ofthe host

cell, and selected by culturing in the presence ofthe appropriate drug for the

selectable marker.

F. Host Cells Containing an Exogenously Supplied AdT Encoding Nucleic

Acid Molecule

The present invention further provides host cells transformed with a nucleic

acid molecule that encodes an AdT protein ofthe present invention, either the entire

heterotrimeric protein or one or more subunits. The host cell can be either prokaryotic

or eukaryotic. Eukaryotic cells useful for expression of an AdT protein are not

limited, so long as the cell line is compatible with cell culture methods and

compatible with the propagation ofthe expression vector and expression of an AdT gene. Preferred eukaryotic host cells include, but are not limited to, yeast, insect and

mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey

or human fibroblastic cell line, the most preferred being cells that do not naturally

express an AdT protein. Any prokaryotic host can be used to express an AdT-encoding recombinant

DNA molecule. The preferred prokaryotic host is E. coli.

Transformation of appropriate cell hosts with an recombinant DNA molecule

ofthe present invention is accomplished by well known methods that typically depend

on the type of vector used and host system employed. With regard to transformation

of prokaryotic host cells, electroporation and salt treatment methods are typically

employed, see, for example, Cohen et al., Proc Acad Sci USA (1972) 69:2110; and

Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor

Laboratory, Cold Spring Harbor, NY (1982). With regard to transformation of

vertebrate cells with vectors containing recombinant DNAs, electroporation, cationic

lipid or salt treatment methods are typically employed, see, for example, Graham et

al., Virol (1973) 52:456; Wigler et al., Proc Natl Acad Sci USA (1979) 76:1373-76.

Successfully transformed cells, i.e., cells that contain an recombinant DNA

molecule ofthe present invention, can be identified by well known techniques. For

example, cells resulting from the introduction of an recombinant DNA ofthe present

invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence ofthe recombinant

DNA using a method such as that described by Southern, J Mol Biol (1975) 98:503, or

Berent et al., Biotech (1985) 3:208 or the proteins produced from the cell assayed via

an immunological method.

G. Production of an AdT Protein Using an recombinant DNA Molecule

The present invention further provides methods for producing an AdT protein

that uses one ofthe AdT encoding nucleic acid molecules herein described. In general

terms, the production of a recombinant AdT protein typically involves the following

steps.

First, a nucleic acid molecule is obtained that encodes an AdT protein, such as

the nucleic acid molecule depicted in Figure 3 (SEQ ID NO:l) or an AdT subunit.

The AdT encoding nucleic acid molecule is then preferably placed in an operable

linkage with suitable control sequences, as described above, to generate an expression

unit containing the AdT encoding sequence. The expression unit is used to transform

a suitable host and the transformed host is cultured under conditions that allow the

production ofthe AdT protein. Optionally the AdT protein is isolated from the

medium or from the cells; recovery and purification ofthe protein may not be

necessary in some instances where some impurities may be tolerated. Each ofthe foregoing steps can be done in a variety of ways. For example, the

desired coding sequences may be obtained from genomic fragments and used directly

in an appropriate host. The construction of expression vectors that are operable in a

variety of hosts is accomplished using an appropriate combination of replicons and

control sequences. The control sequences, expression vectors, and transformation

methods are dependent on the type of host cell used to express the gene and were

discussed in detail earlier. Suitable restriction sites can, if not normally available, be

added to the ends ofthe coding sequence so as to provide an excisable gene to insert

into these vectors. A skilled artisan can readily adapt any host/expression system

known in the art for use with AdT encoding sequences to produce an AdT protein.

H. Identification of Agents that Bind to an AdT Protein

Another embodiment ofthe present invention provides methods for identifying

agents that are agonists or antagonists ofthe AdT proteins herein described.

Specifically, agonists and antagonists of an AdT protein can be identified by the

ability ofthe agent to bind to an AdT protein and/or the ability to inhibit AdT activity.

Activity assays for AdT activity and binding assays using an AdT protein are suitable

for use in high through-put screening methods.

In detail, in one embodiment, an AdT protein is mixed with an agent. After

mixing under conditions that allow association of AdT protein with the agent, the mixture is analyzed to determine if the agent bound the AdT protein. Agonists and

antagonists are identified as being able to bind to an AdT protein. Alternatively or

consecutively, as described below, AdT activity can be directly assessed as a means

for identifying agonists and antagonists of AdT activity.

The AdT protein used in the above assay can be: an isolated and fully

characterized protein, a single subunit of an AdT protein, a partially purified protein, a

cell that has been altered to express an AdT protein or a fraction of a cell that has been

altered to express an AdT protein. Further, the AdT protein can be the entire AdT

protein, a specific fragment ofthe AdT protein or a single subunit ofthe AdT protein.

It will be apparent to one of ordinary skill in the art that so long as the AdT protein

can be assayed for agent binding, e.g., by a shift in molecular weight or activity, as

described in the Examples, the present assay can be used. The AdT protein is

particularly well suited for high through-put screening methods.

The source ofthe AdT protein will be based on the intended use ofthe

modulating agent. For example, microbial AdT protein is used to identify AdT

inhibitors that have bactericidal activity whereas chloroplast derived AdT protein is

used to identify AdT inhibitors that have herbicide activity.

The method used to identify whether an agent binds to an AdT protein will be

based primarily on the nature ofthe AdT protein used. For example, a gel retardation

assay can be used to determine whether an agent binds to a soluble fragment of an AdT protein. Alternatively, immunodetection and biochip technologies can be

adopted for use with an AdT protein. A skilled artisan can readily employ numerous

art-known techniques for determining whether a particular agent binds to an AdT

protein.

Agents can be further tested for the ability to modulate the activity of an AdT

protein using a cell-free assay system or a cellular assay system. Example 1 provides

one such methods that can be used to assay for AdT activity.

As used herein, an agent is said to antagonize AdT activity when the agent

reduces AdT activity. The preferred antagonist will selectively antagonize AdT, not

affecting any other cellular proteins, particularly other proteins involved in translation.

Further, the preferred antagonist will reduce AdT activity by more than 50%, more

preferably by more than 90%, most preferably eliminating all AdT activity.

As used herein, an agent is said to agonize AdT activity when the agent

increases AdT activity. The preferred agonist will increase AdT activity by more than

50%, more preferably by more than 90%, most preferably more than doubling the

level of AdT activity.

The preferred antagonists and agonists will be selective for a specific species,

genus, family, order or kingdom of organisms. Agents can be screened using one

AdT protein, or a combination of AdT proteins, to aid in identifying agents for target

specificity. For example, several different microbial AdT proteins can be used to identify general antimicrobial agents whereas chloroplast derived AdT proteins can be

used to identify herbicide agents.

Agents that are assayed in the above method can be randomly selected or

rationally selected or designed. As used herein, an agent is said to be randomly

selected when the agent is chosen randomly without considering the specific

sequences ofthe AdT protein. An example of randomly selected agents is the use a

chemical library or a peptide combinatorial library, or a growth broth of an organism

or plant extract.

As used herein, an agent is said to be rationally selected or designed when the

agent is chosen on a nonrandom basis that takes into account the sequence ofthe

target site and/or its conformation in connection with the agent's action. Agents can

be rationally selected or rationally designed by utilizing the peptide sequences that

make up the AdT protein. For example, a rationally selected peptide agent can be a

peptide whose amino acid sequence is identical to a fragment of an AdT protein.

The agents ofthe present invention can be, as examples, peptides, small

molecules, and vitamin derivatives, as well as carbohydrates. A skilled artisan can

readily recognize that there is no limit as to the structural nature ofthe agents ofthe

present invention. One class of agents ofthe present invention are peptide agents

whose amino acid sequences are chosen based on the amino acid sequence ofthe AdT protein. Small peptide agents can serve as competitive inhibitors of AdT protein

assembly.

The peptide agents ofthe invention can be prepared using standard solid phase

(or solution phase) peptide synthesis methods, as is known in the art. In addition, the

DNA encoding these peptides may be synthesized using commercially available

oligonucleotide synthesis instrumentation and produced recombinantly using standard

recombinant production systems. The production using solid phase peptide synthesis

is necessitated if non-gene-encoded amino acids are to be included.

Another class of agents ofthe present invention are antibodies immunoreactive

with critical positions ofthe AdT protein. As described above, antibodies are

obtained by immunization of suitable mammalian subjects with peptides, containing

as antigenic regions, those portions ofthe AdT protein intended to be targeted by the

antibodies. Critical regions include the domains identified in Figure 2. Such agents

can be used in competitive binding studies to identify second generation inhibitory

agents.

K. Uses of Agents that Bind to an AdT Protein

As provided in the Background section, the AdT proteins are involved in

protein translation, particularly protein translation in gram positive microorganisms,

fungi and cellular organelles, particularly chloroplasts. Agents that bind an AdT protein and act as an agonist or antagonist can be used to modulate translation in these

organism and serves as a basis for an antibacterial, antifungal or herbicide agents. In

detail, protein translation that requires AdT can be modulated by administering to an

organism an agent that binds to an AdT protein and acts as an agonist or antagonist of

AdT activity.

As used herein, an organism can be any organism, so long as it is desirable to

modulate protein translation in the organism, for example to control the growth of an

infectious agent in a mammalian subject or to act as an herbicide agent. The invention

is particularly useful in the treatment of human subjects for controlling microbial

growth.

As used herein, protein translation that requires AdT refers to protein

translation that would not occur without the presence of an active AdT protein. As

used herein, an agent is said to modulate AdT meditated protein translation when the

agent reduces the degree of protein translation.

The use ofthe AdT modulating agents will be based primarily on the target

AdT protein used to identify the agent as well as the activity/selectivity ofthe agent.

For example, an AdT inhibitory agent, that is used as an antimicrobial agent, is

preferably isolated using one or more microbial AdT proteins. Herbicide agent will

be preferably identified using chloroplast AdT protein as a target. L. Administration of Agonists and Antagonists of an AdT Protein

The administration of agonists and antagonists ofthe AdT protein will be

dependent on their intended purpose. For example, to control microbial growth in a

mammalian subject, an AdT inhibitory agent can be administered via parenteral,

subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal

routes. Alternatively, or concurrently, administration may be by the oral route. The

dosage administered will be dependent upon the age, health, and weight ofthe

recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature

ofthe effect desired. For example, to treat microbial infection, an agent that

modulates AdT activity is administered systemically or locally to the individual being

treated. As described below, there are many methods that can readily be adapted to

administer such agents.

The present invention further provides compositions containing an antagonist

or agonist of an AdT protein that is identified by the methods herein described. The

determination of optimal ranges of effective amounts of each component is within the

skill ofthe art and is based on the intended use.

In addition to the AdT modulating agent, the compositions ofthe present

invention may contain other ingredients, such as suitable pharmaceutically acceptable

carriers comprising excipients and auxiliaries which facilitate processing ofthe active

compounds into preparations which can be used pharmaceutically for delivery to the site of action. Suitable formulations for parenteral administration include aqueous

solutions ofthe active compounds in water-soluble variant, for example, water-soluble

salts. In addition, suspensions ofthe active compounds and as appropriate, oily

injection suspensions may be administered. Suitable lipophilic solvents or vehicles

include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example,

ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances

which increase the viscosity ofthe suspension and include, for example, sodium

carboxymethyl cellulose, sorbitol, and/or dintran. Optionally, the suspension may

also contain stabilizers. Liposomes can also be used to encapsulate the agent for

delivery into the cell.

The pharmaceutical formulation for systemic administration according to the

invention may be formulated for enteral, parenteral or topical administration. Indeed,

all three types of formulations may be used simultaneously to achieve systemic

administration ofthe active ingredient.

Suitable formulations for oral administration include hard or soft gelatin

capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or

inhalations and controlled release variants thereof.

M. Combination Therapy The agents ofthe present invention that modulate AdT activity can be

provided alone, or in combination with another agents that modulate protein synthesis

microbial, fungal or plant growth. For example, an agent ofthe present invention that

reduces microbial AdT activity can be administered in combination with other

antimicrobial agents. As used herein, two agents are said to be administered in

combination when the two agents are administered simultaneously or are administered

independently in a fashion such that the agents will act at the same time.

N. Methods for Identifying the Presence of an AdT protein or gene

The present invention further provides methods for identifying cells or

organisms expressing an AdT protein or an AdT gene. Such methods can be used to

diagnose the presence of an organism that expresses an AdT protein. The methods of

the present invention are particularly useful in the determining the presence of

pathogenic microorganisms. Specifically, the presence of an AdT protein can be

identified by determining whether an AdT protein, or nucleic acid encoding an AdT

protein, is expressed. The expression of an AdT protein can be used as a means for

diagnosing the presence of an organism that relies on AdT mediated translation.

A variety of immunological and molecular genetic techniques can be used to

determine if an AdT protein is expressed/produced in a particular cell or sample. In

general, an extract containing nucleic acid molecules or an extract containing proteins is prepared. The extract is then assayed to determine whether an AdT protein, or an

AdT encoding nucleic acid molecule, is produced in the cell.

For example, to perform a diagnostic test based on nucleic acid molecules, a

suitable nucleic acid sample is obtained and prepared using conventional techniques.

DNA can be prepared, for example, simply by boiling a sample in SDS. The

extracted nucleic acid can then be subjected to amplification, for example by using the

polymerase chain reaction (PCR) according to standard procedures, to selectively

amplify an AdT encoding nucleic acid molecule or fragment thereof. The size or

presence of a specific amplified fragment (typically following restriction

endonuclease digestion) is then determined using gel electrophoresis or the nucleotide

sequence ofthe fragment is determined (for example, see Weber and May Am JHum

Genet (1989) 44:388-339; Davies, J. et al. Nature (1994) 371:130-136)). The

resulting size ofthe fragment or sequence is then compared to the known AdT

proteins encoding sequences, for example via hybridization probe. Using this

method, the presence of an AdT protein can be identified.

To perform a diagnostic test based on proteins, a suitable protein sample is

obtained and prepared using conventional techniques. Protein samples can be

prepared, for example, simply by mixing a sample with SDS followed by salt

precipitation of a protein fraction. The extracted protein can then be analyzed to

determine the presence of an AdT protein using known methods. For example, the presence of specific sized or charged variants of a protein can be identified using

mobility in an electric filed. Alternatively, antibodies can be used for detection

purposes. A skilled artisan can readily adapt known protein analytical methods to

determine if a sample contains an AdT protein.

Alternatively, AdT expression can also be used in methods to identify agents

that decrease the level of expression ofthe AdT gene. For example, cells or tissues

expressing an AdT protein can be contacted with a test agent to determine the effects

ofthe agent on AdT expression. Agents that activate AdT expression can be used as

an agonist of AdT activity whereas agents that decrease AdT expression can be used

as an antagonist of AdT activity.

O. Preparation and Use of Herbicides

As discussed herein, the transamidation pathway is operative in chloroplasts.

The ability to identify AdT inhibitors which specifically inhibit plastid isoforms of

AdT can be useful in designing herbicides that are not toxic or harmful to humans and

animals. Thus, the ability to develop herbicides that inhibit only chloroplast isoforms

of enzymes such as Adt but do not inhibit cytosolic (i.e., the fluid portion ofthe

cytoplasm exclusive of organelles) AdT or human AdT, would provide a new form of

highly effective herbicide that is also less toxic to humans. However, Adt inhibitors

which are not limited to the chloroplasts may also find utility in use as an herbicide. An identified compound which inhibits function ofthe wild-type AdT enzyme

is utilized as an active ingredient in an herbicide. The active ingredient is normally

applied in the form of compositions together with one or more agriculturally

acceptable carriers, and can be applied to the crop area or plant to be treated,

simultaneously or in succession, with further compounds. These additional

compounds can include fertilizers, other herbicides, fungicides, bactericides,

nematicides, or mixtures of several of these preparations, together with further

carriers, surfactants or application-promoting adjuvants. The herbicide may be

applied as a seed coating, a ground spray, incorporated into the soil, or applied

directly to the plant. Preferably, the active ingredient ofthe present invention or an

agrochemical composition which contains at least one ofthe active ingredients ofthe

present invention are applied as a leaf preparation. Methods of herbicide preparation

and application are well known to one skilled in the art.

Resistant mutants to the AdT-inhibiting compound can be identified by

mutagenizing cells or organisms and growing the mutagenized populations in the

presence of a concentration ofthe inhibitor sufficient to inhibit growth ofthe wild-

type cells or organisms, and selecting cells or organisms from the populations that are

able to grow more rapidly than wild-type cells or organisms. Mutagenesis can be

accomplished by any one ofthe means well known to one skilled in the art, including:

chemical mutatgenesis (e.g., ethyl methanesulfonate); ultraviolet radiation; X-ray exposure; and gamma radiation (see, e.g., Watson et al, Recombinant DNA, Second

Edition (1992) Chapter 11:191-211; Freifelder, Molecular Biology (1987) Chapter

11 :293-313). The mutant individuals which have the ability to tolerate or resist the

normally toxic levels ofthe inhibitor are genetically purified, the gene encoding the

mutant AdT is isolated, and the DNA sequence ofthe mutant gene is determined and

translated into a predicted amino acid sequence. The amino acids which differ

between the wild-type AdT enzyme and the mutant AdT enzyme are assumed to be

responsible for the inhibitor-resistant phenotype ofthe newly-identified mutant.

The coding DNA sequence for the mutant AdT can be introduced into the

plant cell in a number of different ways that are well known to those of skill in the art.

Examples of such methods include micro injection, electroporation, Agrobacterium-

mediated transformation, direct gene transfer, and micro projectile bombardment.

Techniques for producing herbicide resistance in plants by incorporating DNA

encoding and expressing enzymes resistant to herbicides are well known (see, e.g.,

U.S. Patent No. 5,145,777; U.S. Patent No. 5,290,926), including techniques for

adding a chloroplast transit sequence upstream from an herbicide gene so that the

protein product is transported into the cholorplasts (Comai et al., Nature (1985)

313:741-744; U.S. Patent No. 4,940,835; U.S. Patent No. 5,188,642). In the same

manner, the gene coding for a mutant AdT may be substituted for one ofthe other

herbicide resistance genes ofthe references. Since AdT performs its function in the chloroplast, it may be particularly relevant to use a plastid transit sequence to ensure

expression in the chloroplast or other plastid as is known in the art.

Following introduction ofthe mutant AdT gene into plant cells and the

regeneration of transformed plants from such cells, conventional methods of plant

husbandry and plant breeding can be used to maintain and increase the transformed

plants. The transformed plants can also be used in conventional hybridization

schemes to produce new plant types which also carry the novel mutant AdT gene (see,

e.g., Fehr and Hadley, Hybridization of Crop Plants (1980); Jensen, Plant Breeding

Methodology (1988); Allard, Principles of Plant Breeding (1960).

Plants which express a gene which is tolerant or resistant to an inhibitor of

AdT can be grown in soil and the herbicide containing the AdT inhibitor can be

applied to inhibit weed growth. Since the weed plants will not be carrying the mutant

AdT gene, the weeds will be susceptible to the herbicide containing the AdT inhibitor.

The following examples are intended to illustrate, but not to limit, aspects of

the present invention.

EXAMPLES

the invention will be further described by reference to the following detailed

examples. These examples are provided for purposes of illustration only, and are not

intended to be limiting unless otherwise specified. Experimental Procedures

Preparation and purification of recombinant Bacillus subtilis GlntRNA^Gln

amidotransferase. E. coli BL21 (DE3) harboring pABC were incubated overnight in

3 mL LB medium (10 g bactotryptone, 5 g yeast extract, 10 g NaCl) with 50 (g/mL

ampicillin at 37°C. The culture was scaled up to 1 L and again allowed to incubate at

37°C overnight. Cells were harvested via centrifugation (4000 x g for 5 minutes at

4°C) and resuspended in 20 mL Buffer A (25 mM Hepes-KOH, pH 7.5, 25 mM KC1,

10 mM MgC12 and 1 mM DTT). This step and all subsequent steps were performed

at 4°C unless otherwise specified. The cells were lysed by sonication (4 x 15

seconds) and centrifuged at 100,000 x g for one hour. The enzyme was then purified

to homogeneity, as determined by SDS-polyacrylamide gel electrophoresis, via a

series of chromato graphic steps using a Pharmacia FPLC system. The supernatant

was first applied to a Q-sepharose (HR 16/10) column (strong anion exchange) and the

activity was eluted by a linear gradient from 0 to 1 M NaCl in Buffer A. The active

fractions from this column were applied to a Superdex-200 (HR 26/100) column (gel

filtration) and the activity was eluted isocratically in Buffer A. The fractions from

this column which contained activity were pooled and applied onto a MonoQ (HR

10/10) column and the activity as eluted with a linear gradient from 150 to 300 mM

NaCl in Buffer A. Active fractions from this column were pooled and dialyzed

against Buffer A + 200 mM NaCl in 50% glycerol for 12 hours and stored at 70°C. In vivo expressed Bacillus subtilis tRNA^Gln isolation and purification. A

3 mL culture of E. coli DH5a pGPl-2/pBTT (encoding tRNAGln) in LB medium

(10 g bactotryptone, 5 g yeast extract, 10 g NaCl) with 50 (g/mL ampicillin and

10 (g/mL kanamycin was incubated at 37°C overnight. The culture was scaled up to

1 L and overnight incubation was repeated. Cells were harvested via centrifugation at

4000 x g for 5 minutes at 4°C and resuspended in 10 mL lysis buffer (20 mM

TrisΗCl, pH 7.4 and 20 mM MgC12). Total nucleic acids were isolated by two

sequential extractions with equal volumes of water saturated phenol followed by

isopropanol precipitation ofthe aqueous phase. The nucleic acid pellet was collected

via centrifugation at 10,000 x g for 15 minutes at 4°C. The pellet was resuspended in

5 mL of 200 mM TrisOAc, pH 9.0 and incubated at 37°C for 1 hour to ensure

complete deacylation ofthe tRNA. The nucleic acids were recovered by ethanol

precipitation followed by centrifugation at 10,000 x g for 15 minutes at 4°C. The

pellet was resuspended in 100 mM NaCl, incubated overnight at 4°C and ethanol

precipitated. The tRNAGln was purified by a two-step anion exchange

chromatography protocol. The nucleic acids were resuspended in 5 mL of Buffer 1

(140 mM NaOAc, pH 4.5) and 1 gm DΕ52 resin/100 OD260 was added. The resin

was washed with 200 mL Buffer A and 150 mL Buffer 2 (140 mM NaOAc, pH 4.5 +

300 mM NaCl) and the tRNA was eluted with 100 mL Buffer 3 (140 mM NaOAc,

pH 4.5 + 1 M NaCl). the nucleic acids were recovered by ethanol precipitation followed by centrifugation at 10,000 x g for 15 minutes at 4°C and resuspended in

10 mM Tris-HCl, pH 7.4, 1 mM MgC12 and 1 mM DTT and applied onto a Pharmacia

MonoQ (HR 10/10) column. The tRNA was eluted with a gradient of 450 to 750 mM

NaCl in 10 mM Tris-HCl, pH 7.4, 1 mM MgC12 and 1 mM DTT. Fractions

containing the tRNAGln, based on ability to be aminoacylated with both Glu and Gin,

were pooled and used as substrates in the amidotransferase assays.

Aminoacylation reactions. The procedure for the formation of radiolabelled

GlntRNA^Gln was adapted from Jahn, D. et al. (1990). Unless otherwise noted, these

reactions were conducted at 37°C in a buffer consisting of 10 mM ATP, 50 mM

Hepes-KOH pH 7.0, 25 mM KCl, 15 mM MgC12, and 5 mM DTT. The concentration

of tRNAGln, recovered from E. coli DH5a harboring the plasmids pGP12 and pBTT

(see above), and L14C(U)-glutamate (300 mCi/mMol) was 10 (M. GluRS was

isolated from B. subtilis and then partially purified by DΕAΕ-sepharose

chromatography. The reactions were allowed to progress for various lengths of time

depending upon the assay. Aliquots from this mixture were then added to the

amidotransferase assay mixtures either directly or following water saturated phenol

extraction, ethanol precipitation, and resuspension in the aminoacylation buffer.

Amidotransferase reactions. The procedure for the formation of

radiolabelled GlntRNA⁰¹" from GlntRNA⁰¹" was adapted from Jahn, D. et al. (1990).

Unless otherwise noted, these reactions were conducted at 37°C in a buffer consisting of 1 mM ATP, 5 mM Hepes-KOH pH 7.0, 2.5 mM KCl, 1.5 mM MgC12, and 0.5 mM

dithiothreitol (DTT). The concentration of L14C(U)-GlutRNA^Gln was 1 (M and the

concentration of Lglutamine was 1 mM. Aliquots (0 to 20 (L) from fractions obtained

during purification ofthe enzyme were added and the mixture was incubated for

various lengths of time depending upon the assay followed by quenching with 10 (L,

3 M NaOAc, pH 5.0. The mixture was extracted with an equal volume of

water-saturated phenol and the aqueous and organic phases were separated by

centrifugation at 15,000 x g at room temperature for 60 seconds. The aqueous phase

was removed, 3x volumes of ethanol were added and the tRNA was precipitated at

70°C for 15 minutes. The precipitated tRNA was recovered by centrifugation at

15,000 x g at 4°C for 15 minutes. The pellet obtained was resuspended in 50 (L 0.01

N KOH and deaminoacylated at 65°C for 10 minutes. The base was neutralized with

1.3 (L, 0.1 N HC1 (to pH ( 6 to 7) and the solution was dried completely under

vacuum. The dried pellet was resuspended in 3 (L double-distilled H2O and spotted

onto a TLC place (cellulose, Aldrich). The front was allowed to migrate 3.5 to 5

hours in one of two solvent systems (A. 20:1:5 isopropyl alcohol: formic acid:water or

B. 2:1:6:6 ammonia:water:chloroform:methanol). The plate was dried at 85°C,

exposed to an activated phosphoroimaging plate ((12 hours) and the image was

analyzed using MacBas v2.0. In this way, the conversion of Glu to Gin was

measured. Example 1

Characterization of DNA Fragment Encoding AdT

Three genes; one transcript of correct size hybridizing with three probes is

provided in Figure 3 (SEQ ID NO:l). Open reading frames for each ofthe subunits is

provided.

Example 2

Characterization of B. subtilis AdT Protein

The amino acid sequence of B. subtilis AdT is encoded by the nucleotide

sequence of Figure 3 (SEQ ID NO:l).

The molecular weights ofthe three subunits is computed to be: 53.039 Kd, A

subunit; 53.314 Kd, B subunit; and 10.859 Kd, C subunit. The amino acid sequences

of each ofthe subunits A, B and C are provided in SEQ ID NOs: 4, 6 and 8,

respectively.

The sizes ofthe subunits were confirmed via polyacrylamide gel

electrophoresis.

Example 3

Preparation of polyclonal antiserum containing anti- AdT antibodies

A polyclonal antiserum containing anti- AdT antibodies was obtained by

administered to rabbits recombinant AdT (trimeric protein) to rabbits following known methods. Incubation ofthe antiserum with a B. subtilis extract containing AdT

protein completely inhibited AdT activity.

Using polyacrylamide gel electrophoresis, the antiserum was shown to contain

antibodies immunoreactive to the assembled AdT protein. The polyclonal antisera

was significantly less immunoreactive to non-assembled, individual subunits.

Example 4

Production and purification of AdT

Example 5

Identifying inhibitors of AdT activity

Purified amidotransferase is used in an assay to identify inhibitors of AdT

activity. The assay used to identify inhibitors of AdT activity comprises:

(a) incubating a first sample of AdT and its substrate;

(b) measuring an uninhibited reactivity ofthe AdT from step (a);

(c) incubating a first sample of AdT and its substrate in the presence of a

second sample comprising an inhibitor compound;

(d) measuring an inhibited reactivity ofthe AdT from step (c); and, (e) comparing the inhibited reactivity to the uninhibited reactivity ofthe

AdT.

Inhibitors of AdT identified using this process are utilized as antibacterial,

antifungal and herbicidal agents.

Example 6

Identification of inhibitor-resistant AdT mutants

Purified amidotransferase and an identified inhibitor of AdT is used in an

assay to identify inhibitor-resistant AdT mutants. The assay used to identify

inhibitor-resistant AdT mutants comprises :

(a) incubating a first sample of AdT and its substrate in the presence of a

second sample comprising an AdT inhibitor;

(b) measuring an unmutated reactivity ofthe AdT from step (a);

(c) incubating a first sample of a mutated AdT and its substrate in the

presence of a second sample comprising an AdT inhibitor;

(d) measuring a mutated reactivity ofthe mutated AdT from step (c); and,

(e) comparing the mutated reactivity to the unmutated reactivity ofthe

AdT.

Inhibitor-resistant AdT mutants identified using this process are utilized in the

production of cells and organisms resistant to AdT inhibitors. Example 7

Diagnostic assays

Nucleic acids for diagnosis are obtained from cells or tissues. Genomic DNA

may be used directly for dectection or may be amplified enzymatically by using PCR

or other amplification techniques prior to analysis. The genomic DNA can be

compared to the polynucleotide coding for amidotransferase as provided in SEQ ID

NO:l . Deletions and insertions can be detected by a change in size ofthe amplified

product in comparison to SEQ ID NO: 1. Point mutations can be identified by

hybridizing amplified DNA to labeled AdT polynucleotide sequences. Perfectly

matched sequences can be distinguished from mismatched duplexes by RNASE

digestion or by differences in melting temperatures. DNA sequence differences may

also be detected by alterations in the electrophoretic mobility ofthe DNA fragments

in gels, with or without denaturing agents, or by direct DNA sequencing. Sequence

changes at specific locations also may be revealed by nuclease protection assays, such

as RNase and SI protection or a chemical cleavage method.

Cells carrying mutations or polymorphisms in the gene ofthe invention may

also be detected at the DNA level by a variety of techniques. For example, RTPCR

can be used to detect mutations. It is particularly preferred to used RTPCR in

conduction with automated detection systems, such as, for example, GeneScan. RNA

or cDNA may also be used for the same purpose, PCR or RTPCR. As an example,

PCR primers complementary to the nucleic acid encoding AdT can be used to identify and analyze mutations. These primers may be used for amplifying AdT DNA isolated

from a sample derived from an organism. The invention also provides these primers

with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end. The primers may

be used to amplify the gene isolated from an infected individual such that the gene

may then be subject to various techniques for elucidation ofthe DNA sequence. In

this way, mutations in the AdT DNA sequence may be detected and used to diagnose

infection and to serotype or classify the infectious agent.

Increased or decreased expression of AdT polynucleotide can be measured

using any ofthe methods well known in the art for the quantitation of polynucleotides,

such as, for example, amplification, PCR, RTPCR, RNase protection, Northern

blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the intention for detecting

over-or under- expression of AdT protein compared to normal control tissue samples.

Assay techniques that can be sued to determine levels of AdT protein, in a sample

derived from a host are well-known to those of skill in the art. Such assay methods

include radioimmunoassay, competitive-binding assays, Western Blot analysis and

ELISA assays.

Example 8

Production of transformed plants

A mutated AdT-encoding DNA sequence that confers resistance to AdT

inhibitors is isolated from an inhibitor-resistant AdT mutant using mutagenesis and

isolation techniques well known to one of skill in the art. The coding sequence for the

mutant AdT gene is then introduced into a plant cell and whole transformed plants are

regenerated from the transformed plant cell using a number of different techniques

well known to those of skill in the art. The transformed plants are used in

conventional plant breeding schemes to produce new varieties of plants which also

carry the mutant AdT gene. Crop plants carrying the mutant AdT gene are grown in

production and an herbicide comprising an AdT inhibitor is applied to the crop to

control weeds.

Although the present invention has been described in detail with reference to

examples above, it is understood that various modifications can be made without

departing from the spirit ofthe invention. All cited references referred to in the

application are hereby incorporated by reference. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(l) APPLICANT: Soil, Dieter

(ll) TITLE OF INVENTION: GLU-TRAN AMIDOTRANSFERASE - A NOVEL ESSENTIAL TRANSLATIONAL COMPONENT

(m) NUMBER OF SEQUENCES: 8

(lv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: MORGAN, LEWIS & BOCKIUS LLP

(B) STREET: 1800 M Street, N.W.

(C) CITY: Washington

(D) STATE: D.C.

(E) COUNTRY: USA

(F) ZIP: 20036-5869

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk ιB) COMPUTER: IEM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi! CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US Unassigned

(B) FILING DATE: 03-FEB-1998

(C) CLASSIFICATION:

(vn) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/037,275

(B) FILING DATE: 03-FEB-1997

(vin) ATTORNEY/AGENT INFORMATION:

(A) NAME: Adler, Reid G.

(B) REGISTRATION NUMBER: 30,988

(C) REFERENCE/DOCKET NUMBER: 044574-5024-WO

(lx, TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 202-467-7000

(B) TELEFAX: 202-467-7176

(2) INFORMATION FOR SEQ ID NO:l: x. ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3495 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: cDNA

(lx) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: ;joιn(1..54, 58..390, 394..1866, 1870..3303, 3310

..3321, 3325..3348, 3352..3429, 3433..3471, 3475 ..3480, 3484..3495) ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GAA TTC GAT CCT GTC TCA AGG CGT TTT GTT GCT TTA AAG GGC TTG TTT 48 Glu Phe Asp Pro Val Ser Arg Arg Phe Val Ala Leu Lys Gly Leu Phe 1 5 10 15

TTG ATA TGA TCA GTA TTA TAT GAC TTA ACG GAG AAA TAT GTG GAG GTG 96 Leu lie Ser Val Leu Tyr Asp Leu Thr Glu Lys Tyr Val Glu Val 20 25 30

GAT CAT ATG TCA CGA ATT TCA ATA GAA GAA GTA AAG CAC GTT GCG CAC 144 Asp His Met Ser Arg lie Ser lie Glu Glu Val Lys His Val Ala His 35 40 45

CTT GCA AGA CTT GCG ATT ACT GAA GAA GAA GCA AAA ATG TTC ACT GAA 192 Leu Ala Arg Leu Ala He Thr Glu Glu Glu Ala Lys Met Phe Thr Glu 50 55 60

CAG CTC GAC AGT ATC ATT TCA TTT GCC GAG GAG CTT AAT GAG GTT AAC 240 Gin Leu Asp Ser He He Ser Phe Ala Glu Glu Leu Asn Glu Val Asn 65 70 75

ACA GAC AAT GTG GAG CCT ACA ACT CAC GTG CTG AAA ATG AAA AAT GTC 288 Thr Asp Asn Val Glu Pro Thr Thr His Val Leu Lys Met Lys Asn Val 80 85 90 95

ATG AGA GAA GAT GAA GCG GGT AAA GGT CTT CCG GTT GAG GAT GTC ATG 336 Met Arg Glu Asp Glu Ala Gly Lys Gly Leu Pro Val Glu Asp Val Met 100 105 110

AAA AAT GCG CCT GAC CAT AAA GAC GGC TAT ATT CGT GTG CCA TCA ATT 384 Lys Asn Ala Pro Asp His Lys Asp Gly Tyr He Arg Val Pro Ser He 115 120 125

CTG GAC TAA AGG AGG GAC ACA AGA ATG TCA TTA TTT GAT CAT AAA ATC 432 Leu Asp Arg Arg Asp Thr Arg Met Ser Leu Phe Asp His Lys He 130 135 140

ACA GAA TTA AAA CAG CTC ATA CAT AAA AAA GAG ATT AAG ATT TCT GAT 480 Thr G1 Leu Lys Gin Leu He His Lys Lys Glu He Lys He Ser Asp 145 150 155

CTG GTT GAT GAA TCT TAT AAA CGC ATC CAA GCG GTT GAT GAT AAG GTA 528 Leu Vai Asp Glu Ser Tyr Lys Arg He Gin Ala Val Asp Asp Lys Val 160 165 170

CAA GCC TTT TTG GCA TTA GAT GAA GAA AGA GCG CGC GCA TAC GCG AAG 576 Gin Ala Phe Leu Ala Leu Asp Glu Glu Arg Ala Arg Ala Tyr Ala Lys 175 180 185 190

GAG CTT GAT GAG GCG GTT GAC GGC CGT TCT GAG CAC GGT CTT CTT TTC 624 Glu Leu Asp Glu Ala Val Asp Gly Arg Ser Glu His Gly Leu Leu Phe 195 200 205

GGT ATG CCG ATC GGC GTA AAA GAT AAT ATC GTA ACA AAA GGG CTG CGC 672 Gly Met Pro He Gly Val Lys Asp Asn He Val Thr Lys Gly Leu Arg 210 215 220 ACA ACA TGC TCC AGC AAA ATT CTC GAA AAC TTT GAT CCG ATT TAC GAT 720 Thr Thr Cys Ser Ser Lys He Leu Glu Asn Phe Asp Pro He Tyr Asp 225 230 ^" 235

GCT ACT GTC GTT CAG CGC CTT CAA GAC GCT GAA GCG GTC ACA ATC GGA 768 Ala Thr Val Val Gin Arg Leu Gin Asp Ala Glu Ala Val Thr He Gly 240 245 250

AAA CTG AAC ATG GAC GAA TTC GCC ATG GGG TCA TCT ACA GAA AAC TCA 816 Lys Leu Asn Met Asp Glu Phe Ala Met Gly Ser Ser Thr Glu Asn Ser 255 260 265 270

GCT TAC AAG CTG ACG AAA AAC CCT TGG AAC CTG GAT ACA GTT CCC GGC 864 Ala Tyr Lys Leu Thr Lys Asn Pro Trp Asn Leu ASD Thr Val Pro Gly 275 280 ^" 285

GGT TCA AGC GGC GGA TCT GCA GCT GCG GTT GCT GCG GGA GAA GTT CCG 912 Gly Ser Ser Gly Gly Ser Ala Ala Ala Val Ala Ala Gly Glu Val Pro 290 295 300

TTT TCT CTT GGA TCT GAC ACA GGC GGC TCC ATC CGT CAG CCG GCA TCT 960 Phe Ser Leu Gly Ser Asp Thr Gly Gly Ser He Arg Gin Pro Ala Ser 305 310 315

TTC TGC GGC GTT GTC GGA TTA AAA CCT ACA TAC GGA CGT GTA TCT CGT 1008 Phe Cys Gly Val Val Gly Leu Lys Pro Thr Tyr Gly Arg Val Ser Arg 320 325 330

TAC GGC CTG GTC GCA TTT GCG TCT TCA TTG GAC CAA ATC GGA CCG ATT 1056 Tyr Gly Leu Val Ala Phe Ala Ser Ser Leu Asp Gin He Gly Pro He 335 ^" 340 345 350

ACA CG? ACG GTT GAG GAT AAC GCG TTT TTA CTT CAA GCG ATT TCC GGC 1104 Thr Arg Thr Val Glu Asp Asn Ala Phe Leu Leu Gin Ala He Ser Gly 355 360 365

GTA GAC AAA ATG GAC TCT ACG AGT GCA AAT GTG GAC GTG CCT GAT TTT 1152 Val Asp Lys Met Asp Ser Thr Ser Ala Asn Val Asp Val Pro Asp Phe 370 375 ^' 380

CTT TCT TCA TTA ACT GGC GAC ATC AAA GGA CTG AAA ATC GCC GTT CCG 1200 Leu Ser Ser Leu Thr Gly Asp He Lys Gly Leu Lys He Ala Val Pro 385 390 395

AAA GAA TAC CTT GGT GAA GGT GTC GGC AAA GAA GCG AGA GAA TCT GTC 1248 Lys Glu Tyr Leu Gly Glu Gly Val Gly Lys Glu Ala Arg Glu Ser Val 400 405 410

TTG GCA GCG CTG AAA GTC CTT GAA GGT CTC GGC GCT ACA TGG GAA GAA 1296 Leu Ala Ala Leu Lys Val Leu Glu Gly Leu Gly Ala Thr Trp Glu Glu 415 420 425 430

GTG TCT CTT CCG CAC AGT AAA TAC GCG CTT GCG ACA TAT TAC CTG CTG 1344 Val Ser Leu Pro His Ser Lys Tyr Ala Leu Ala Thr Tyr Tyr Leu Leu 435 440 445

TCA TCT TCT GAA GCG TCA GCG AAC CTT GCA CGC TTT GAC GGC ATC CGC 1392 Ser Ser Ser Glu Ala Ser Ala Asn Leu Ala Arg Phe Asp Gly He Arg 450 455 46C 1440

Tyr Gly Tyr Arg Thr Asp Asn Ala Asp Asn Leu He Asp Leu Tyr Lys 465 470 475

CAA ACG CGC GCT GAA GGT TTC GGA AAT GAA GTC AAA CGC CGC ATC ATG 1488

Gin Thr Arg Ala Glu Gly Phe Gly Asn Glu Val Lys Arg Arg He Met 480 485 490

CTC GGA ACG TTT GCT TTA AGC TCA GGC TAC TAC GAT GCG TAC TAC AAA 1536

Leu Gly Thr Phe Ala Leu Ser Ser Gly Tyr Tyr Asp Ala Tyr Tyr Lys

495 500 505 510

AAA GCG CAA AAA GTG CGT ACG TTG ATT AAG AAG GAT TTC GAG GAC GTA 1584

Lys Ala Gin Lys Val Arg Thr Leu He Lys Lys Asp Phe Glu Asp Val 515 520 525

TTT GAA AAA TAT GAT GTT ATT GTT GGA CCG ACT ACA CCG ACA CCT GCG 1632

Phe Glu Lys Tyr Asp Val He Val Gly Pro Thr Thr Pro Thr Pro Ala 530 535 540

TTT AAA ATC GGT GAA AAC ACG AAG GAT CCG CTC ACA ATG TAC GCA AAC 1680

Phe Lys He Gly Glu Asn Thr Lys Asp Pro Leu Thr Met Tyr Ala Asn 545 550 555

GAT ATC TTA ACG ATT CCG GTC AAC CTT GCG GCG TAC CGG GAA TCA GGT 1728

Asp He Leu Thr He Pro Val Asn Leu Ala Ala Tyr Arg Glu Ser Gly 560 565 570

GCC ATG CGG TTA GCA GAC GGA CTT CCG CTC GGC CTG CAA ATC ATC GGA 1776

Ala Met Arg Leu Ala Asp Gly Leu Pro Leu Gly Leu Gin He He Gly

575 580 585 590

AAA CAC TTT GAT GAA GCA CTG TAT ACC GCG TTG CTC ATG CAT TTG AAC 1824

Lys His Phe Asp Glu Ala Leu Tyr Thr Ala Leu Leu Met His Leu Asn 595 600 605

AAG CAA CAG ACC ATC ATA AAG CAA AAC CTG AAC TGT AAG GGG 1866

Lys Gin Gin Thr He He Lys Gin Asn Leu Asn Cys Lys Gly 610 615 620

TGA AAA GAA TTG AAC TTT GAA ACG GTA ATC GGA CTT GAA GTC CAC GTT 1914

Lys Glu Leu Asn Phe Glu Thr Val He Gly Leu Glu Val His Val 625 630 635

GAG AAA ACA AAA TCA AAA ATT TTC TCA AGC TCT CCA ACG CCA TTC 1962

Glu Leu Lys Thr Lys Ser Lys He Phe Ser Ser Ser Pro Thr Pro Phe 640 645 650

GGC GCG GAG GCG AAT ACG CAG ACA AGC GTT ATT GAC CTC GGA TAT CCG 2010

Gly Ala Glu Ala Asn Thr Gin Thr Ser Val He Asp Leu Gly Tyr Pro 655 660 665

GGC GTC CTG CCT GTT CTG AAC AAA GAA GCC GTT GAA TTC GCA ATG AAA 2058

Gly Val Leu Pro Val Leu Asn Lys Glu Ala Val Glu Phe Ala Met Lys 670 675 680

GCC GCT ATG GCG CTC AAC TGT GAG ATC GCA ACG GAT ACG AAG TTT GAC 2106

Ala Ala Met Ala Leu Asn Cys Glu He Ala Thr Asp Thr Lys Phe Asp 685 690 695 CGC AAA AAC TAT TTC TAT CCT GAC AAC CCG AAA GCG TAT CAG ATT TCT 2154 Arg Lys Asn Tyr Phe Tyr Pro Asp Asn Pro Lys Ala Tyr Gin He Ser 700 ^" 705 710 ^" 715

CAA TTT GAT AAG CCA ATC GGC GAA AAC GGC TGG ATC GAA ATT GAA GTC 2202 Gin Phe Asp Lys Pro He Gly Glu Asn Gly Trp He Glu He Glu Val 720 725 730

GGC GGC AAA ACA AAA CGC ATC GGC ATC ACG CGC CTT CAT CTT GAA GAG 2250 Gly Gly Lys Thr Lys Arg He Gly He Thr Arg Leu His Leu Glu Glu 735 ^' 740 745

GAT GCC GGA AAA CTG ACG CAT ACG GGC GAC GGC TAT TCT CTT GTT GAC 2298 Asp Ala Gly Lys Leu Thr His Thr Gly Asp Gly Tyr Ser Leu Val Asp 750 755 760

TTC AAC CGT CAA GGA ACG CCG CTT GTT GAG TNC GTA TCA GAG CCG GAC 2346 Phe Asn Arg Gin Gly Thr Pro Leu Val Glu Xaa Val Ser Glu Pro Asp 765 770 775

ATC CGC ACG CCG GAA GAA NCG TAC GCA TAT CTT GAA AAG CTG AAA TCC 2394 He Arg Thr Pro Glu Glu Xaa Tyr Ala Tyr Leu Glu Lys Leu Lys Ser 780 785 790 795

ATC ATC CAA TAT ACA GGC GTT TCT GAC TGT AAA ATG GAA GAA GGC TCA 2442 He He Gin Tyr Thr Gly Val Ser Asp Cys Lys Met Glu Glu Gly Ser 800 805 810

CTT CGC TGT GAC GCC AAT ATC TCT CTT CGT CCG ATC GGC CAA GAG GAA 2490 Leu Arg Cys Asp Ala Asn He Ser Leu Arg Pro He Gly Gin Glu Glu 815 820 825

TTC GGC ACA AAA ACA GAA TTG AAA AAC TTG AAC TCC TTT GCG TTT GTT 2538 Phe Gly Thr Lys Thr Glu Leu Lys Asn Leu Asn Ser Phe Ala Phe Val 830 835 840

CAA AAA GGC CTT GAG CAT GAA GAA AAA CGC CAG GAG CAG GTT CTT CTT 2586 Gin Lys Gly Leu Glu His Glu Glu Lys Arg Gin Glu Gin Val Leu Leu 845 850 855

TCC GGC TTC TTC ATC CAG CAA GAA ACT CGC CGT TAT GAT GAA GCA ACG 2634 Ser Gly Phe Phe He Gin Gin Glu Thr Arg Arg Tyr Asp Glu Ala Thr 860 865 870 875

AAG AAA ACC ATT CTT ATG CGT GTC AAA GAG GGA TCT GAC GAC TAC CGT 2682 Lys Lys Thr He Leu Met Arg Val Lys Glu Gly Ser Asp Asp Tyr Arg 880 885 890

TAC TTC CCA GAG CCA GAT CTA GTC GAG CTC TAC ATT GAT GAT GAA TGG 2730 Tyr Phe Pro Glu Pro Asp Leu Val Glu Leu Tyr He Asp Asp Glu Trp 895 900 905

AAG GAA CGC GTA AAA GCA AGC ATT CCT GAG CTT CCG GAT GAG CGC CGC 2778 Lys Glu Arg Val Lys Ala Ser He Pro Glu Leu Pro Asp Glu Arg Arg 910 915 920

AAG CGT TAT ATC GAA GAG CTT GGC TTC GCT GCA TAT GAC GCA ATG GTT 2826 Lys Arg Tyr He Glu Glu Leu Gly Phe Ala Ala Tyr Asp Ala Met Val 925 930 935 CTG ACG CTG ACA AAA GAA ATG GCT GAT TTC TTC GAA GAA ACC GTT CAA 2874 Leu Thr Leu Thr Lys Glu Met Ala Asp Phe Phe Glu Glu Thr Val Gin 940 945 950 955

AAA GGC GCT GAA GCT AAA CAA GCG TCT AAC TGG CTG ATG GGT GAA GTG 2922 Lys Glv Ala Glu Ala Lys Gin Ala Ser Asn Trp Leu Met Gly Glu Val 960 965 970

TCA GCT TAC CTA AAC GCA GAA CAA AAA GAG CTT GCC GAT GTT GCC CTG 2970 Ser Ala Tyr Leu Asn Ala Glu Gin Lys Glu Leu Ala Asp Val Ala Leu 975 980 ^* 985

ACA CCT GAA GGC CTT GCC GGC ATG ATC AAA TTG ATT GAA AAA GGA ACC 3018 Thr Pro Glu Gly Leu Ala Gly Met He Lys Leu He Glu Lys Gly Thr 990 995 1000

ATT TCT TCT AAG ATC GCG AAG AAA GTG TTT AAA GAA TTG ATT GAA AAA 3066 He Ser Ser Lys He Ala Lys Lys Val Phe Lys Glu Leu He Glu Lys 1005 1010 1015

GGC GGC GAC GCT GAG AAG ATT GTG AAA GAG AAA GGC CTT GTT CAG ATT 3114 Gly Gly Asp Ala Glu Lys He Val Lys Glu Lys Gly Leu Val Gin He 1020 1025 1030 1035

TCT GAC GAA GGC GTG CTT CTG AAG CTT GTC ACT GAG GCG CTT GAC AAC 3162 Ser Asp Glu Gly Val Leu Leu Lys Leu Val Thr Glu Ala Leu Asp Asn 1040 1045 1050

AAT CCT CAA TCA ATC GAA GAC TTT AAA AAC GGA AAA GAC CGC GCG ATC 3210 Asn Pro Gin Ser He Glu Asp Phe Lys Asn Gly Lys Asp Arg Ala He 1055 1060 1065

GGC TTC CTA GTC GGA CAG ATT ATG AAA GCG TCC AAA GGA CAA GCC AAC 3258 Gly Phe Leu Val Gly Gin He Met Lys Ala Ser Lys Gly Gin Ala Asn 1070 ^" 1075 1080

CCG CCG ATG GTC AAC AAA ATT CTG CTT GAA GAA ATT AAA AAA CGC 3303

Pro Pro Met Val Asn Lys He Leu Leu Glu Glu He Lys Lys Arg 1085 1090 1095

TAATAA AAA AGC AGC CCT TAG AGG CTG CTT TTT TTA TGG TCA AAT 3348

Lys Ser Ser Pro Arg Leu Leu Phe Leu Trp Ser Asn 1100 1105 1110

TGA GAT AAA GAC AAG ATG AGG GCC CGA AGC CTT TCA ACT TCT TTG TCG 3396 Asp Lys Asp Lys Met Arg Ala Arg Ser Leu Ser Thr Ser Leu Ser 1115 1120 1125

TTG GTT CCG GCC AAA TTG GAC AGC ATG CCT TTA TAA TCG GCT TGC GCG 3444 Leu Val Pro Ala Lvs Leu Asp Ser Met Pro Leu Ser Ala Cys Ala 1130 1135 1140

GTT TAT CCT GAG TCA ATT CTT CCT CGA TAA GAT AAG TGA CAC GGT GAT 3492 Val Tyr Pro Glu Ser He Leu Pro Arg Asp Lys His Gly Asp 1145 1150

ATC 3495

He

1155 (2) INFORMATION FOR SEQ ID NO: 2:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1155 ammo acids (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Glu Pne Asp Pro Val Ser Arg Arg Phe Val Ala Leu Lys Gly Leu Phe 1 5 10 15

Leu He Ser Val Leu Tyr Asp Leu Thr Glu Lys Tyr Val Glu Val Asp 20 25 30

His Met Ser Arg He Ser He Glu Glu Val Lys His Val Ala His Leu 35 40 45

Ala Arg Leu Ala He Thr Glu Glu Glu Ala Lys Met Phe Thr Glu Gin 50 55 60

Leu Asc Ser He He Ser Phe Ala Glu Glu Leu Asn Glu Val Asn Thr 65 70 75 80

Asp Asn Val Glu Pro Thr Thr His Val Leu Lys Met Lys Asn Val Met 85 90 95

Arg Glu Asp Glu Ala Gly Lys Gly Leu Pro Val Glu Asp Val Met Lys 100 105 110

Asn Ala Pro Asp riis Lys Asp Gly Tyr He Arg Val Pro Ser He Leu 115 120 125

Asp Arg Arg Asp Thr Arg Met Ser Leu Phe Asp His Lys He Thr Glu 130 135 140

Leu Lys Gin Leu He His Lys Lys Glu He Lys He Ser Asp Leu Val 145 150 155 160

Asp Glu Ser Tyr Lys Arg He Gin Ala Val Asp Asp Lys Val Gin Ala 165 170 175

Phe Le- Ala Leu Asp Glu Glu Arg Ala Arg Ala Tyr Ala Lys Glu Leu 180 185 190

Asp Glu Ala Val Asp Gly Arg Ser Glu His Gly Leu Leu Phe Gly Met 195 200 205

Pro He Gly Val Lys Asp Asn He Val Thr Lys Gly Leu Arg Tnr Thr 210 215 220

Cys Ser Ser Lys He Leu Glu Asn Phe Asp Pro He Tyr Asp Ala Thr 225 230 ^" 235 240

Val Val Gin Arg Leu Gin Asp Ala Glu Ala Val Thr He Gly Lys Leu 245 250 255

Asn Met Asp Glu Phe Ala Met Gly Ser Ser Thr Glu Asn Ser Ala Tyr 260 265 270 Lys Leu Thr Lys Asn Pro Trp Asn Leu Asp Thr Val Pro Gly Gly Ser 275 280 285

Ser Gly Gly Ser Ala Ala Ala Val Ala Ala Gly Glu Val Pro Phe Ser 290 295 300

Leu Gly Ser Asp Thr Gly Gly Ser He Arg Gin Pro Ala Ser Phe Cys 305 310 315 320

Gly Val Val Gly Leu Lys Pro Thr Tyr Gly Arg Val Ser Arg Tyr Gly 325 330 335

Leu Val Ala Phe Ala Ser Ser Leu Asp Gin He Gly Pro He Thr Arg 340 345 350

Thr Val Glu Asp Asn Ala Phe Leu Leu Gin Ala He Ser Gly Val Asp 355 360 365

Lys Met Asp Ser Thr Ser Ala Asn Val Asp Val Pro Asp Phe Leu Ser 370 375 380

Ser Leu Thr Gly Asp He Lys Gly Leu Lys He Ala Val Pro Lys Glu 385 390 395 400

Tyr Leu Gly Glu Gly Val Gly Lys Glu Ala Arg Glu Ser Val Leu Ala 405 410 415

Ala Leu Lys Val Leu Glu Gly Leu Gly Ala Thr Trp Glu Glu Val Ser 420 425 430

Leu Pro His Ser Lys Tyr Ala Leu Ala Thr Tyr Tyr Leu Leu Ser Ser 435 440 445

Ser Glu Ala Ser Ala Asn Leu Ala Arg Phe Asp Gly He Arg Tyr Gly 450 455 460

Tyr Arg Thr Asp Asn Ala Asp Asn Leu He Asp Leu Tyr Lys Gin Thr 465 ^" 470 475 480

Arg Ala Glu Gly Phe Gly Asn Glu Val Lys Arg Arg He Met Leu Gly 485 490 495

Thr Phe Ala Leu Ser Ser Gly Tyr Tyr Asp Ala Tyr Tyr Lys Lys Ala 500 505 510

Gin Lys Val Arg Thr Leu He Lys Lys Asp Phe Glu Asp Val Phe Glu 515 520 525

Lys Tyr Asp Val He Val Gly Pro Thr Thr Pro Thr Pro Ala Phe Lys 530 535 540

He Gly Glu Asn Thr Lys Asp Pro Leu Thr Met Tyr Ala Asn Asp He 545 550 555 560

Leu Thr He Pro Val Asn Leu Ala Ala Tyr Arg Glu Ser Gly Ala Met 565 570 575

Arg Leu Ala Asp Gly Leu Pro Leu Gly Leu Gin He He Gly Lys His 580 585 590 Phe Asp Glu Ala Leu Tyr Thr Ala Leu Leu Met His Leu Asn Lys Gin 595 600 605

Gin Thr He He Lys Gin Asn Leu Asn Cys Lys Gly Lys Glu Leu Asn 610 615 620

Phe Glu Thr Val He Gly Leu Glu Val His Val Glu Leu Lys Thr Lys 625 630 635 640

Ser Lys He Phe Ser Ser Ser Pro Thr Pro Phe Gly Ala Glu Ala Asn 645 650 655

Thr Gin Thr Ser Val He Asp Leu Gly Tyr Pro Gly Val Leu Pro Val 660 665 670

Leu Asn Lys Glu Ala Val Glu Phe Ala Met Lys Ala Ala Met Ala Leu 675 680 685

Asn Cys Glu He Ala Thr Asp Thr Lys Phe Asp Arg Lys Asn Tyr Phe 690 695 700

Tyr Pro Asp Asn Pro Lys Ala Tyr Gin He Ser Gin Phe Asp Lys Pro 705 710 715 720

He Gly Glu Asn Gly Trp He Glu He Glu Val Gly Gly Lys Thr Lys 725 730 735

Arg He Gly He Thr Arg Leu His Leu Glu Glu Asp Ala Gly Lys Leu 740 745 750

Thr His Thr Gly Asp Gly Tyr Ser Leu Val Asp Phe Asn Arg Gin Gly 755 760 765

Thr Pro Leu Val Glu Xaa Val Ser Glu Pro Asp He Arg Thr Pro Glu 770 775 780

Glu Xaa Tyr Ala Tyr Leu Glu Lys Leu Lys Ser He He Gin Tyr Thr 785 790 795 800

Gly Vai Ser Asp Cys Lys Met Glu Glu Gly Ser Leu Arg Cys Asp Ala 805 810 815

Asn He Ser Leu Arg Pro He Gly Gin Glu Glu Phe Gly Thr Lys Thr 820 825 830

Glu Leu Lys Asn Leu Asn Ser Phe Ala Phe Val Gin Lys Gly Leu Glu 835 840 845

His Glu Glu Lys Arg Gin Glu Gin Val Leu Leu Ser Gly Phe Phe He 850 855 860

Gin Gin Glu Thr Arg Arg Tyr Asp Glu Ala Thr Lys Lys Thr He Leu 865 870 875 880

Met Arg Val Lys Glu Gly Ser Asp Asp Tyr Arg Tyr Phe Pro Glu Pro 885 890 895

Asp Leu Val Glu Leu Tyr He Asp Asp Glu Trp Lys Glu Arg Val Lys 900 905 910 Ala Ser He Pro Glu Leu Pro Asp Glu Arg Arg Lys Arg Tyr He Glu 915 920 925

Glu Leu Gly Phe Ala Ala Tyr Asp Ala Met Val Leu Thr Leu Thr Lys 930 935 940

Glu Met Ala Asp Phe Phe Glu Glu Thr Val Gin Lys Gly Ala Glu Ala 945 950 955 960

Lys Gin Ala Ser Asn Trp Leu Met Gly Glu Val Ser Ala Tyr Leu Asn 965 970 975

Ala Glu Gin Lys Glu Leu Ala Asp Val Ala Leu Thr Pro Glu Gly Leu 980 985 990

Ala Gly Met He Lys Leu He Glu Lys Gly Thr He Ser Ser Lys He 995 1000 1005

Ala Lys Lys Val Phe Lys Glu Leu He Glu Lys Gly Gly Asp Ala Glu 101C 1015 1020

Lys He Val Lys Glu Lys Gly Leu Val Gin He Ser Asp Glu Gly Val 1025 1030 1035 1040

Leu Leu Lys Leu Val Thr Glu Ala Leu Asp Asn Asn Pro Gin Ser He 1045 1050 1055

Glu Asp Phe Lys Asn Gly Lys Asp Arg Ala He Gly Phe Leu Val Gly 1060 1065 1070

Gin He Met Lys Ala Ser Lys Gly Gin Ala Asn Pro Pro Met Val Asn 1075 1080 1085

Lys He Leu Leu Glu Glu He Lys Lys Arg Lys Ser Ser Pro Arg Leu 1090 1095 1100

Leu Phe Leu Trp Ser Asn Asp Lys Asp Lys Met Arg Ala Arg Ser Leu 1105 1110 1115 1120

Ser Thr Ser Leu Ser Leu Val Pro Ala Lys Leu Asp Ser Met Pro Leu 1125 1130 ^" 1135

Ser Ala Cys Ala Val Tyr Pro Glu Ser He Leu Pro Arg Asp Lys His 1140 1145 1150

Gly Asp He 1155

(2) INFORMATION FOR SEQ ID NO: 3: ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1461 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

;ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS ( B ) LOCATION : 1 . . 1458

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

ATG TCA TTA TTT GAT CAT AAA ATC ACA GAA TTA AAA CAG CTC ATA CAT 48 Met Ser Leu Phe Asp His Lys He Thr Glu Leu Lys Gin Leu He His 1 5 10 15

AAA AAA GAG ATT AAG ATT TCT GAT CTG GTT GAT GAA TCT TAT AAA CGC 96 Lys Lys Glu He Lys He Ser Asp Leu Val Asp Glu Ser Tyr Lys Arg 20 25 30

ATC CAA GCG GTT GAT GAT AAG GTA CAA GCC TTT TTG GCA TTA GAT GAA 144 He Gin Ala Val Asp Asp Lys Val Gin Ala Phe Leu Ala Leu Asp Glu 35 40 45

GAA AGA GCG CGC GCA TAC GCG AAG GAG CTT GAT GAG GCG GTT GAC GGC 192 Glu Arg Ala Arg Ala Tyr Ala Lys Glu Leu Asp Glu Ala Val Asp Gly 50 55 60

CGT TCT GAG CAC GGT CTT CTT TTC GGT ATG CCG ATC GGC GTA AAA GAT 240 Arg Ser Glu His Gly Leu Leu Phe Gly Met Pro He Gly Val Lys Asp 65 70 75 80

AAT ATC GTA ACA AAA GGG CTG CGC ACA ACA TGC TCC AGC AAA ATT CTC 288 Asn He Val Thr Lys Gly Leu Arg Thr Thr Cys Ser Ser Lys He Leu 85 90 95

GAA AAC TTT GAT CCG ATT TAC GAT GCT ACT GTC GTT CAG CGC CTT CAA 336 Glu Asn Phe Asp Pro He Tyr Asp Ala Thr Val Val Gin Arg Leu Gin 100 105 110

GAC GCT GAA GCG GTC ACA ATC GGA AAA CTG AAC ATG GAC GAA TTC GCC 384 Asp Ala Glu Ala Val Thr He Gly Lys Leu Asn Met Asp Glu Phe Ala 115 120 125

ATG GGG TCA TCT ACA GAA AAC TCA GCT TAC AAG CTG ACG AAA AAC CCT 432 Met Gly Ser Ser Thr Glu Asn Ser Ala Tyr Lys Leu Thr Lys Asn Pro

130 135 140

TGG AAC CTG GAT ACA GTT CCC GGC GGT TCA AGC GGC GGA TCT GCA GCT 480 Trp Asn Leu Asp Thr Val Pro Gly Gly Ser Ser Gly Gly Ser Ala Ala 145 150 155 160

GCG GTT GCT GCG GGA GAA GTT CCG TTT TCT CTT GGA TCT GAC ACA GGC 528 Ala Val Ala Ala Gly Glu Val Pro Phe Ser Leu Gly Ser Asp Thr Gly 165 170 175

GGC TCC ATC CGT CAG CCG GCA TCT TTC TGC GGC GTT GTC GGA TTA AAA 576 Gly Ser He Arg Gin Pro Ala Ser Phe Cys Gly Val Val Gly Leu Lys 180 185 190

CCT ACA TAC GGA CGT GTA TCT CGT TAC GGC CTG GTC GCA TTT GCG TCT 624 Pro Thr Tyr Gly Arg Val Ser Arg Tyr Gly Leu Val Ala Phe Ala Ser 195 200 205

TCA TTG GAC CAA ATC GGA CCG ATT ACA CGT ACG GTT GAG GAT AAC GCG 672 Ser Leu Asp Gin He Gly Pro He Thr Arg Thr Val Glu Asp Asn Ala 210 215 220 TTT TTA CTT CAA GCG ATT TCC GGC GTA GAC AAA ATG GAC TCT ACG AGT 720 Phe Leu Leu Gin Ala He Ser Gly Val Asp Lys Met Asp Ser Thr Ser 225 230 235 240

GCA AAT GTG GAC GTG CCT GAT TTT CTT TCT TCA TTA ACT GGC GAC ATC 768 Ala Asn Val Asp Val Pro Asp Phe Leu Ser Ser Leu Thr Gly Asp He 245 250 255

AAA GGA CTG AAA ATC GCC GTT CCG AAA GAA TAC CTT GGT GAA GGT GTC 816 Lys Gly Leu Lys He Ala Val Pro Lys Glu Tyr Leu Gly Glu Gly Val 260 265 270

GGC AAA GAA GCG AGA GAA TCT GTC TTG GCA GCG CTG AAA GTC CTT GAA 864 Gly Lys Glu Ala Arg Glu Ser Val Leu Ala Ala Leu Lys Val Leu Glu 275 280 285

GGT CTC GGC GCT ACA TGG GAA GAA GTG TCT CTT CCG CAC AGT AAA TAC 912 Gly Leu Gly Ala Thr Trp Glu Glu Val Ser Leu Pro His Ser Lys Tyr 290 295 300

GCG CTT GCG ACA TAT TAC CTG CTG TCA TCT TCT GAA GCG TCA GCG AAC 960 Ala Leu Ala Thr Tyr Tyr Leu Leu Ser Ser Ser Glu Ala Ser Ala Asn 305 310 315 320

CTT GCA CGC TTT GAC GGC ATC CGC TAC GGC TAC CGC ACA GAC AAC GCG 1008 Leu Ala Arg Phe Asp Gly He Arg Tyr Gly Tyr Arg Thr Asp Asn Ala 325 330 335

GAT AAC CTG ATC GAC CTT TAC AAG CAA ACG CGC GCT GAA GGT TTC GGA 1056 Asp Asn Leu He Asp Leu Tyr Lys Gin Thr Arg Ala Glu Gly Phe Gly 340 345 350

AAT GAA GTC AAA CGC CGC ATC ATG CTC GGA ACG TTT GCT TTA AGC TCA 1104 Asn Glu Val Lys Arg Arg He Met Leu Gly Thr Phe Ala Leu Ser Ser 355 360 365

GGC TAC TAC GAT GCG TAC TAC AAA AAA GCG CAA AAA GTG CGT ACG TTG 1152 Gly Tyr Tyr Asp Ala Tyr Tyr Lys Lys Ala Gin Lys Val Arg Thr Leu 370 375 380

ATT AAG AAG GAT TTC GAG GAC GTA TTT GAA AAA TAT GAT GTT ATT GTT 1200 He Lys Lys Asp Phe Glu Asp Val Phe Glu Lys Tyr Asp Val He Val 385 390 395 400

GGA CCG ACT ACA CCG ACA CCT GCG TTT AAA ATC GGT GAA AAC ACG AAG 1248 Gly Pro Thr Thr Pro Thr Pro Ala Phe Lys He Gly Glu Asn Thr Lys 405 410 415

GAT CCG CTC ACA ATG TAC GCA AAC GAT ATC TTA ACG ATT CCG GTC AAC 1296 Asp Pro Leu Thr Met Tyr Ala Asn Asp He Leu Thr He Pro Val Asn 420 425 430

CTT GCG GCG TAC CGG GAA TCA GGT GCC ATG CGG TTA GCA GAC GGA CTT 1344 Leu Ala Ala Tyr Arg Glu Ser Gly Ala Met Arg Leu Ala Asp Gly Leu 435 440 445

CCG CTC GGC CTG CAA ATC ATC GGA AAA CAC TTT GAT GAA GCA CTG TAT 1392 Pro Leu Gly Leu Gin He He Gly Lys His Phe Asp Glu Ala Leu Tyr 450 455 460 ACC GCG TTG CTC ATG CAT TTG AAC AAG CAA CAG ACC ATC ATA AAG CAA 1440

Thr Ala Leu Leu Met His Leu Asn Lys Gin Gin Thr He He Lys Gin

465 470 475 480

AAC CTG AAC TGT AAG GGG TGA 1461

Asn Leu Asn Cys Lys Gly 485

(2) INFORMATION FOR SEQ ID NO: 4:

^'i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 486 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Ser Leu Phe Asp His Lys He Thr Glu Leu Lys Gin Leu He His 1 5 10 15

Lys Lys Glu He Lys He Ser Asp Leu Val Asp Glu Ser Tyr Lys Arg 20 25 30

He Gin Ala Val Asp Asp Lys Val Gin Ala Phe Leu Ala Leu Asp Glu 35 40 45

Glu Arg Ala Arg Ala Tyr Ala Lys Glu Leu Asp Glu Ala Val Asp Gly 50 55 60

Arg Ser Glu His Gly Leu Leu Phe Gly Met Pro He Gly Val Lys Asp 65 70 75 80

Asn He Val Thr Lys Gly Leu Arg Thr Thr Cys Ser Ser Lys He Leu 85 90 95

Glu Asn Phe Asp Pro He Tyr Asp Ala Thr Val Val Gin Arg Leu Gin 100 105 110

Asp Ala Glu Ala Val Thr He Gly Lys Leu Asn Met Asp Glu Phe Ala 115 120 125

Met Glv Ser Ser Thr Glu Asn Ser Ala Tyr Lys Leu Thr Lys Asn Pro 130 135 140

Trp Asn Leu Asp Thr Val Pro Gly Gly Ser Ser Gly Gly Ser Ala Ala 145 150 155 160

Ala Val Ala Ala Gly Glu Val Pro Phe Ser Leu Gly Ser Asp Thr Gly 165 170 175

Gly Ser He Arg Gin Pro Ala Ser Phe Cys Gly Val Val Gly Leu Lys 180 185 190

Pro Thr Tyr Gly Arg Val Ser Arg Tyr Gly Leu Val Ala Phe Ala Ser 195 200 205

Ser Leu Asp Gin He Gly Pro He Thr Arg Thr Val Glu Asp Asn Ala 210 215 220 Phe Leu Leu Gin Ala He Ser Gly Val Asp Lys Met Asp Ser Thr Ser 225 230 235 240

Ala Asn Val Asp Val Pro Asp Phe Leu Ser Ser Leu Thr Gly Asp He 245 250 255

Lys Gly Leu Lys He Ala Val Pro Lys Glu Tyr Leu Gly Glu Gly Val 260 265 270

Gly Lys Glu Ala Arg Glu Ser Val Leu Ala Ala Leu Lys Val Leu Glu 275 280 285

Gly Leu Gly Ala Thr Trp Glu Glu Val Ser Leu Pro His Ser Lys Tyr 290 295 300

Ala Leu Ala Thr Tyr Tyr Leu Leu Ser Ser Ser Glu Ala Ser Ala Asn 305 310 315 320

Leu Ala Arg Phe Asp Gly He Arg Tyr Gly Tyr Arg Thr Asp Asn Ala 325 330 335

Asp Asn Leu He Asp Leu Tyr Lys Gin Thr Arg Ala Glu Gly Phe Gly 340 345 350

Asn Glu Val Lys Arg Arg He Met Leu Gly Thr Phe Ala Leu Ser Ser 355 360 365

Gly Tyr Tyr Asp Ala Tyr Tyr Lys Lys Ala Gin Lys Val Arg Thr Leu 370 375 380

He Lys Lys Asp Phe Glu Asp Val Phe Glu Lys Tyr Asp Val He Val 385 390 395 400

Gly Pro Thr Thr Pro Thr Pro Ala Phe Lys He Gly Glu Asn Thr Lys 405 410 415

Asp Pro Leu Thr Met Tyr Ala Asn Asp He Leu Thr He Pro Val Asn 420 425 430

Leu Ala Ala Tyr Arg Glu Ser Gly Ala Met Arg Leu Ala Asp Gly Leu 435 440 445

Pro Leu Gly Leu Gin He He Gly Lys His Phe Asp Glu Ala Leu Tyr 450 455 460

Thr Ala Leu Leu Met His Leu Asn Lys Gin Gin Thr He He Lys Gin 465 470 475 480

Asn Leu Asn Cys Lys Gly 485

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1431 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA ( ix ) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 1..1428

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

TTG AAC TTT GAA ACG GTA ATC GGA CTT GAA GTC CAC GTT GAG TTA AAA 48 Leu Asn Phe Glu Thr Val He Gly Leu Glu Val His Val Glu Leu Lys 1 5 10 15

ACA AAA TCA AAA ATT TTC TCA AGC TCT CCA ACG CCA TTC GGC GCG GAG 96 Thr Lys Ser Lys He Phe Ser Ser Ser Pro Thr Pro Phe Gly Ala Glu 20 25 30

GCG AAT ACG CAG ACA AGC GTT ATT GAC CTC GGA TAT CCG GGC GTC CTG 144 Ala Asn Thr Gin Thr Ser Val He Asp Leu Gly Tyr Pro Gly Val Leu 35 40 45

CCT GTT CTG AAC AAA GAA GCC GTT GAA TTC GCA ATG AAA GCC GCT ATG 192 Pro Vai Leu Asn Lys Glu Ala Val Glu Phe Ala Met Lys Ala Ala Met =0 55 60

GCG CTC AAC TGT GAG ATC GCA ACG GAT ACG AAG TTT GAC CGC AAA AAC 240 Ala Leu Asn Cys Glu He Ala Thr Asp Thr Lys Phe Asp Arg Lys Asn 65 70 75 80

TAT TTC TAT CCT GAC AAC CCG AAA GCG TAT CAG ATT TCT CAA TTT GAT 288 Tyr Phe Tyr Pro Asp Asn Pro Lys Ala Tyr Gin He Ser Gin Phe Asp 85 90 95

AAG CCA ATC GGC GAA AAC GGC TGG ATC GAA ATT GAA GTC GGC GGC AAA 336 Lys Pro He Gly Glu Asn Gly Trp He Glu He Glu Val Gly Gly Lys 100 105 110

ACA AAA CGC ATC GGC ATC ACG CGC CTT CAT CTT GAA GAG GAT GCC GGA 384 Thr Lys Arg He Gly He Thr Arg Leu His Leu Glu Glu Asp Ala Gly 115 120 125

AAA CTG ACG CAT ACG GGC GAC GGC TAT TCT CTT GTT GAC TTC AAC CGT 432 Lys Leu Thr His Thr Gly Asp Gly Tyr Ser Leu Val Asp Phe Asn Arg 130 135 140

CAA GGA ACG CCG CTT GTT GAG TNC GTA TCA GAG CCG GAC ATC CGC ACG 480 Gin Gly Thr Pro Leu Val Glu Xaa Val Ser Glu Pro Asp He Arg Thr 145 150 155 160

CCG GAA GAA NCG TAC GCA TAT CTT GAA AAG CTG AAA TCC ATC ATC CAA 528 Pro Glu Glu Xaa Tyr Ala Tyr Leu Glu Lys Leu Lys Ser He He Gin 165 170 175

TAT ACA GGC GTT TCT GAC TGT AAA ATG GAA GAA GGC TCA CTT CGC TGT 576 Tyr Thr Gly Val Ser Asp Cys Lys Met Glu Glu Gly Ser Leu Arg Cys 180 185 190

GAC GCC AAT ATC TCT CTT CGT CCG ATC GGC CAA GAG GAA TTC GGC ACA 624 Asp Ala Asn He Ser Leu Arg Pro He Gly Gin Glu Glu Phe Gly Thr 195 200 205 AAA ACA GAA TTG AAA AAC TTG AAC TCC TTT GCG TTT GTT CAA AAA GGC 672 Lys Thr Glu Leu Lys Asn Leu Asn Ser Phe Ala Phe Val Gin Lys Gly 210 215 220

CTT GAG CAT GAA GAA AAA CGC CAG GAG CAG GTT CTT CTT TCC GGC TTC 720 Leu Glu His Glu Glu Lys Arg Gin Glu Gin Val Leu Leu Ser Gly Phe 225 230 235 240

TTC ATC CAG CAA GAA ACT CGC CGT TAT GAT GAA GCA ACG AAG AAA ACC 768 Phe He Gin Gin Glu Thr Arg Arg Tyr Asp Glu Ala Thr Lys Lys Thr 245 ^' 250 255

ATT CTT ATG CGT GTC AAA GAG GGA TCT GAC GAC TAC CGT TAC TTC CCA 816 He Leu Met Arg Val Lys Glu Gly Ser Asp Asp Tyr Arg Tyr Phe Pro 260 265 270

GAG CCA GAT CTA GTC GAG CTC TAC ATT GAT GAT GAA TGG AAG GAA CGC 864 Glu Pro Asp Leu Val Glu Leu Tyr He Asp Asp Glu Trp Lys Glu Arg 275 280 285

GTA AAA GCA AGC ATT CCT GAG CTT CCG GAT GAG CGC CGC AAG CGT TAT 912 Val Lys Ala Ser He Pro Glu Leu Pro Asp Glu Arg Arg Lys Arg Tyr 290 295 300

ATC GAA GAG CTT GGC TTC GCT GCA TAT GAC GCA ATG GTT CTG ACG CTG 960 He Glu Glu Leu Gly Phe Ala Ala Tyr Asp Ala Met Val Leu Thr Leu 305 310 315 320

ACA AAA GAA ATG GCT GAT TTC TTC GAA GAA ACC GTT CAA AAA GGC GCT 1008 Thr Lys Glu Met Ala Asp Phe Phe Glu Glu Thr Val Gin Lys Gly Ala 325 330 335

GAA GCT AAA CAA GCG TCT AAC TGG CTG ATG GGT GAA GTG TCA GCT TAC 1056 Glu Ala Lys Gin Ala Ser Asn Trp Leu Met Gly Glu Val Ser Ala Tyr 340 345 350

CTA AAC GCA GAA CAA AAA GAG CTT GCC GAT GTT GCC CTG ACA CCT GAA 1104 Leu Asn Ala Glu Gin Lys Glu Leu Ala Asp Val Ala Leu Thr Pro Glu 355 360 365

GGC CTT GCC GGC ATG ATC AAA TTG ATT GAA AAA GGA ACC ATT TCT TCT 1152 Gly Leu Ala Gly Met He Lys Leu He Glu Lys Gly Thr He Ser Ser 370 375 380

AAG ATC GCG AAG AAA GTG TTT AAA GAA TTG ATT GAA AAA GGC GGC GAC 1200 Lys He Ala Lys Lys Val Phe Lys Glu Leu He Glu Lys Gly Gly Asp 385 390 395 400

GCT GAG AAG ATT GTG AAA GAG AAA GGC CTT GTT CAG ATT TCT GAC GAA 1248 Ala Glu Lys He Val Lys Glu Lys Gly Leu Val Gin He Ser Asp Glu 405 410 415

GGC GTG CTT CTG AAG CTT GTC ACT GAG GCG CTT GAC AAC AAT CCT CAA 1296 Gly Val Leu Leu Lys Leu Val Thr Glu Ala Leu Asp Asn Asn Pro Gin 420 425 430

TCA ATC GAA GAC TTT AAA AAC GGA AAA GAC CGC GCG ATC GGC TTC CTA 1344 Ser He Glu Asp Phe Lys Asn Gly Lys Asp Arg Ala He Gly Phe Leu 435 440 445 GTC GGA CAG ATT ATG AAA GCG TCC AAA GGA CAA GCC AAC CCG CCG ATG 1392 Val Gly Gin He Met Lys Ala Ser Lys Gly Gin Ala Asn Pro Pro Met 450 455 460

GTC AAC AAA ATT CTG CTT GAA GAA ATT AAA AAA CGC TAA 1431

Val Asn Lys He Leu Leu Glu Glu He Lys Lys Arg 465 470 475

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 476 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Leu Asn Phe Glu Thr Val He Gly Leu Glu Val His Val Glu Leu Lys 1 5 10 15

Thr Lys Ser Lys He Phe Ser Ser Ser Pro Thr Pro Phe Gly Ala Glu 20 25 30

Ala Asn Thr Gin Thr Ser Val He Asp Leu Gly Tyr Pro Gly Val Leu 35 40 45

Pro Val Leu Asn Lys Glu Ala Val Glu Phe Ala Met Lys Ala Ala Met 50 55 60

Ala Leu Asn Cys Glu He Ala Thr Asp Thr Lys Phe Asp Arg Lys Asn 65 70 75 80

Tyr Phe Tyr Pro Asp Asn Pro Lys Ala Tyr Gin He Ser Gin Phe Asp 85 90 95

Lys Pro He Gly Glu Asn Gly Trp He Glu He Glu Val Gly Gly Lys 100 105 110

Thr Lys Arg He Gly He Thr Arg Leu His Leu Glu Glu Asp Ala Gly 115 120 125

Lys Leu Thr His Thr Gly Asp Gly Tyr Ser Leu Val Asp Phe Asn Arg 130 135 140

Gin Gly Thr Pro Leu Val Glu Xaa Val Ser Glu Pro Asp He Arg Thr 145 150 155 160

Pro Glu Glu Xaa Tyr Ala Tyr Leu Glu Lys Leu Lys Ser He He Gin 165 170 175

Tyr Thr Gly Val Ser Asp Cys Lys Met Glu Glu Gly Ser Leu Arg Cys 180 185 190

Asp Ala Asn He Ser Leu Arg Pro He Gly Gin Glu Glu Phe Gly Thr 195 200 205

Lys Thr Glu Leu Lys Asn Leu Asn Ser Phe Ala Phe Val Gin Lys Gly 210 215 220 Leu Glu His Glu Glu Lys Arg Gin Glu Gin Val Leu Leu Ser Gly Phe 225 230 235 240

Phe He Gin Gin Glu Thr Arg Arg Tyr Asp Glu Ala Thr Lys Lys Thr 245 250 255

He Leu Met Arg Val Lys Glu Gly Ser Asp Asp Tyr Arg Tyr Phe Pro 260 265 270

Glu Pro Asp Leu Val Glu Leu Tyr He Asp Asp Glu Trp Lys Glu Arg 275 280 285

Val Lys Ala Ser He Pro Glu Leu Pro Asp Glu Arg Arg Lys Arg Tyr 290 295 300

He Glu Glu Leu Gly Phe Ala Ala Tyr Asp Ala Met Val Leu Thr Leu 305 310 315 320

Thr Lys Glu Met Ala Asp Phe Phe Glu Glu Thr Val Gin Lys Gly Ala 325 330 335

Glu Ala Lys Gin Ala Ser Asn Trp Leu Met Gly Glu Val Ser Ala Tyr 340 345 350

Leu Asn Ala Glu Gin Lys Glu Leu Ala Asp Val Ala Leu Thr Pro Glu 355 360 365

Gly Leu Ala Gly Met He Lys Leu He Glu Lys Gly Thr He Ser Ser 370 375 380

Lys He Ala Lys Lys Val Phe Lys Glu Leu He Glu Lys Gly Gly Asp 385 390 395 400

Ala Glu Lys He Val Lys Glu Lys Gly Leu Val Gin He Ser Asp Glu 405 410 415

Gly Val Leu Leu Lys Leu Val Thr Glu Ala Leu Asp Asn Asn Pro Gin 420 425 ^* 430

Ser He Glu Asp Phe Lys Asn Gly Lys Asp Arg Ala He Gly Phe Leu 435 440 445

Val Gly Gin He Met Lys Ala Ser Lys Gly Gin Ala Asn Pro Pro Met 450 455 460

Val Asn Lys He Leu Leu Glu Glu He Lys Lys Arg 465 470 475

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 291 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION: 1..288

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

ATG TCA CGA ATT TCA ATA GAA GAA GTA AAG CAC GTT GCG CAC CTT GCA 48 Met Ser Arg He Ser He Glu Glu Val Lys His Val Ala His Leu Ala 1 5 10 15

AGA CTT GCG ATT ACT GAA GAA GAA GCA AAA ATG TTC ACT GAA CAG CTC 96 Arg Leu Ala He Thr Glu Glu Glu Ala Lys Met Phe Thr Glu Gin Leu 20 25 30

GAC AGT ATC ATT TCA TTT GCC GAG GAG CTT AAT GAG GTT AAC ACA GAC 144 Asp Ser He He Ser Phe Ala Glu Glu Leu Asn Glu Val Asn Thr Asp 35 40 45

AAT GTG GAG CCT ACA ACT CAC GTG CTG AAA ATG AAA AAT GTC ATG AGA 192 Asn Val Glu Pro Thr Thr His Val Leu Lys Met Lys Asn Val Met Arg 50 55 60

GAA GAT GAA GCG GGT AAA GGT CTT CCG GTT GAG GAT GTC ATG AAA AAT 240 Glu Asp Glu Ala Gly Lys Gly Leu Pro Val Glu Asp Val Met Lys Asn 65 70 75 80

GCG CCT GAC CAT AAA GAC GGC TAT ATT CGT GTG CCA TCA ATT CTG GAC 288 Ala Pro Asp His Lys Asp Gly Tyr He Arg Val Pro Ser He Leu Asp 85 90 95

TAA 291

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 96 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Met Ser Arg He Ser He Glu Glu Val Lys His Val Ala His Leu Ala 1 5 10 15

Arg Leu Ala He Thr Glu Glu Glu Ala Lys Met Phe Thr Glu Gin Leu 20 25 30

Asp Ser He He Ser Phe Ala Glu Glu Leu Asn Glu Val Asn Thr Asp 35 40 45

Asn Val Glu Pro Thr Thr His Val Leu Lys Met Lys Asn Val Met Arg 50 55 60

Glu Asp Glu Ala Gly Lys Gly Leu Pro Val Glu Asp Val Met Lys Asn 65 70 75 80

Ala Pro Asp His Lys Asp Gly Tyr He Arg Val Pro Ser He Leu Asp 85 90 95

Claims (1)

1. Claims

   1. An isolated polynucleotide encoding an amidotransferase (AdT) protein.

   2. An isolated polynucleotide comprising a polynucleotide sequence selected

   from the group consisting of:

   (a) a polynucleotide having at least a 70% identity to a polynucleotide

   encoding a polypeptide encoded by SEQ ID NO:l ;

   (b) a polynucleotide which is complementary to the polynucleotide of (a);

   and,

   (c) a polynucleotide comprising at least 15 sequential bases ofthe

   polynucleotide of (a) or (b).

   3. The polynucleotide of claim 2 wherein the polynucleotide is DNA.

   4. The polynucleotide of claim 2 wherein the polynucleotide is RNA.

   5. The polynucleotide of claim 3 comprising the nucleotides 103 to 3306 set

   forth in SEQ ID NO: 1.

   6. A vector comprising the DNA of claim 3.

   7. A host cell comprising the vector of claim 6.

   8. A process for producing a polypeptide comprising expressing from the host

   cell of claim 7 a polypeptide encoded by said DNA.

   9. A process for producing a cell which expresses a polypeptide comprising

   transforming or transfecting the cell with the vector of claim 6 such that the

   cell expresses the polypeptide encoded by the DNA contained in the vector.

   10. A process for producing an amidotransferase polypeptide or amidotransferase

   fragment comprising culturing a host cell of claim 7 under conditions

   sufficient for the production of said polypeptide or fragment.

   11. A polypeptide comprising an amino acid sequence which is at least 70%

   identical to a polypeptide encoded by nucleotides 103 to 3306 set forth in SEQ

   ID NO:l.

   12. An antibody, or antibody fragment containing an antigen binding site, wherein

   said antibody binds to a polypeptide of claim 11.

   13. An antagonist which inhibits the activity of the polypeptide of claim 11.

   14. A method for the treatment of an individual having need of amidotransferase

   comprising administering to the individual a therapeutically effective amount

   of the polypeptide of claim 11.

   15. The method of claim 14 wherein said therapeutically effective amount of the

   polypeptide is administered by providing to the individual DNA encoding said

   polypeptide and expressing said polypeptide in vivo.

   16. A method for the treatment of an individual having need to inhibit

   amidotransferase polypeptide comprising administering to the individual a

   therapeutically effective amount ofthe antagonist of claim 13.

   17. A process for diagnosing a disease related to expression of the polypeptide of

   claim 11 comprising determining a nucleic acid sequence encoding said

   polypeptide.

   18. A diagnostic process comprising analyzing for the presence ofthe polypeptide

   of claim 11 in a sample derived from a host.

   19. A method for identifying compounds which bind to and inhibit an activity of a

   polypeptide of claim 11 comprising:

   (a) incubating a first sample ofthe polypeptide and its substrate; (b) measuring an uninhibited reactivity ofthe polypeptide from step (a);

   (c) incubating a first sample ofthe polypeptide and its substrate in the

   presence of a second sample comprising an inhibitor compound;

   (d) measuring an inhibited reactivity ofthe polypeptide from step (c); and,

   (e) comparing the inhibited reactivity to the uninhibited reactivity of the

   polypeptide.

   20. A method for identifying compounds which bind to and inhibit an activity of a

   polypeptide of claim 11 comprising:

   (a) contacting a cell expressing on the surface thereof a binding site for the

   polypeptide, said binding being associated with a second component

   capable of providing a detectable signal in response to the binding of a

   compound to said binding site, with a compound to be screened under

   conditions to permit binding to the binding site; and

   (b) determining whether the compound binds to and activates or inhibits

   the binding by detecting the presence or absence of a signal generated

   from the interaction ofthe compound with the binding site.

   21. A method for identifying inhibitor-resistant AdT mutants comprising:

   (a) incubating a first sample of wild-type AdT and its substrate in the

   presence of a second sample comprising an AdT inhibitor;

   (b) measuring an unmutated reactivity ofthe AdT from step (a); (c) incubating a first sample of a mutated AdT and its substrate in the

   presence of a second sample comprising an AdT inhibitor;

   (d) measuring a mutated reactivity ofthe mutated AdT from step (c); and,

   (e) comparing the mutated reactivity to the unmutated reactivity ofthe

   wild-type AdT.

   22. A method for inducing an immunological response in a mammal which

   comprises inoculating the mammal with amidotransferase, or a fragment or

   variant thereof, adequate to produce antibody to protect said animal from

   disease.

   23. A method of inducing immunological response in a mammal which comprises,

   through gene therapy, delivering a gene encoding an amidotransferase

   fragment or a variant thereof, for expressing amidotransferase, or a fragment

   or a variant thereof in vivo in order to induce an immunological response to

   produce antibody to protect said animal from disease.

   24. An immunological composition comprising a DNA which codes for and

   expresses an amidotransferase polynucleotide or protein coded therefrom

   which, when introduced into a mammal, induces an immunological response

   in the mammal to a given amidotransferase polynucleotide or protein coded

   therefrom.

   25. An isolated polynucleotide comprising a polynucleotide sequence selected

   from the group consisting of:

   (a) a polynucleotide having at least a 70% identity to a polynucleotide

   encoding a polypeptide comprising SEQ ID NO:4;

   (b) a polynucleotide which is complementary to the polynucleotide of (a);

   and,

   (c) a polynucleotide comprising at least 15 sequential bases ofthe

   polynucleotide of (a) or (b).

   26. A vector comprising the polynucleotide of claim 25.

   27. A host cell comprising the vector of claim 26.

   28. A process for producing a polypeptide comprising expressing from the host

   cell of claim 27 a polypeptide encoded by said polynucleotide.

   29. A polypeptide comprising an amino acid sequence which is at least 70%

   identical to the amino acid set forth in SEQ ID NO:4.

   30. An antibody, or antibody fragment containing an antigen binding site, wherein

   said antibody binds to a polypeptide of claim 29.

   31. An antagonist which inhibits the activity of the polypeptide of claim 29.

   32. An isolated polynucleotide comprising a polynucleotide sequence selected

   from the group consisting of:

   (a) a polynucleotide having at least a 70% identity to a polynucleotide

   encoding a polypeptide comprising SEQ ID NO:6;

   (b) a polynucleotide which is complementary to the polynucleotide of (a);

   and,

   (c) a polynucleotide comprising at least 15 sequential bases ofthe

   polynucleotide of (a) or (b).

   33. A vector comprising the polynucleotide of claim 32.

   34. A host cell comprising the vector of claim 33.

   35. A process for producing a polypeptide comprising expressing from the host

   cell of claim 34 a polypeptide encoded by said polynucleotide.

   36. A polypeptide comprising an amino acid sequence which is at least 70%

   identical to the amino acid set forth in SEQ ID NO:6.

   37. An antibody, or antibody fragment containing an antigen binding site, wherein

   said antibody binds to a polypeptide of claim 36.

   38. An antagonist which inhibits the activity ofthe polypeptide of claim 36.

   39. An isolated polynucleotide comprising a polynucleotide sequence selected

   from the group consisting of:

   (a) a polynucleotide having at least a 70% identity to a polynucleotide

   encoding a polypeptide comprising SEQ ID NO:8;

   (b) a polynucleotide which is complementary to the polynucleotide of (a);

   and,

   (c) a polynucleotide comprising at least 15 sequential bases ofthe

   polynucleotide of (a) or (b).

   40. A vector comprising the polynucleotide of claim 39.

   41. A host cell comprising the vector of claim 40.

   42. A process for producing a polypeptide comprising expressing from the host

   cell of claim 41 a polypeptide encoded by said polynucleotide.

   43. A polypeptide comprising an amino acid sequence which is at least 70%

   identical to the amino acid set forth in SEQ ID NO:8.

   44. An antibody, or antibody fragment containing an antigen binding site, wherein

   said antibody binds to a polypeptide of claim 43.

   45. An antagonist which inhibits the activity of the polypeptide of claim 43.

   46. An isolated heterotrimeric protein comprising subunits A, B, and C, wherein:

   said subunit A has an amino acid sequence selected from the group consisting

   of SEQ ID NO:4, an allelic variant of SEQ ID NO:4, a conservative substitution

   variant of SEQ ID NO:4, and an amino acid sequence that is encoded by a nucleic

   acid molecule that hybridizes under stringent conditions to a nucleic acid molecule

   encoding SEQ ID NO:4 and encodes a subunit of a heterotrimeric amidotransferase;

   said subunit B has an amino acid sequence selected from the group consisting

   of SEQ ID NO:6, an allelic variant of SEQ ID NO:6, a conservative substitution

   variant of SEQ ID NO:6, and an amino acid sequence that is encoded by a nucleic

   acid molecule that hybridizes under stringent conditions to a nucleic acid molecule

   encoding SEQ ID NO:6 and encodes a subunit of a heterotrimeric amidotransferase;

   said subunit C has an amino acid sequence selected from the group consisting

   of SEQ ID NO:8, an allelic variant of SEQ ID NO:8, a conservative substitution

   variant of SEQ ID NO:8, and an amino acid sequence that is encoded by a nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid molecule

   encoding SEQ ID NO:8 and encodes a subunit of a heterotrimeric amidotransferase.

   47. An antibody, or antibody fragment containing an antigen binding site, wherein

   said antibody binds to a protein of claim 46.

   48. An isolated nucleic acid molecule that encodes a protein of claim 46.

   49. A recombinant host that has been altered to contain a nucleic acid molecule of

   claim 48.

   50. A method for producing an AdT protein comprising the step of culturing the

   host of claim 49 under conditions in which said introduced nucleic acid molecule is

   expressed.

   51. A method to identify an agent that blocks translation, said method comprising

   the steps of:

   (a) contacting an agent with an AdT protein, or a subunit thereof; and,

   (b) determining whether said agent binds to said AdT protein or said

   subunit;

   wherein said translation blocking agent is identified as being able to bind to said AdT

   protein, or said subunit.

   52. The method of claim 51 , wherein said AdT protein comprises a heterotrimeric

   protein consisting of an A, B and C subunit, wherein:

   said subunit A has an amino acid sequence selected from the group consisting

   of SEQ ID NO:4, an allelic variant of SEQ ID NO:4, a conservative substitution

   variant of SEQ ID NO:4, and an amino acid sequence that is encoded by a nucleic

   acid molecule that hybridizes under stringent conditions to a nucleic acid molecule

   encoding SEQ ID NO:4 and encodes a subunit of a heterotrimeric amidotransferase;

   said subunit B has an amino acid sequence selected from the group consisting

   of SEQ ID NO:6, an allelic variant of SEQ ID NO:6, a conservative substitution

   variant of SEQ ID NO: 6, and an amino acid sequence that is encoded by a nucleic

   acid molecule that hybridizes under stringent conditions to a nucleic acid molecule

   encoding SEQ ID NO:6 and encodes a subunit of a heterotrimeric amidotransferase;

   said subunit C has an amino acid sequence selected from the group consisting

   of SEQ ID NO:8, an allelic variant of SEQ ID NO:8, a conservative substitution

   variant of SEQ ID NO:8, and an amino acid sequence that is encoded by a nucleic

   acid molecule that hybridizes under stringent conditions to a nucleic acid molecule

   encoding SEQ ID NO:8 and encodes a subunit of a heterotrimeric amidotransferase.

   53. The method of claim 52, wherein a single subunit of said AdT protein is used,

   and:

   if subunit A is used, said subunit A has an amino acid sequence selected from

   the group consisting of SEQ ID NO:4, an allelic variant of SEQ ID NO:4, a conservative substitution variant of SEQ ID NO:4, and an amino acid sequence that is

   encoded by a nucleic acid molecule that hybridizes under stringent conditions to a

   nucleic acid molecule encoding SEQ ID NO: 4 and encodes a subunit of a

   heterotrimeric amidotransferase;

   if subunit B is used, said subunit B has an amino acid sequence selected from

   the group consisting of SEQ ID NO:6, an allelic variant of SEQ ID NO:6, a

   conservative substitution variant of SEQ ID NO:6, and an amino acid sequence that is

   encoded by a nucleic acid molecule that hybridizes under stringent conditions to a

   nucleic acid molecule encoding SEQ ID NO:6 and encodes a subunit of a

   heterotrimeric amidotransferase;

   if subunit C is used, said subunit C has an amino acid sequence selected from

   the group consisting of SEQ ID NO:8, an allelic variant of SEQ ID NO:8, a

   conservative substitution variant of SEQ ID NO: 8, and an amino acid sequence that is

   encoded by a nucleic acid molecule that hybridizes under stringent conditions to a

   nucleic acid molecule encoding SEQ ID NO: 8 and encodes a subunit of a

   heterotrimeric amidotransferase.

   54. The method of claim 51 , wherein said agent is further tested for the ability to

   block the activity of said AdT protein.

   55. The method of claim 54, wherein said AdT activity is tested in a cell free

   system.

   56. The method of claim 54, wherein said AdT activity is tested in a cellular

   system.

   57. A method to identify an agent that blocks translation, said method comprising

   the steps of:

   (a) contacting an agent with one or more ofthe subunits of an AdT

   protein;

   (b) incubating the three subunits of an AdT protein under conditions in

   which said subunits would associate to form an active AdT protein, wherein at least

   one of said subunits is from step (a);

   (c) determining whether said agent blocks the association of said three

   subunits;

   wherein said translation blocking agent is identified as being able to block the

   association ofthe subunits of said AdT protein.

   58. The method of claim 57, wherein said AdT protein comprises a heterotrimeric

   protein consisting of an A, B and C subunit, wherein:

   said subunit A has an amino acid sequence selected from the group consisting

   of SEQ ID NO:4, an allelic variant of SEQ ID NO:4, a conservative substitution

   variant of SEQ ID NO:4, and an amino acid sequence that is encoded by a nucleic

   acid molecule that hybridizes under stringent conditions to a nucleic acid molecule

   encoding SEQ ID NO:4 and encodes a subunit of a heterotrimeric amidotransferase; said subunit B has an amino acid sequence selected from the group consisting

   of SEQ ID NO:6, an allelic variant of SEQ ID NO:6, a conservative substitution

   variant of SEQ ID NO:6, and an amino acid sequence that is encoded by a nucleic

   acid molecule that hybridizes under stringent conditions to a nucleic acid molecule

   encoding SEQ ID NO: 6 and encodes a subunit of a heterotrimeric amidotransferase;

   said subunit C has an amino acid sequence selected from the group consisting

   of SEQ ID NO:8, an allelic variant of SEQ ID NO:8, a conservative substitution

   variant of SEQ ID NO:8, and an amino acid sequence that is encoded by a nucleic

   acid molecule that hybridizes under stringent conditions to a nucleic acid molecule

   encoding SEQ ID NO: 8 and encodes a subunit of a heterotrimeric amidotransferase.

   59. The method of claim 57, wherein said agent is further tested for the ability to

   block the activity of said AdT protein.

   60. The method of claim 59, wherein said AdT protein activity is tested in a cell

   free system.

   61. The method of claim 59, wherein said AdT protein activity is tested in a

   cellular system.

   62. A method to identify an agent that blocks translation, said method comprising

   the steps of: (a) contacting an agent with an AdT protein;

   (b) determining whether said agent blocks the activity of said AdT protein.

   63. The method of claim 62, wherein said AdT protein comprises a heterotrimeric

   protein consisting of an A, B and C subunit, wherein:

   said subunit A has an amino acid sequence selected from the group consisting

   of SEQ ID NO:4, an allelic variant of SEQ ID NO:4, a conservative substitution

   variant of SEQ ID NO:4, and an amino acid sequence that is encoded by a nucleic

   acid molecule that hybridizes under stringent conditions to a nucleic acid molecule

   encoding SEQ ID NO:4 and encodes a subunit of a heterotrimeric amidotransferase;

   said subunit B has an amino acid sequence selected from the group consisting

   of SEQ ID NO:6, an allelic variant of SEQ ID NO:6, a conservative substitution

   variant of SEQ ID NO:6, and an amino acid sequence that is encoded by a nucleic

   acid molecule that hybridizes under stringent conditions to a nucleic acid molecule

   encoding SEQ ID NO:6 and encodes a subunit of a heterotrimeric amidotransferase;

   said subunit C has an amino acid sequence selected from the group consisting

   of SEQ ID NO:8, an allelic variant of SEQ ID NO:8, a conservative substitution

   variant of SEQ ID NO:8, and an amino acid sequence that is encoded by a nucleic

   acid molecule that hybridizes under stringent conditions to a nucleic acid molecule

   encoding SEQ ID NO: 8 and encodes a subunit of a heterotrimeric amidotransferase.

   64. The method of claim 63, wherein said AdT protein activity is tested in a cell

   free system.

   65. The method of claim 63, wherein said AdT protein activity is tested in a

   cellular system.

   66. A method to block translation of a protein within a cell, comprising the step of

   contacting said cell with an amount of an agent that binds to an AdT protein, or a

   subunit thereof, sufficient to block said translation.

   67. The method of claim 66, wherein said agent binds to a subunit of said AdT

   and blocks the association of said subunits.

   68. The method of claim 66, wherein said agent is used as an antibacterial agent.

   69. The method of claim 66, wherein said agent is used as an antifungal agent.

   70. The method of claim 66, wherein said agent is used as a herbicide.

   71. An isolated polynucleotide that codes for a mutant AdT which confers

   resistance to an inhibitor of wild-type AdT.

   72. A vector comprising the polynucleotide of claim 71.

   73. A host cell comprising the vector of claim 72.

   74. The host cell of claim 73 wherein the host cell comprises a plant cell.

   75. A process for producing a polypeptide comprising expressing from the host

   cell of claim 73 a polypeptide encoded by said polynucleotide.

   76. A process for producing a cell which expresses a polypeptide comprising

   transforming or transfecting the cell with the vector of claim 73 such that the cell

   expresses the polypeptide encoded by the polynucleotide contained in the vector.

   77. A process for producing a plant which comprises a gene for resistance to an

   AdT inhibitor, said process comprising regenerating a plant from the plant cell of

   claim 74.

   78. A process of plant husbandry comprising:

   (a) planting a plant which comprises a gene for resistance to an AdT

   inhibitor;

   (b) applying a herbicide which comprises an AdT inhibitor.