Receptor kinase, BIN1 Number:6,765,085 from the United States Patent and Trademark Office (PTO) owispatent A novel plant steroid receptor, BIN1, is provided, as well as polynucleotides encoding BIN1. The function of BIN1, an extracellular steroid-binding receptor, is provided as well as its primary binding affinity with brassinolide. BIN1 polypeptide is useful in promoting increased plant yield and/or increased plant biomass.<H Receptor, kinase, Receptor, kinase, 6765085.html, Patent, pto, 6765085

Title: Receptor kinase, BIN1

Abstract: A novel plant steroid receptor, BIN1, is provided, as well as polynucleotides encoding BIN1. The function of BIN1, an extracellular steroid-binding receptor, is provided as well as its primary binding affinity with brassinolide. BIN1 polypeptide is useful in promoting increased plant yield and/or increased plant biomass.

Patent Number: 6,765,085 Issued on 07/20/2004 to Chory, et al.

Inventors: Chory; Joanne (Del Mar, CA), Li; Jianming (Ann Arbor, MI)
Assignee: The Salk Institute for Biological Studies (La Jolla, CA)

Appl. No.: 09/823,394

Filed: March 30, 2001

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
881706	Jun., 1997	6245969

Current U.S. Class: 530/350 ; 530/300

Current International Class: C07K 14/415 (20060101); C12N 15/82 (20060101); G01N 33/74 (20060101); A61K 38/00 (20060101)

Field of Search: 530/350,300

References Cited [Referenced By]

U.S. Patent Documents


6245969	June 2001	Chory et al.

Foreign Patent Documents


WO 95/18230	Jul., 1995	WO
WO 96/34627	Nov., 1996	WO

Other References

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Clouse, et al., A Brassinosteroid-Insensitive Mutant in Arabidopsis thaliana Exhibits Multiple Defects in Growth and Development, Plant Physiol 111:671-678 (1996). .
Li et al., Conservation of function between mammalian and plant steroid 5.alpha.-reductases, Proc. Natl. Acad. Sci. USA 94:3554-3559 (1997). .
Song et al., A Receptor Kinase-Like Protein Encoded by the Rice Disease Resistance Gene Xa21, Science 270:1804-1806 (1995). .
Li et al. (1997) A putative leucine-Rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell. 90:929-938. .
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PC Morris et al. (1995) GenBank Accession #F13577. .
TE Weier et al. (1982) Botany. 315-319. .
Asami et al. (2000) Characterization of brassinazole, a triazole-type brassinosteroid biosynthesis inhibitor. Plant Plysiology. 123:93-99. .
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Primary Examiner: Kemmerer; Elizabeth
Attorney, Agent or Firm: Knobbe, Martens, Olson & Bear LLP

Government Interests

This invention was made with Government support under Grant No. DIR 9116923 awarded by the National Science Foundation and Grant No. 93-373019125 awarded by the U.S. Department of Agriculture. The Government has certain rights in this invention.

Parent Case Text

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent application Ser. No. 08/881,706, filed Jun. 24, 1997, now U.S. Pat. No. 6,245,969, which is hereby incorporated by reference in its entirety.

Claims

What is claimed is:

1. A substantially purified Brassinosteroid 1 plasma membrane receptor (BIN1) polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO: 2, wherein said fragment binds to brassinosteroids.

2. The fragment of claim 1, wherein said fragment has an amino acid sequence corresponding to about amino acid residues 588 to 649 of SEQ ID NO: 2.

3. Substantially purified Brassinosteroid 1 plasma membrane receptor (BIN1) polypeptide having the amino acid sequence of SEQ ID NO: 2, or a variant thereof, wherein a single ammo acid is replaced by another, and wherein said polypeptide has receptor kinase activity and is a receptor for brassinosteroids.

4. The BIN1 polypeptide of claim 3, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2.

5. The polypeptide of claim 3, wherein said polypeptide has a molecular weight of approximately 130 kD, as determined by SDS-PAGE.

6. The BIN1 polypeptide of claim 3, wherein said receptor kinase activity is activated by brassinolide.

7. The BIN1 polypeptide of claim 3, wherein said polypeptide has a brassinosteroid binding affinity of approximately K.sub.d =7.4+0.9 nM to 10.8+3.2 nM.

8. The BIN1 polypeptide of claim 3, wherein the Alanine at position 1031 is replaced by Threonine.

9. The BIN1 polypeptide of claim 3, wherein the Threonine at position 750 is replaced by an Isoleucine.

10. The BIN1 polypeptide of claim 3, wherein said variant is a conservative variant.

Description

FIELD OF THE INVENTION

The present invention relates generally to plant genetic engineering, and specifically to both a novel gene whose polypeptide functions as a receptor of brassinolide and is useful for producing genetically engineered plants characterized as having a phenotype of increased crop yield, enhanced disease resistance and longer-lived vegetative growth phase and to the receptor function of Brassinosteroid 1 (BIN1) plasma membrane receptor.

BACKGROUND OF THE INVENTION

The brassinosteroids are a unique class of biologically active natural products that possess plant steroidal hormone activity. Their low effective concentrations for use on crops make them environmentally safe and those brassinosteroids used on a large scale are generally non-toxic. At the physiological level, brassinosteroids elicit many changes and could represent a new class of hormones in plants. The economic aspects of the brassinosteroids may have worldwide effects. For example, the brassinosteroids can be used as plant protectants from both pesticide and environmental adversity. In addition, brassinosteroids appear to be useful for insect control. Further, brassinosteroids may regulate some stage of the reproductive cycle in plants, thereby providing the means to increase or decrease the reproductive process. For example, in certain horticultural crops, it may be desirable to eliminate the flowering process to ensure continuous production of other tissues such as leaves, bulbs and other storage organs. This modulation of the reproductive process could be important in the control of certain seed bearing weeds, where cessation of the flowering cycle eliminates future generations. Brassinosteroids also appear to stimulate root growth, and external application causes no deformity of plants.

Brassinosteroids qualify for classification as biochemical pesticides. Such pesticides are generally distinguished from conventional chemical pesticides by their unique modes of action, low effective concentration, target species, and specificity. Historically, the brassinosteroids have not been used in actual agricultural applications due to the expense involved in producing them as well as the difficulty in purifying them.

It is known that once hormones, such as glucocorticoid, enter a cell, they bind to specific receptor proteins, thereby creating a ligand/receptor complex. The binding of the hormone to the receptor is believed to initiate an allosteric alteration of the receptor protein. As a result, it is believed that the ligand/receptor complex is capable of binding with high affinity to certain specific sites on the chromatin nucleic acid. Such sites, which are known as response elements, modulate expression of nearby target gene promoters.

Recent evidence indicates that in addition to intracellular, genomic effects, steroids also exhibit non-genomic effects, ie., they affect the surface of cells and alter ion permeability, as well as release of neurohormones and neurotransmitters. Steroids such as estrogens and adrenal steroids and their naturally produced and synthetic analogs have shown membrane effects. In view of the foregoing, it appears that steroids may cause synergistic interactions between non-genomic and genomic responses resulting in alterations in neural activity or certain aspects of oocyte and spermatozoa maturation, for example.

Most multicellular organisms use steroids as signalling molecules for physiological and developmental regulation. Two different modes of steroid actions have been described in animal systems: the well-studied gene regulation response mediated by nuclear receptors, and the rapid non-genomic responses mediated by proposed membrane-bound receptors. See Beato, M., Herrlich, P. & Schutz, G. Steroid hormone receptors: many actors in search of a plot. Cell 83, 851-7 (1995); Mangelsdorf, D. J. et al. The nuclear receptor superfamily: The second decade. Cell 83, 835-839 (1995); Wehling, M. Specific, nongenomic actions of steroid hormones. Annu. Rev. Physiol. 59, 365-393 (1997); and Schmidt, B. M., Gerdes, D., Feuring, M., Falkenstein, E., Christ, M., Wehling, M. Rapid, nongenomic steroid actions: A new age? Front Neuroendocrinol 21, 57-94 (2000). Plant genomes do not appear to encode members of the nuclear receptor superfamily. For these reasons, it would be important to identify any new brassinosteriod receptors.

SUMMARY OF THE INVENTION

Although steroid hormones are important for animal development, the physiological role of plant steroids is largely unknown. The present invention is based on the discovery of the BIN1 gene, which encodes a polypeptide that functions as a receptor kinase which binds with brassinosteroids.

In one embodiment, the invention provides a trans-membrane receptor kinase, BIN1 polypeptide, comprising a plant steroid receptor of brassinsteroids. This BIN1 polypeptide has an active binding region having a 70 amino acid island region as an extracellular domain receptor. The 70 amino acid island region is required for brassinosteriod binding to the receptor on the cell membrane.

In another embodiment, the invention provides a BIN1 polypeptide wherein the polypeptide function in the brassinolide response pathway; has brassinolide-binding activity; an extracellular location of its functional binding site; brassinolide-binding activity which co-immunoprecipitates with BIN1; and trans-membrane receptor kinase activity that transduces steroid signals across the plasma membrane.

In yet another embodiment, the invention provides a BIN1 receptor kinase that has binding affinity of approximately K.sub.d =7.4.+-.0.9 nM to 10.8.+-.3.2 nM depending on the number of BIN1 binding sites B.sub.MAX =2.66 pmole mg.sup.-1 membrane protein. The BIN1 immunoprecipitated binding activity has a similar disassociation constant (K.sub.d =15.2.+-.5 nM) as determined for membrane fractions.

Still another embodiment of the invention provides a BIN1 mutant with missense mutations in the kinase domain (BIN1-104, A1031T) or in a region of the extracellular domain near the transmembrane domain (BIN1-102, T750-I) wherein the brassinolide binding activity is similar to the wild type and the biosynthetic mutant det2. Another BIN1 mutant with missense mutations (BIN1-6, G644-D) and a mutation causes a premature translation step (BIN1-116, Q583-stop), both in the 7 amino acid island region, wherein the brassinolide binding activity is greatly reduced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the nucleotide (A,B) and deduced amino acid (C) sequences of BIN1 of the invention (SEQ ID NO: I and SEQ ID NO:2, respectively).

FIGS. 2A-F shows that overexpression of a BIN1-GFP fusion protein increases cell elongation and increases the number of BL binding sites in membrane fractions. a. Proteins from wild-type and BIN1-GFP transgenic plants probed with anti-BIN1N antibody after western blotting. N, a cleaved fragment of BIN1's extracellular domain. Asterisks, non-specific bands. b. Wild-type and BIN1-GFP plants grown on 2 .mu.M brassinazole in the dark for 6 days. c. A wild-type plant, a BIN1-GFP transgenic plant, and a mutant plant overexpressing the DWF4 gene (35SE-DWF4) grown in 0 hr light, 15 hr dark cycles for 45 days. d. Specific [.sup.3 H]-BL binding to microsomal fractions of wild-type (WT) and BIN1-GFP plants was determined by subtracting the binding in the presence of 100-fold unlabeled BL from the total biding in the absence of cold competitor. Representative Data of one of three repeat experiments are shown. e. Scatchard plot of the biding data in d. The K.sub.d values were calculated from data of three experiments, with correlation coefficient R.sup.2 =0.998 for BIN1-GFP and 0.983 for wild type samples. f. Competition for [.sup.3 H]BL binding to membrane fractions of BIN1-GFP plants by brassinolide, castasterone, ecdysone, and 2,3,22-23-)-tetramethyl BL. Structures of the competitors are shown.

FIG. 3 shows that BIN1 binds to BL. a. Proteins immunoprecipitated with anti-GFP antibodies from extracts of wild-type of BIN1-GFP plants were assayed for [.sup.3 H]BL binding activity I the absence (open bars) or presence (filled bars) of 5 .mu.M unlabeled BL. No protein, binding assays with protein A beads as control. b. Specific and saturation [.sup.3 H]BL binding to immunoprecipitated BIN1-GFP protein. c. Mutations in the extracellular domain of BIN1, but not the kinase domain, reduce BL binding. Specific [.sup.3 H]BL binding to microsomal fractions of wild type (WT), det2, BIN1-6, BIN1-116, BIN1-104, and BIN1-102 mutant plants. Data were normalized to the relative BIN1 protein levels determined by quantitative western blotting (d), except for BIN1-116. d. Protein immunoblot showing the BIN1 protein levels in the membrane fractions used in the binding assays of c. Varying loading of the wild-type sample was used to generate a standard curve, which was used to determine the relative level of BIN1 in the BIN1 mutant samples (6 .mu.l/lane).

FIG. 4 shows that BL induces phosphorylation of BIN1 in plants. a. Five-day old wild-type seedlings grown in the dark on medium containing 1 .mu.M brassinazole were untreated (-), treated with 1 .mu.M BL in water (BL), or with water only (H.sub.2 O) for 1 hour. Proteins were analyzed by 4% SDS-PAGE, blotted, and probed with anti-BIN1N antibody. b. Proteins of the BL treated wild-type sample were treated with alkaline phosphatase (CIP) or protein phosphatase 1 (PP1), in the presence (+) or absence (-) of phosphatase inhibitors (PI), and analyzed by western blotting as in a. c. Mutations in either the 70-aa island or the kinase domain abolish BL-activation of BIN1 phosphorylation. The det2, BIN1-6, and BIN1-117 mutant seedlings were treated and analyzed as in a. N, cleavage product of BIN1's extracellular domain.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a novel steroid receptor kinase, BIN1, which is involved in the pathway for synthesis of the plant steroid hormone, brassinolide. Overexpression of BIN1 in transgenic plants provides plants characterized as having enhanced disease resistance, increased plant yield or vegetative biomass, and increased seed yield. As used herein, the term "yield" or "increased plant yield" refers to increased plant biomass or seed yield relative to wild-type biomass.

BIN1 Polypeptides and Polynucleotides

In a first embodiment, the present invention provides substantially pure BIN1 polypeptide. BIN1 polypeptide is exemplified by the amino acid sequence shown in FIGURE I and SEQ ID NO:2. BIN1 polypeptide is characterized as having a predicted molecular weight of 130 kDa as determined by SDS-PAGE, and functioning in the brassinolide response pathway.

The term "substantially pure" as used herein refers to BIN1 polypeptide which is substantially free of other proteins, lipids, carbohydrates, or other materials with which it is naturally associated. One skilled in the art can purify BIN1 using standard techniques for protein purification. The substantially pure polypeptide will yield a single major band of about 130 kDa on a denaturing polyacrylamide gel. The purity of the BIN1 polypeptide can also be determined by amino-terminal amino acid sequence analysis.

The invention includes functional BIN1 polypeptide as well as functional fragments thereof. As used herein, the term "functional polypeptide" refers to a polypeptide which possesses biological function or activity which is identified through a defined functional assay and which is associated with a particular biologic, morphologic, or phenotypic alteration in the cell. The term "functional fragments of BIN1 polypeptide", refers to all fragments of BIN1 that retain Bin1 activity, e.g., receptor protein kinase activity or the ability to bind brassinosteroids. Biologically functional fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell. An example of a functional fragment of BIN1 is a polypeptide including from about amino acid residue 588 to 649 of SEQ ID NO:2. This fragment includes the brassinosteroid binding domain of BIN1 polypeptide. Another functional fragment of BIN1 is a polypeptide including from about amino acid residue 831 to 1196 of SEQ ID NO:2. This fragment includes the protein kinase domain of BIN1 polypeptide.

The receptor protein kinase activity of BIN1 and the role of BIN1 in the brassinolide response pathway can be utilized in bioassays to identify biologically active fragments of BIN1 polypeptide or related polypeptides. For example, BIN1 may not only bind brassinosteroids, but other hormones as well, therefore an assay can be performed to detect BIN1 binding activity. In addition, inhibitors of BIN1 can be used to cause loss of BIN1 function resulting in, for example, male sterile plants, reduced stature, reduced yield, etc. Moreover, inhibition of BIN1 may be useful in horticulture for creating dwarf varieties.

Minor modifications of the BIN1 primary amino acid sequence may result in proteins which have substantially equivalent activity to the BIN1 polypeptide described herein in SEQ ID NO:2 (FIG. 1). Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by such BIN1 modifications are included herein as long as the peptide possesses BIN1 biological activity (i.e., receptor protein kinase activity). Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its activity. Deletion can lead to the development of a smaller active molecule which could have broader utility. For example, it may be possible to remove amino or carboxy terminal amino acids required for BIN1 activity.

For example, a less active form of BIN1 has an amino acid change at residue 611 from glycine to glutamic acid. This mutant form has reduced affinity for the steroid. Other mutants can be produced which activate enzymatic activity. For example, a mutant can be produced such that the kinase domain is expressed, thereby allowing constitutive kinase activity.

BIN1 polypeptide includes amino acid sequences substantially the same as the sequence set forth in SEQ ID NO:2. The term "substantially the same" refers to amino acid sequences that retain the activity of BIN1 as described herein, e.g., receptor protein kinase activity. The BIN1 polypeptides of the invention include conservative variations of the polypeptide sequence. The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine, or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.

In another aspect, the invention provides isolated polynucleotides encoding BIN1 polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The BIN1 gene has been mapped to a 5-kb interval on Arabidopsis chromosome 4. The BIN1 transcript contains a single, long open reading frame that encodes 1196 amino acid protein. The term "isolated" as used herein includes polynucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which it is naturally associated. Polynucleotide sequences of the invention include nucleic acid, cDNA and RNA sequences which encode BIN1. It is understood that polynucleotides encoding all or varying portions of BIN1 are included herein, as long as they encode a polypeptide with BIN1 activity. Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides as well as splice variants. For example, portions of the mRNA sequence may be altered due to alternate RNA splicing patterns or the use of alternate promoters for RNA transcription. Moreover, BIN1 polynucleotides of the invention include polynucleotides having alterations in the nucleic acid sequence which still encode functional BIN1. Alterations in BIN1 nucleic acid include but are not limited to intragenic mutations (e.g., point mutation, nonsense (stop), antisense, splice site, and frameshift) and heterozygous or homozygous deletions. Detection of such alterations can be done by standard methods known to those of skill in the art including sequence analysis, Southern blot analysis, PCR based analyses (e.g., multiplex PCR, sequence tagged sites (STSs)) and in situ hybridization. Invention polynucleotide sequences also include antisense sequences. The polynucleotides of the invention include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of BIN1 polypeptide encoded by such nucleotide sequences retains BIN1 receptor protein kinase activity. A "functional polynucleotide" denotes a polynucleotide which encodes a functional polypeptide as described herein. In addition, the invention also includes a polynucleotide encoding a polypeptide having the biological activity of the amino acid sequence set forth in SEQ ID NO:2 and having at least one epitope for an antibody immunoreactive with BIN1 polypeptide.

As used herein, the terms polynucleotides and nucleic acid sequences of the invention refer to nucleic acid, RNA, and cDNA sequences.

Polynucleotides encoding BIN1 include the nucleotide sequence set forth in FIGURE I (SEQ ID NO: 1), as well as nucleic acid sequences complementary to that sequence. Complementary sequences may include antisense polynucleotides. When the sequence is RNA, the deoxyribonucleotides A, G, C, and T of FIG. 1C are replaced by ribonucleotides A, G, C, and U, respectively. Also included in the invention are fragments ("probes") of the above-described nucleic acid sequences that are at least 15 bases in length, which is sufficient to permit the probe to selectively hybridize to nucleic acid that encodes the amino acid sequence set forth in FIGURE I (SEQ ID NO: 2). "Selective hybridization" as used herein refers to hybridization under moderately stringent or highly stringent physiological conditions (See, for example, the techniques described in Maniatis et al., 1989 Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., incorporated herein by reference), which distinguishes related from unrelated BIN1 nucleotide sequences.

Specifically disclosed herein is a cDNA sequence for BIN1. Figure I shows the complete cDNA and deduced protein sequences (SEQ ID NOs: 1 and 2, respectively). It is understood that homologs of the plant BIN1 are included herein and can be identified, for example, by using plant BIN1 nucleic acid probes based on SEQ ID NO: 1.

In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. nucleic acid) of the hybridizing regions can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.

An example of progressively higher stringency conditions is as follows: 2.times.SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2.times.SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2.times.SSC/0.1% SDS at about 42.degree. C. (moderate stringency conditions); and 0.1.times.SSC at about 68.degree. C. (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. Optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.

Nucleic acid sequences of the invention can be obtained by several methods. For example, the nucleic acid can be isolated using hybridization or computer-based techniques which are well known in the art. Such techniques include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences; 2) antibody screening of expression libraries to detect cloned nucleic acid fragments with shared structural features; 3) polymerase chain reaction (PCR) on genomic nucleic acid or cDNA using primers capable of annealing to the nucleic acid sequence of interest; 4) computer searches of sequence databases for similar sequences; and 5) differential screening of a subtracted nucleic acid library.

Screening procedures which rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available. Oligonucleotide probes, which correspond to a part of the BIN1 sequence encoding the protein in question, can be synthesized chemically. This requires that short, oligopeptide stretches of the amino acid sequence must be known. The nucleic acid sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. It is possible to perform a mixed addition reaction when the sequence is degenerate. This includes a heterogeneous mixture of denatured double-stranded nucleic acid. For such screening, hybridization is preferably performed on either single-stranded nucleic acid or denatured double-stranded nucleic acid. Hybridization is particularly useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present. In other words, by using stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA clone by the hybridization of the target nucleic acid to a single probe in the mixture which is its complete complement (Wallace, et al., Nucl. Acid Res., 9:879, 1981). Alternatively, a subtractive library, as illustrated herein is useful for elimination of non-specific cDNA clones.

When the amino acid sequence is not known, the direct synthesis of nucleic acid sequences is not possible and the method of choice is the synthesis of cDNA sequences. Among the standard procedures for isolating cDNA sequences of interest is the formation of plasmid- or phage-carrying cDNA libraries which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned. In cases where significant portions of the amino acid sequence of a polypeptide are known, the production of labeled single or double-stranded nucleic acid or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in nucleic acid/nuclei acid hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single-stranded form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).

A cDNA expression library, such as lambda gt11, can be screened indirectly for BIN1 peptides using antibodies specific for BIN1. Such antibodies can be either polyclonal or monoclonal and used to detect expression product indicative of the presence of BIN1 cDNA.

Detection of alterations in BIN1 nucleic acid (e.g., point mutation, nonsense (stop), missense, splice site, and frameshift) and heterozygous or homozygous deletions can be effected by standard methods known to those of skill in the art including sequence analysis, Southern blot analysis, PCR based analyses (e.g., multiplex PCR, sequence tagged sites (STSs)) and in situ hybridization. Such proteins can be analyzed by standard SDS-PAGE and/or immunoprecipitation analysis and/or Western blot analysis, for example.

Nucleic acid sequences encoding BIN1 can be expressed in vitro by nucleic acid transfer into a suitable host cell. "Host cells" are cells in which a vector can be propagated and its nucleic acid expressed. The term "host cells" also includes any progeny or graft material, for example, of the parent host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used. Methods of stable transfer, meaning that the foreign nucleic acid is continuously maintained in the host, are known in the art.

In the present invention, the BIN1 polynucleotide sequences may be inserted into a recombinant expression vector. The terms "recombinant expression vector" or "expression vector" refer to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the BIN1 genetic sequence. Such expression vectors contain a promoter sequence which facilitates the efficient transcription of the inserted BIN1 sequence. The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.

Methods which are well known to those skilled in the art can be used to construct expression vectors containing the BIN1 coding sequence and appropriate transcriptional/translational control signals. Such methods include in vitro recombinant nucleic acid techniques, synthetic techniques, and in vivo recombination/genetic techniques. (See, for example, the techniques described in Maniatis et al., 1989 Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.)

A variety of host-expression vector systems may be utilized to express the BIN1 coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage nucleic acid, plasmid nucleic acid, or cosmid nucleic acid expression vectors containing the BIN1 coding sequence; yeast transformed with recombinant yeast expression vectors containing the BIN1 coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the BIN1 coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the BIN1 coding sequence; or animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccinia virus) containing the BIN1 coding sequence, or transformed animal cell systems engineered for stable expression.

Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al., 1987, Methods in Enzymology 153:516-544). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage .gamma., plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. When cloning in mammalian systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., the retroviral long terminal repeat; adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant nucleic acid or synthetic techniques may also be used to provide for transcription of the inserted BIN1 coding sequence.

In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology of the Yeast Saccharomyces, 1982, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II. A constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol. 11, A Practical Approach, Ed. D M Glover, 1986, IRL Press, Wash., D.C.). Alternatively, vectors may be used which promote integration of foreign nucleic acid sequences into the yeast chromosome.

Eukaryotic systems, and preferably mammalian expression systems, allow for proper post-translational modifications of expressed mammalian proteins to occur. Eukaryotic cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation, and advantageously, plasma membrane insertion of the gene product may be used as host cells for the expression of BIN1.

Mammalian expression systems which utilize recombinant viruses or viral elements to direct expression may be engineered. For example, when using adenovirus expression vectors, the BIN1 coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. Alternatively, the vaccinia virus 7.5K promoter may be used. (e.g., see, Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79: 7415-7419; Mackett et al., 1984, J. Virol. 49: 857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. USA 79: 4927-4931). Of particular interest are vectors based on bovine papilloma virus which have the ability to replicate as extrachromosomal elements (Sarver, et al., 1981, Mol. Cell. Biol. 1: 486). Shortly after entry of this nucleic acid into mouse cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression. These vectors can be used for stable expression by including a selectable marker in the plasmid, such as, for example, the neo gene. Alternatively, the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the BIN1 gene in host cells (Cone & Mulligan, 1984, Proc. Natl. Acad. Sci. USA 81:6349-6353). High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothionein IIA promoter and heat shock promoters.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with BIN1 cDNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stabily integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. For example, following the introduction of foreign nucleic acid, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. A number of selection systems may be used, including, but not limited to the herpes simplex virus thymidine kinase gene (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase gene (Szybalska & Szybalski, 1962, Proc. Nat. Acad. Sci USA 48:2026), and the adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22: 817) genes can be employed in tk-, hgprt.sup.- or aprt.sup.- cells respectively. Additionally, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl Acad. Sci. USA 77: 3567; O'Hare, et al., 1981, Proc. Natl. Acad Sci. USA 78:1527); the gpt gene, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad Sci. USA 78: 2072; the neo gene, which confers resistance to the aminoglycoside G418 (Colberre-Garapin, et al., 1981, J Mol. Biol. 150: 1); and the hygro gene, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30: 147) genes. Recently, additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, 1988, Proc. Natl. Acad: Sci. USA 85: 8047); and ODC (omithine decarboxylase) which confers resistance to the omithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-omithine, DFMO (McConlogue L., 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.).

When the host is a eukaryote, transfection of nucleic acid may be accomplished by employing as calcium phosphate co-precipitates and conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors. Eukaryotic cells can also be cotransformed with nucleic acid sequences encoding a BIN1 polypeptide of the invention, and a second foreign nucleic acid molecule encoding a selectable phenotype. Another method employs a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein. (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

Isolation and purification of recombinantly expressed polypeptides, or fragments thereof, provided by the invention, may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.

BIN1 is a Plasma Membrane Receptor for Plant Steroids

To test whether BL is the ligand that directly activates the BIN1 receptor kinase, we first analyzed the effect of overexpression of BIN1 on BL binding activity in membrane fractions. Transgenic Arabidopsis plants overexpressing a fusion protein of BIN1 and Green Fluorescent Protein (BIN1-GFP) showed reduced inhibition of hypocotyl growth by a BR biosynthesis inhibitor. (FIGS. 2a, b) See Asami, T. et al. Characterization of brassinazole, a triazole-type brassinosteroid biosynthesis inhibitor, Plant Physiol 123, 93-100 (2000). They also had longer petioles, similar to plants overexpressing the BR biosynthetic enzyme DWF4. (FIG. 2c) These phenotypes are consistent with the interpretation that overexpression of the BIN1-GFP protein increases the response of Arabidopsis to BRs. We observed a dramatic increase of BL binding activity in the membrane fractions of the BIN1-GFP transgenic plants (FIG. 2d). The increase of binding was due to an increase of binding sites (B.sub.max =2.66 pmole mg.sup.-1 membrane protein compared to 0.23 pmole mg.sup.-1 membrane protein), with similar binding affinities (K.sub.d =7.4.+-.0.9 nM compared to 10.8.+-.3.2 nM) (FIG. 1e). Such K.sub.d values are consistent with physiological concentrations of BL and coincide with the BL concentration that induces 50% of the maximum growth response in BL-deficient mutants.

The specificity of the BL binding activity was determined by comparing the relative binding affinity for several steroid compounds in binding competition assays as described below in Example 3. (FIG. 2F). Binding [.sup.3 H]-BL to the membrane fraction of BIN1-GFP plants was effectively competed by unlabelled BL (50% inhibition concentration, IC.sub.50. 80 nM), less effectively by castasterone (CS) (IC.sub.50. 340 nM), and not competed by 2,3,22,23-O-tetramethylBL (Me-BL) (IC.sub.50.>10 .mu.M) and ecdysone (IC.sub.50.>10 .mu.M). The relative binding affinity (RBA, ratio between IC.sub.50. of a competitor and that of BL) of CS is about 4-5 times lower than BL, and this is consistent with CS being about 5 times less active than BL in bioassays. The lack of competition by Me-BL and ecdysone is consistent with their lack of biological activity in plants. Such specificity and high affinity for biologically active brassinosteroids indicate that this BL binding activity accounts for the BL-induced biological responses.

A specific BL binding activity was detected after the BIN1-GFP proteins were immunoprecipitated using anti-GFP antibodies (FIGS. 3a, b) as described in Example 3. No specific BL binding activity was immunoprecipitated from wild-type Arabidopsis plants using the same antibodies (FIG. 3a), indicating that the BL binding activity is specific to the BIN1-GFP protein. The immunoprecipitated binding activity has a similar disassociation constant (K.sub.d =15.2.+-.5 nM) as determined for membrane fractions (FIG. 3b). These results demonstrate that BIN1 either binds BL directly or is a limiting component of a receptor complex for BL in plant cells.

Mutations in large numbers of BIN1 alleles implicate the functional importance of the cytoplasmic kinase domain and the 70-aa island of BIN1's extracellular domain. We found that BIN1 mutants with missense mutations in the kinase domain (BIN1-104, A 1031-T) or in a region of the extracellular domain near the transmembrane domain (BIN1-102, T750-I) have BL binding activities similar to wild type and the biosynthetic mutant det2 (FIGS. 3c, d). In contrast, a missense mutation (BIN1-6, G644-D) and a mutation that causes a premature translation stop (BIN1-116, Q583-stop), both in the 70-aa island region of BIN1, greatly reduced the BL binding activity (FIGS. 3c, d). These results provide direct evidence that the 70-amino acid island region of BIN1's extracellular domain is required for BL binding to the receptor on the cell membrane.

We tested whether BL-binding activities leading to BIN1's kinase receptor activation involves auto-phosphorylation which can lead to a change of mobility in SDS-polyacrylamide gel electrophoresis (PAGE). These methods are described in Example 4 below. Arabidopsis seedlings grown in the presence of the BR biosynthetic inhibitor brassinazole were treated with BL and analyzed by immunoblotting (FIG. 4). Treatment of wild-type seedlings with 1 .mu.M BL for 1 hour caused a shift of BIN1 from a faster to a slower migrating band, compared with untreated sample or sample treated with mock solution (FIG. 3a). Phosphatase treatment of the BL-treated samples in the absence, but not in the presence of phosphatase inhibitors, shifted the slower band back to the fast migrating band, indicating that the shift of mobility represents BIN1 phosphorylation (FIG. 4b). Such BL-induced BIN1 phosphorylation was also observed in the BL biosynthetic mutant det2, but not in the BIN1-6 and BIN1117 mutants (FIG. 4c), which contain mutations that abolish the BL-binding activity (FIG. 3c) and in vitro kinase activity, respectively. Therefore, BL induction of BIN1 phosphorylation appears to require both the BL-binding and kinase activities of BIN1. These results indicate that interaction of BL with the extracellular domain of BIN1 leads to activation of BIN1's kinase.

Our identification of the receptor kinase BIN1 as a plant steroid receptor illustrates the function of a member of the largest family of receptor kinases in Arabidopsis. The Arabidopsis genome sequence revealed 174 LRR-receptor kinases, of which only a few are known for their biological functions and only one, CLV1, has been characterized at the biochemical level. See Initiative, T.A.G. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature in press (2000); Trotochaud, A. E., Jeong, S. & Clark, S. E. CLAVATA3, a multimeric ligand for the CLAVARA1 receptor-kinase. Science 289, 613-7 (2000); Torii, K. U. et al. The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats. The Plant Cell 8, 735-746 (1996); and Jinn, T. L., Stone, J. M. & Walker, J. C. HAESA, an Arabidopsis leucine-rich repeat receptor kinase, controls floral organ abscission. Genes Dev. 14, 108-17 (2000). The mechanism by which BIN1 kinase is activated by ligand binding may be shared by other LRR-receptor kinases, as suggested by the BR activation of a BIN1-Xa21 chimeric receptor. See He, Z. et al. Perception of brassinosteroids by the extracellular domain of the receptor kinase BIN1. Science 288, 2360-3 (2000). However, BIN1 appears to differ from CIV1, which has recently been shown to require its own kinase activity for binding to its peptide ligand. See Trotochaud, A. E., Jeong, S. & Clark, S. E. CLAVATA3, a multimeric ligand for the CLAVARA1 receptor-kinase. Science 289, 613-7 (2000).

Our results also reveal a new mechanism of steroid signalling. Steroid hormones are generally known to pass freely across plasma membranes into animal cells, where they bind to members of the nuclear receptor superfamily of ligand-dependent transcription factors. See Beato, M., Herrlich, P. &Schutz, G. Steroid hormone receptors: many actors in search of a plot. Cell 83, 851-7 (1995); and Mangelsdorf, D. J. et al. The nuclear receptor superfamily: The second decade. Cell 83, 835-839 (1995). In contrast, the Arabidopsis genome does not appear to encode members of this family of proteins. The near identical phenotypes of BIN1 to BR-biosynthetic mutants and our results presented here seem to indicate that plants perceive steroids at the cell surface and that BIN1 is likely to be the primary BR receptor in Arabidopsis. Such a cell-surface signalling mechanism may not be unique to plant steroids. In fact, membrane-initiated steroid responses have been observed in many animal systems, and signalling molecules such as calcium, inositol phosphates, cAMP, G proteins and various kinases have been implicated. However, little is known about the membrane-bound steroid receptors that initiate these signalling cascades in animal cells.

BIN1 Antibodies

Embodiments of the invention also include antibodies immunoreactive with the BIN1 polypeptide or antigenic fragments thereof. Antibodies of the invention are useful for modulating BIN1 ligand binding, for example. Antibodies directed against peptides derived from the extracellular domain of BIN1 are preferred (e.g., peptides contained in the domain from about amino acid 588 to 649 of SEQ ID NO:2). Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art (Kohler, et al., Nature, 256:495, 1975).

The preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green et al., Production of Polyclonal Antisera, in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages 1-5 (Humana Press 1992); Coligan et al., Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in CURRENT PROTOCOLS IN IMMUNOLOGY, section 2.4.1 (1992), which are hereby incorporated by reference.

The preparation of monoclonal antibodies likewise is conventional. See, for example, Kohler & Milstein, Nature 256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et al., ANTIBODIES: A LABORATORY MANUAL, page 726 (Cold Spring Harbor Pub. 1988), which are hereby incorporated by reference. Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by analyzing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e.g., Coligan et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al, Purification of Immunoglobulin G (IgQ), in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (Humana Press 1992). Methods of in vitro and in vivo multiplication of monoclonal antibodies is well-known to those skilled in the art. Multiplication in vitro may be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPM1 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages. Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies. Large scale hybridoma, cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stiffer reactor, or in immobilized or entrapped cell culture. Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, e.g., osyngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.

Therapeutic applications for antibodies disclosed herein are also part of the present invention. With increasing evidence that steroids affect the cell surface and alter ion permeability, as well as the release of neurohormones and neurotransmitters, antibodies to extracellular receptors such as BIN1 may have therapeutic applications. Antibodies of the present invention may also be derived from subhuman primate antibody. General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Publication WO 91/11465 (1991) and Losman et al., Int. J Cancer 46:310 (1990), which are hereby incorporated by reference.

Alternatively, a therapeutically useful anti-BIN1 antibody may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al., Proc. Natl. Acad. Sci. USA 86:3833 (1989), which is hereby incorporated in its entirety by reference. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321: 522 (1986); Riechmann et al., Nature 332: 323 (1988); Verhoeyen et al., Science 239: 1534 (1988); Carter et al., Proc. Nat. Acad. Sci. USA 89: 4285 (1992); Sandhu, Crit. Rev. Biotech. 12: 437 (1992); and Singer et al., J Immunol. 150: 2844 (1993), which are hereby incorporated by reference.

Antibodies of the invention also may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al, METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 119 (199 1); Winter et at., Ann. Rev. Immunol. 12: 433 (1994), which are hereby incorporated by reference. Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, Calif.).

In addition, antibodies of the present invention may be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens and can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856 (1994); and Taylor et al., Int. Immuno. 6:5 79 (1994), which are hereby incorporated by reference.

Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of nucleic acid encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab'monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. No. 4,036,945 and No. 4,331,647, and references contained therein. These patents are hereby incorporated in their entireties by reference. See also Nisonhoff et al., Arch. Biochem. Biophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959); Edelman et al., METHODS IN ENZYMOLOGY, VOL. 1, page 422 (Academic Press 1967); and Coligan et al. at sections 2.8.1-2.8. 10 and 2.10.1-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

For example, Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al., Proc. Nat 7 A cad. Sci. USA 69:2659 (1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu, supra. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising nucleic acid sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 97 (1991); Bird et al., Science 242:423-426 (1988); Ladner et al., U.S. Pat. No. 4,946,778; Pack et al., BiolTechnology 11: 1271-77 (1993); and Sandhu, supra.

Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991).

The term "antibody" as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab')2, and Fv which are capable of binding to an epitopic determinant present in BIN1 polypeptide. Such antibody fragments retain some ability to selectively bind with its antigen or receptor.

As used in this invention, the term "epitope" refers to an antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

Antibodies which bind to the BIN1 polypeptide of the invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen. For example, it may be desirable to produce antibodies that specifically bind to the N- or C-terminal domains of BIN1. The polypeptide or peptide used to immunize an animal which is derived from translated cDNA or chemically synthesized which can be conjugated to a carrier protein, if desired. Such commonly used carriers which are chemically coupled to the immunizing peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.

Invention polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound. Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan, et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994, incorporated by reference).

It is also possible to use the anti-idiotype technology to produce invention monoclonal antibodies which mimic an epitope. For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the "image" of the epitope bound by the first monoclonal antibody.

Genetically Modified Plants and Methods of Making

In another embodiment, the invention provides a method for producing a genetically modified plant characterized as having increased yield as compared to a plant which has not been genetically modified (e.g., a wild-type plant). The term "yield" has been previously defined herein. The invention method comprises the steps of introducing at least one nucleic acid sequence encoding BIN1, into a plant cell to obtain a transformed plant cell wherein the nucleic acid sequence is operably associated with a pro