# Patent

## Title
Vector system for selection of genes encoding secreted proteins and membrane-bound proteins

## Abstract

The subject invention concerns novel vectors for the rapid and robust selection for cDNA sequences that encode secreted or membrane-bound proteins. The invention also pertains to methods for cloning secreted or membrane-bound proteins, including proteins encoded by novel members of gene families.

## Background
CROSS-REFERENCE TO RELATED APPLICATION(S)
This application claims the benefit of provisional patent application Ser. No. 60/288,046, filed May 2, 2001, which is hereby incorporated by reference in its entirety, including all nucleic acid sequences, amino acid sequences, figures, tables, and drawings.


The subject invention was made with government support under a research project supported by National Institutes of Health Grant No. AI23338. The government may have certain rights in this invention.


BACKGROUND OF THE INVENTION
Proteins destined for transport into or across cell membranes are usually translated with a signal sequence that directs the newly synthesized protein to the appropriate membrane translocation system. The primary structure of signal sequences is highly variable among different proteins. Signal sequences that target proteins for export from the cytosol generally contain a short stretch (7–20 residues) of hydrophobic amino acids. In most cases, the signal sequence is located at the amino terminus of a nascent protein and is proteolytically removed on the trans side of the membrane (e.g. lumen of endoplasmic reticulum, bacterial periplasm, intercisternal space of mitochondria and chloroplasts), although examples of mature proteins containing uncleaved or internal signal sequences have been described. Export signal sequences may be interchanged among different proteins, even proteins of different species of organisms.
Many secreted proteins interact with target cells to bring about physiological responses such as growth, differentiation and/or activation. These activities make secreted proteins biologically interesting molecules that are potentially valuable as therapeutics or as targets for ligands. Of the estimated 60,000 to 100,000 human genes, about 25% carry a signal peptide and about 4% are secreted extracellularly. Clearly, approaches to rapidly and accurately identifying secreted proteins are important components of gene-based drug discovery programs.
With advances in techniques for sequencing cDNAs, many expressed sequence tags (ESTs) have been generated which have enhanced the process of identifying novel secreted proteins as compared to the conventional reverse genetics approaches. However, ESTs are small random cDNA sequences and thus it becomes hard to identify secretion signal sequence that is normally present in the 5′ end of cDNA encoding secreted protein. Moreover, after an EST carrying a potential secretion signal sequence is identified based on the homology search, it has to be authenticated in a functional assay. Thus a means for selection for the biochemical function of the proteins encoded by inserted cDNA would greatly simplify the process of obtaining novel secreted genes.
Secretion signal trap is one such method to clone 5′ ends of cDNAs encoding secreted proteins from a random cDNA library. Generally, signal trapping relies on secretion of a reporter polypeptide by signal sequences present in a cDNA library. The secreted reporter polypeptide may then be detected by a variety of assays based upon, for example, growth selection, enzymatic activity, or immune reactivity. Examples of signal trap cloning procedures include those in U.S. Pat. No. 5,536,637 and Klein et al. Proc. Natl. Acad. Sci. USA 93, 7108–13 (1996), which describe signal trap cloning in yeast using the yeast invertase polypeptide as a reporter. Furthermore, Imai et al. J. Biol. Chem. 271, 21514–21 (1996) describes signal trap cloning in mammalian cells using CD4 as a reporter and identifying signal sequences by screening for surface expression of CD4 antigen. In addition, U.S. Pat. No. 5,525,486, Shirozu et al. Genomics 37, 273–80 (1996) and Tashiro et al. Science 261, 600–03 (1993) describe signal trap cloning in mammalian cells and identify signal sequences by screening for surface expression of IL-2 receptor fusion proteins. None of these references teaches cloning in prokaryotic cells.
Signal sequence trapping using mammalian cells has disadvantages, including low transfection efficiency, relatively expensive culture medium, and difficult recovery of vector-borne cDNA sequences from cells that have been transfected. Signal sequence trapping using yeast cells also has the disadvantage of slow growth time as compared to bacterial cells. Further, methods for molecular cloning in yeast cells are generally more complicated than bacterial methods. By contrast, bacterial cells have the advantages of fast doubling times, high transformation efficiencies, and ease of use, as compared to both mammalian and yeast cells, accommodating a wider range of experience levels in the laboratory.
U.S. Pat. No. 5,037,760 describes signal trap cloning in Bacillus using α-amylase and β-lactamase as reporter genes. This patent teaches vectors for identifying secretory signal sequences from DNA fragments of unicellular microorganisms. It does not teach identifying signal sequences in complex eukaryotic organisms.
Sibakov et al. (1991) Appl. Environ. Microbiol. 57: 341–48 and Chubb et al. (1998) Microbiology 144: 1619–29 describe cloning of prokaryotic signal sequences using β-lactamase fusions. Sibakov, et al. and Chubb, et al. do not describe a screening strategy for detection of eukaryotic signal sequences using selection in a prokaryotic system.
Kolmar et al. (1992) J. Mol. Biol. 228: 359–365, Seehaus et al Gene 114: 235–37, Sutter et al. Mol. Microbiol. 6: 2201–2208, and Palzkill et al. (1994) J. Bacteriol. 176: 563–68 utilize β-lactamase fusions in the study of specific biological processes rather than as a means of cloning novel cDNAs on a large scale.
Chen and Leder (1999) Nucleic Acids Res. 27: 1219–22 and Lee et al. (1999) J. Bacteriol. 181: 5790–99 utilize color change from alkaline phosphatase activity during colony formation as a screening mechanism. Thus, a subjective determination of color changes is required for selection using these systems.
Although many of the above references describe the utility of fusions of various cDNA sequences to a β-lactamase sequence, none present a library-screening strategy for detection of eukaryotic signal sequences using selection in a prokaryotic system. Further, none of the aforementioned systems incorporate a single, degenerate primer-based polymerase chain reaction (PCR) strategy designed to clone novel gene family members.
Thus, there is a need to develop alternative approaches for rapid and accurate identification of novel secreted eukaryotic proteins using bacterial host cells.
BRIEF SUMMARY OF THE INVENTION
The present invention relates to a vector system that allows rapid and robust selection for cDNA sequences that encode secreted or membrane-bound proteins. More particularly, the present invention pertains to vectors comprising a reporter gene (such as β-lactamase) lacking a functional signal sequence; a selectable marker gene (such as neomycin phosphotransferase), wherein the reporter gene and selectable marker gene are operably linked to a promoter sequence (such as the lac promoter); and a multiple cloning site. Optionally, the vectors of the subject invention can further comprise a SLIP sequence, a plurality of thymidine nucleotides that allows for all three frames of any cloned cDNA to be fused to the reporter gene, thereby increasing the efficiency of cloning cDNAs for secreted or membrane-bound proteins.
The invention also relates to a method for cloning novel members of a gene family using plasmid vectors of the present invention. The method includes providing a vector of the subject invention. Preferably, the vector is linearized. The vector can be linearized, for example, with one or more restriction enzymes in order to produce a “sticky end” for ligation to a candidate nucleic acid sequence encoding a potential secreted or membrane-bound protein. The vector comprises DNA encoding a reporter gene lacking a functional signal sequence. The method further includes cutting the candidate nucleic acid sequence with one or more restriction enzymes in order to produce a compatible “sticky end” for ligation to the linearized vector and ligating the candidate nucleic acid sequence to the linearized vector, thereby forming a ligation product. Bacterial cells can then be transformed with the ligation product and colonies can be selected based on expression of the reporter gene functionally linked to the gene encoding the secreted or membrane-bound protein. The method can further include determining the nucleic acid sequence within the transformants from the selected colonies and determining the amino acid sequence based on the nucleic acid sequence.
In order to improve the overall efficiency of cloning of cDNAs that encode secreted proteins or membrane-bound proteins (such as membrane-bound receptors), as well as to identify homologous genes possessing only minimal sequence relatedness, the present inventors have engineered unique plasmid-based selection vectors and developed a cloning strategy that utilizes such vectors, wherein only minimal information about the gene of interest is necessary. This cloning strategy has been validated with a number of known members of the Ig gene superfamily (IgSF) and has led to the identification of a novel V region-containing, presumably bifunctional gene in amphioxus (Branchiostoma floridae), a protochordate (cephalochordate) species that lacks an adaptive immune system.



BRIEF DESCRIPTION OF THE DRAWINGS
The following is a brief description of the drawings that are presented only for the purposes of further illustrating the invention and not for the purposes of limiting same.
FIG. 1 outlines a strategy for the cloning of novel members of a gene family using the plasmid vector G7311 of the present invention. First-strand cDNA is synthesized using the SMART system (CLONTECH). First strand synthesis is performed in the presence of an oligoribonucleotide, SMART-5′, that anneals to a nontemplated stretch of oligo(dC) residues added by reverse transcriptase (RT) to the end of the nascent cDNA. The RT enzyme completes the first strand of cDNA by adding nucleotides complementary to the SMART sequence. Polymerase chain reaction (PCR) is then performed on the cDNA using (1) an oligonucleotide corresponding to the SMART-5′ sequence, which contains an SfiI recognition sequence (5′-GGCCNNNN^NGGCC (SEQ ID NO. 6)) and (2) a degenerate oligonucleotide, e.g., YXC-Sfi, corresponding to a putative conserved motif of three to five amino acids plus an SfiI recognition sequence. The SfiI sites in the PCR primers are asymmetric and allow directional cloning of PCR products into the Amptrap vector at corresponding SfiI sites. After selection of E. coli transformants on ampicillin, colonies can be evaluated for insert size using colony PCR. Inserts of the anticipated size range can be sequenced directly, and the source colonies can be archived for future use.
FIG. 2 is a map of the Amptrap vector G7311 of the present invention.
FIG. 3 is a map of the Amptrap vector G7637 of the present invention.
FIGS. 4A and 4B are maps of the phage vectors λ7311 and λ7637, respectively, of the present invention.
FIGS. 5A and 5B show the complete gene sequence of an Amptrap vector G7311 (SEQ ID NO:1) of the present invention.
FIGS. 6A and 6B show the complete gene sequence of an Amptrap vector G7637 (SEQ ID NO:2) of the present invention.
FIGS. 7A–7D show a strategy for cloning of R. eglanteria MHC Class II. FIG. 7A shows a priming strategy based on two conserved codon positions that occur in MHC I, MHC II and β2m. FIG. 7B shows agarose gel analysis of the 5′-RACE PCR products; size standard is ΦX174/Hae III. FIG. 7C shows the sizing of inserts from ampicillin resistant colonies. FIG. 7D shows the results of sequencing of eight size-selected clones. (*) indicates products selected for sequencing; size standard indicated.
FIGS. 8A–8E show the cloning of a novel IgSF gene from B. floridae. FIG. 8A shows sequence motifs that served as a basis for primer design. FIG. 8B (1–7) shows agarose gel analysis of 5′-RACE PCR products that were formed using individual Amptrap primers; (8) of FIG. 8B is a product formed with only a 5′ primer (SMART-DNA primer: 5′-AAGCAGTGGTATCAACGCAGAGT-3′ (SEQ ID NO. 7)); (6) of FIG. 8B is a size standard. FIG. 8C shows sizing of inserts from ampicillin resistant colonies by PCR; (*) indicates products selected for sequencing, note the length variation in products. FIG. 8D shows a schematic of amplicon G7977, containing a partial Ig-encoding sequence. FIG. 8E shows a schematic of full-length cDNA G9119.
FIGS. 9A–9E show structural aspects of a V region-containing chitin binding protein (V-CBP) and the presence of V-CBP1 mRNA in B. floridae. FIG. 9A shows, a schematic representation of V-CBP. FIG. 9B shows a ClustalW alignment of the three V-CBP proteins described herein (SEQ ID NOs. 3–5). FIG. 9C–9E show in situ hybridization to mRNA in serial transverse sections of adult B. floridae intestine. FIG. 9C shows hematoxylin and eosin staining. FIG. 9D shows in situ hybridization using a antisense RNA probe corresponding to V-CBP1 (note staining in scattered cells). FIG. 9E shows in situ hybridization using a sense (control) RNA probe corresponding to V-CBP1.



BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO. 1 is the nucleotide sequence (G7311) of a vector of the subject invention.
SEQ ID NO. 2 is the nucleotide sequence (G7637) of a vector of the subject invention.
SEQ ID NOs. 3–5 are portions of V-CBP proteins, as shown in the CrustalW alignment in FIG. 9B.
SEQ ID NO. 6 is the recognition sequence of the SfiI endonuclease.
SEQ ID NOs. 7–15 are primers that were utilized to identify new proteins, using the methods of the subject invention.
DETAILED DISCLOSURE OF THE INVENTION
The present invention relates to a vector system that allows rapid and robust selection for cDNA sequences that encode secreted or membrane-bound proteins even when gene families are highly divergent and share only limited regions of sequence identity.
The present invention pertains to vectors comprising a reporter gene (such as β-lactamase) lacking a functional signal sequence; a selectable marker gene (such as neomycin phosphotransferase gene), wherein the reporter gene and the selectable marker gene are operably linked to a promoter (such as a lac promoter); and a multiple cloning site. Optionally, the vectors of the subject invention can further comprise a SLIP sequence, a plurality of thymidine nucleotides that allows for all three frames of any cloned cDNA to be fused to the reporter gene, thereby increasing the efficiency of cloning cDNAs for secreted or membrane-bound proteins.
The invention also relates to a method for cloning novel members of a gene family using plasmid vectors of the present invention. The method includes providing a vector of the subject invention. Preferably, the vector is linearized. The vector can be linearized, for example, with one or more restriction enzymes in order to produce a “sticky end” for ligation to a candidate nucleic acid sequence encoding a potential secreted or membrane-bound protein. The vector comprises DNA encoding a reporter gene lacking a functional signal sequence. The method further includes cutting the candidate nucleic acid sequence with one or more restriction enzymes in order to produce a compatible “sticky end” for ligation to the linearized vector and ligating the candidate nucleic acid sequence to the linearized vector, thereby forming a ligation product. Bacterial cells can then be transformed with the ligation product and colonies can be selected based on expression of the reporter gene functionally linked to the gene encoding the secreted or membrane-bound protein. The method can further include determining the nucleic acid sequence within the transformants from the selected colonies and determining the amino acid sequence based on the nucleic acid sequence.
The present inventors have developed a novel strategy for cloning cDNAs encoding any secreted or membrane-bound proteins based on the use of a plasmid that contains a reporter gene lacking a functional signal sequence. Preferably, the reporter gene is a β-lactamase gene in which the start and signal peptide codons have been deleted. Resistance to β-lactam antibiotics (e.g., ampicillin) can be achieved after the introduction of an in-frame signal peptide sequence from a directionally cloned cDNA. This selection system, termed “Amptrap”, efficiently selects mRNAs with intact 5′ regions and can be used in conjunction with a degenerate 5′-RACE strategy that requires knowledge of only a single target motif corresponding to as few as three amino acids. Amptrap has been validated in a number of systems and has proven to be highly efficient in the recovery of orthologs of known immune receptors as well as novel forms of immune-related genes. An unusual secreted gene product from a protochordate in which the N terminus consists of two immunoglobulin (Ig) variable (V) domains and the C terminus is a chitin-binding domain has been identified and characterized. Consideration of such molecules is important in discerning the genetic mechanisms that have diversified both innate and adaptive receptors.
The methods and vectors of the subject invention can be utilized for cloning cDNAs encoding any secreted or membrane-bound proteins from a vast array of eukaryotic organisms, including vertebrates and invertebrates. For example, the methods and vectors of the subject invention can be utilized to identify secreted or membrane-bound proteins of reptiles, birds, fish, amphibians, and mammals, such as rodents and humans. The methods and vectors of the subject invention are suitable in a number of potential applications, particularly those that are normally hampered by knowledge of only minimal structural interrelatedness and/or by low concentrations of mRNA that would not be represented in standard EST (expressed sequence tag) libraries.
The methods of the subject invention can be carried out using a plasmid vector comprising a reporter gene lacking a functional signal sequence. For example, the reporter gene can encode a β-lactamase enzyme in which the N-terminal signal peptide has been deleted. The absence of this region precludes the secretion of β-lactamase and results in sensitivity to β-lactam antibiotics (e.g., ampicillin). Secretion of β-lactamase is restored if a cDNA sequence that is inserted 5′ and in-frame to the β-lactamase coding sequence encodes both a methionine start codon (ATG) and a signal peptide immediately downstream from the start codon. Advantageously, the cloning of cDNAs that encode intracellular proteins, nuclear proteins, or any other sequence that does not encode a signal peptide, can be selectively eliminated by growth in a selective medium (a β-lactam antibiotic, such as ampicillin). Such irrelevant sequences can drastically reduce the efficiency of recovery of target clones in degenerate, low stringency PCR amplifications.
A selective, directed cloning strategy, which represents a method of the subject invention, and requires only minimal a priori sequence information, is shown in FIG. 1. In the first step, cDNA is synthesized. Chemical synthesis of nucleic acid sequences can be accomplished using methods well known in the art, such as those set forth by Engels et al., Angew. Chem. Intl. Ed., 28:716–734 (1989), CLONTECH's SMART cDNA synthesis manual (www.clontech.com), and Wells et al. Gene, 34:315 (1985), the disclosures of which are hereby incorporated by reference. These methods include the phosphotriester, phosphoramidite and H-phosphonate methods of nucleic acid sequence synthesis. Large nucleic acid sequences, for example those larger than about 100 nucleotides in length, can be synthesized as several fragments and ligated together. A preferred method is polymer-supported synthesis using standard phosphoramidite chemistry. The SMART system (CLONTECH) is based on the non-templated addition of polyC to nascent cDNA by reverse transcriptase. The double-stranded cDNA sequences that are produced contain a common, specific anchor sequence at their 5′ ends. Using the SMART system, a 5′-RACE PCR reaction is performed in which the specific (SMART) anchor sequence also serves as the 5′ primer-binding site and is coupled with a 3′ degenerate antisense primer that complements a short region of predicted amino acid sequence identity. Following PCR amplification, amplicons can be cloned directionally into the vector using one or more restriction enzymes. For example, asymmetric Sfi I sites can be utilized. SfiI enzymes are type II restriction endonucleases having two binding surfaces which act cooperatively to grasp two copies of its 13 base pair recognition sequence, 5′-GGCCnnnn↓nGGCC (SEQ ID NO. 6). Only those clones that contain a start codon and signal sequence, fused in-frame to the codons complemented by the 3′ PCR primer, will grow on the β-lactam antibiotic. In many applications, the approximate distance between a single conserved priming site and the N-terminal signal peptide can be predicted, thus permitting size selection and further elimination of irrelevant amplicons. PCR amplicons in the range of  ̃200→800 base pairs (bp) have been cloned and selected successfully using the methods of the subject invention.
Conventional cDNA cloning vectors allow a cDNA sequence to be propagated in a host cell, usually a bacterium or yeast, after insertion of the cDNA into a plasmid at a specific site. Modern vectors allow sequencing of the cDNA inserts by placing primer binding sites both 5′ and 3′ to the inserted DNA. Subsets of these vectors are also designed for other specific purposes, such as expression of the inserted cDNA sequence in either bacterial or eukaryotic cells by the addition of promoter sequences 5′ to the insert. Although these vectors have allowed investigators to clone a large variety of novel sequences from almost any organism, most common, commercially available vectors do not provide a means of selection for the biochemical function of the proteins encoded by inserted cDNA. Because of this condition, searches for transcripts encoding proteins with specific functions or properties can become cumbersome due to the large number of extraneous insertion events that must be screened in order to isolate rare clones of interest. Selection for biochemical functions of the inserted sequences can be valuable in an experiment designed to identify cDNA sequences encoding proteins with a specific biochemical property, such as kinases, DNA-binding proteins, or membrane-bound receptors.
The inventors have designed a new vector system, the Amptrap, which allows rapid and robust selection for cDNA sequences encoding proteins that are secreted or bound to lipid membranes. Using this system, an investigator can rapidly narrow a large pool of cDNA inserts to only those sequences that encode such proteins, while excluding any sequences that encode cytoplasmic proteins, nuclear proteins, or incomplete membrane protein segments. Because the system is based on selection rather than simple screening, clones encoding irrelevant proteins are deleted from the experiment and do not appear in the pool of colonies for analysis, reducing the risk of false positives. All cDNA sequences isolated using this method must contain a methionine start codon in addition to a secretion signal sequence, eliminating isolation of 5′-truncated cDNA sequences. The vector set can accommodate cDNA library construction, either in plasmids or lambda phage.
The Amptrap vectors described in this disclosure, G7311 (FIG. 2) and G7637 (FIG. 3), are plasmids designed to allow direct, robust selection for cDNA sequences that encode secreted or membrane-bound proteins. Both vectors contain a sequence encoding a mature β-lactamase enzyme that lacks a sequence of twenty-three largely hydrophobic amino acids at the N-terminus of the protein, the signal peptide, that directs export of the wildtype protein into the bacterial periplasmic space. Without this signal peptide, β-lactamase cannot be secreted and remains within the bacterial cell.
Because the β-lactamase enzyme must be secreted into the periplasmic space of the bacterium in order to confer resistance to β-lactam antibiotics such as ampicillin, a bacterium bearing G7311 or G7637 is ampicillin-sensitive. However, if a cDNA sequence inserted 5′ to the β-lactamase sequence contains both a methionine start codon (ATG) and codons for a signal peptide immediately 3′ to the initiation sequence that can be fused in frame to the β-lactamase coding sequence, secretion of β-lactamase is restored and the host clone will express ampicillin resistance. If the cDNA fails in either of these two requirements, the bacterium will remain ampicillin-sensitive and the clone will not be propagated upon selection.
The G7637 vector is similar to the G7311 vector, except for the addition of a sequence of 13 thymidine residues at the 5′ region of the β-lactamase coding region (the “SLIP” sequence: CLONTECH). This region allows slippage of the transcription and translation machinery of the cell such that peptides encoded by all three frames of any cDNA become fused to β-lactamase, thus removing the requirement for proper in-frame fusion of an open reading frame in the cDNA to the β-lactamase sequence, and increasing the efficiency of selection for signal sequences.
In order to facilitate construction of large cDNA libraries, the plasmid vectors G7311 and G7637 have been inserted into phage lambda-based vectors to form λ7311 (FIG. 4A) and λ7637 (FIG. 4B). Derived from CLONTECH's λTripleX (www.clontech.com), the λ7311 and λ7637 phage vectors contain loxP recombination sequences that allow in vivo plasmid excision.
In addition to the disclosed vectors, the inventors have designed a strategy for the cloning of novel members of a gene family using the plasmid vectors. A strategy using the plasmid vector G7311 is shown in FIG. 1. In this system, cDNA is synthesized using CLONTECH's SMART system (CLONTECH, 1020 East Meadow Circle, Palo Alto, Calif. 94303-4230, USA, available at www.clontech.com/smart/), which produces double-stranded cDNA sequences containing a common, specific anchor sequence at their 5′ ends. This anchor sequence is used as a 5′ primer binding site in a PCR reaction, coupled with a 3′ degenerate antisense primer based on amino acids thought to be conserved throughout a given gene family. By performing PCR, directionally cloning the amplicons into the G7311 vector, and then selecting on ampicillin, only those sequences that contain a start codon and signal sequence, fused in frame to the codons dictated by the 3′ PCR primer, will be propagated in bacterial colonies. Other 5′RACE primers can also be utilized in the present invention. If domains of a particular size are expected from the PCR amplification, size selection can be used to screen out clones that depart from the expected insert size.
SMART stands for Switch Mechanism At 5′ end of the RNA Transcript. SMART cDNA synthesis begins with just nanograms of either total or poly A+ RNA. A modified oligo(dT) primer is used to prime the first-strand reaction. When reverse transcriptase (RT) reaches the 5′ end of the mRNA, the enzyme's terminal transferase activity adds a few deoxycytidine (dC) nucleotides. The 3′ end of the SMART oligonucleotide anneals with the (dC) stretch, forming an extended template. RT then switches templates and replicates the oligonucleotide. The resulting single-stranded (ss) cDNA contains the complete 5′ end of the mRNA template, as well as the sequence complementary to the SMART oligonucleotide, called the SMART anchor. This anchor, together with the modified oligo(dT) sequence, serves as a universal priming site for long-distance (LD) PCR, primer extension, or RACE amplification.
All steps in this method provide very strong tools for the elimination of undesirable or artifactual sequences. Using this system, primers corresponding to motifs containing as few as two known amino acids have produced successful amplification and targeted cloning of a cDNA sequence encoding major histocompatibility complex class II, a member of a specific family of membrane-bound proteins. Thus, because of the relaxed requirements for degenerate priming sites, this strategy allows amplification and cloning of novel gene family members based on only very limited knowledge of conserved motifs.
The PCR strategy described above, while applicable to other signal trap vectors, allows a very easy and robust way to clone sequences using the SfiI sites in the Amptrap. SfiI is a very rare cutter in DNA, cutting once every 65,536 bases in theory, and also leaves unique ends after cutting because it has a “separated” recognition site (5′-GGCCNNNN^NGGCC (SEQ ID NO. 6)). Therefore, the inventors' SfiI-containing vector coupled with the inventors' PCR method is the most powerful approach to clone secreted/membrane proteins with short, specific amino acid motifs. Other restriction enzymes that provide for incorporation of inserts into the vector, including directional cloning of inserts, can also be used in the present invention.
Amptrap-based selection for cDNAs allows cloning and selection to occur in bacterial cells, which are very amenable to DNA transformation and propagation, and are preferable to yeast in many experiments. Because the mechanism of the Amptrap system can operate by antibiotic resistance rather than color change from alkaline phosphatase activity during colony formation (as described by Chen and Leder Nucleic Acids Res. 27: 1219–22 (1999) and Lee, et al. J. Bacteriol. 181: 5790–99(1999)), screens for secreted/membrane proteins using Amptrap are more convenient and potentially more robust, as only those colonies containing signal-positive cDNA inserts will survive in the selection. The requirement for subjective determination of color changes using the alkaline phosphatase system is eliminated.
The vectors of the subject invention can carry a constitutively expressed neomycin phosphotransferase gene, which confers resistance to antibiotics such as kanamycin and neomycin, thus allowing selection of KanR-AmpR doubly resistant clones, as described in the Examples section. Advantageously, if an inserted ORF contains a methionine start codon coupled to a signal peptide that is in frame with the β-lactamase ORF, secretion of β-lactamase is restored and transformed bacterial clones acquire a KanR AmpR doubly resistant phenotype, allowing their direct selection on Kan+Amp medium.
The subject invention is exemplified by using Escherichia coli strain DH10B as the cloning host. However, any prokaryotic cell (including other E. coli strains) capable of accommodating recombinant DNA propagation without rearrangement could be used in the present invention.
The utility of the methods and vectors of the subject invention can be expanded to include cloning directed at antigenic epitopes for which an amino acid sequence can be inferred. This technique would extend to include antigens present on novel infectious agents, tumor-specific antigens, and other structures that are not necessarily encoded in known genomes and other structures that are not necessarily encoded in known genomes. For example, cDNA from cells infected with a virus that is novel but related antigenically to other, previously characterized viruses could be isolated and prepared for Amptrap cloning. Degenerate primers designed to amplify conserved sequences from the novel virus could be produced after analysis of protein sequences from the other, known members of its family. If amplicons can be generated successfully from the cDNA of the novel virus, they would provide immediate molecular probes for the cloning of its entire genome, thus aiding in the eventual isolation of the pathogen. Alternatively, analysis of Amptrap libraries from various tumors or tumor cell lines could provide a survey of secreted or membrane-bound protein sequences in cancerous tissues, thus aiding in searches for antigens or other factors expressed specifically or at high levels in certain tumors. Such antigens may be attractive therapeutic targets.
Recombinant DNA techniques used herein are generally set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989); by Ausubel et al., eds Current Protocols in Molecular Biology, Current Protocols Press, (1994); and by Berger and Kimmel, Methods in Enzymology: Guide to Molecular Cloning Techniques, Vol. 152, Academic Press, Inc., San Diego, Calif., (1987), the disclosures of which are hereby incorporated by reference. For example, nucleic acids and/or vectors can be introduced into host cells by well-known methods, such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection. Preferably, according to the methods of the subject invention, the host cells are transformed with nucleic acids and/or vectors via electroporation.
Both protein and nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman [1988] Proc. Natl. Acad. Sci. USA 85(8):2444–2448; Altschul et al. [1990] J. Mol. Biol. 215(3):403–410; Thompson et al. [1994] Nucleic Acids Res. 22(2):4673–4680; Higgins et al. [1996] Methods Enzymol. 266:383–402; Altschul et al. [1990] J. Mol. Biol. 215(3):403–410; Altschul et al. [1993] Nature Genetics 3:266–272).
Various restriction enzymes can be used to cleave or cut nucleic acids according to the methods of the subject invention. Preferably, type II restriction endonucleases are utilized. For example, endonucleases such as EcoRI, BamHI, HindIII, XhoI, NotI, SacI, SacII, and SalI can be utilized. More preferably, the SfiI endonuclease is utilized according to the methods of the present invention.
As used herein, the term “secreted protein” refers to a polypeptide that is extruded from the cell through the cell membrane. Secreted proteins include, but are not limited to, those polypeptides containing a signal sequence that are directed into the endoplasmic reticulum, or other organelles and subsequently directed out of the cell through a vesicle. Many secreted proteins, such as cytokines and hormones, are of therapeutic importance.
As used herein, the term “membrane-bound protein” refers to a polypeptide that is directed to a membrane-bound organelle and/or the cell membrane, and is not immediately secreted from the cell but remains associated with the membrane for a time. Therefore, membrane-bound proteins are inclusive of external membrane proteins (which are entirely outside of the cell membrane but bound to it by weak molecular attractions, such as ionic, hydrogen, and/or Van der Waals forces) and intrinsic membrane proteins that are embedded in the membrane. Membrane-bound proteins include, for example, integral membrane proteins, transmembrane proteins (which are amphipathic, having hydrophobic and hydrophilic regions and, therefore, having one or more membrane-spanning domains, such as type I and type II transmembrane proteins and multipass transmembrane receptors), peripheral membrane proteins, and lipid-anchored proteins. Many membrane-bound proteins are glycoproteins. Many membrane-bound proteins are receptors, such as the epidermal growth factor (EGF) receptor and G protein (guanine nucleotide binding proteins) coupled receptors.
As used herein, the term “reporter gene” refers to a nucleic acid sequence encoding a gene product (reporter molecule) that allows the presence of a vector (carrying a foreign nucleic acid sequence, such as a foreign gene) to be identified in eukaryotic or prokaryotic cells. Examples include the amp (ampicillin resistance) gene, β-lactamase, and genes encoding a chromogenic molecule, such as BCIP (5-bromo-4-chloro-3-indooylphosphate) or alkaline phosphatase. Only cells carrying the reporter gene can grow in the presence of the appropriate drug (the antibiotics neomycin and ampicillin, for example). Preferably, the reporter gene is one in which the reporter molecule encoded by the reporter gene must be secreted outside of the cell in order to operate. Any reporter gene that would allow signal sequence rescue by selection can be utilized.
As used herein, the term “selectable marker gene” refers to a nucleic acid sequence encoding a gene product (selectable marker molecule) that can be utilized to detect initial transformants. Therefore, the selectable marker gene can be constitutively expressed by a promoter sequence within the vector construct. Preferably, the selectable marker gene can be expressed independently from the reporter gene. More preferably, the reporter gene is operably linked to a first promoter sequence and the selectable marker gene is operably linked to a second (separate) promoter sequence. Examples of selectable marker genes include a neomycin-resistance gene (such as neomycin phosphotransferase), tetracycline-resistance gene, chloramphenicol-resistance gene (such as chloramphenicol acetyl transferase (CAT)), and bleomycin-resistance gene.
As used herein, the term “operably linked” refers to the functional and positional relationship between a nucleic acid sequence and a regulatory sequence. Polynucleotide sequences may be “operably linked” to regulatory sequences such as promoters and enhancers. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is “operably linked” to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is “operably linked” to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is “operably linked” to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers can be used in accordance with conventional practice.
As used herein, the terms “signal sequence”, “leader sequence”, or “signal peptide”, refer to a sequence (e.g., about 7 to about 20 residues) added to the amino-terminal end of a polypeptide chain that forms an amphipathic helix allowing the nascent polypeptide to migrate in or through cellular membranes such as the endoplasmic reticulum or the cell membrane. The signal sequence is generally cleaved from the polypeptide after the protein has crossed the membrane. As used herein, the term “signal sequence” may be used generically to refer to the signal peptide on a polypeptide chain, or to the nucleotides encoding the signal peptide.
As used herein, the term “sequencing” refers to the determination of the order of the repeating units in a nucleic acid sequence (the nucleotides in a DNA molecule) or a polypeptide sequence (the amino acids of a protein). For example, in the case of DNA, copies of the DNA to be sequenced can be made and labeled with fluorescent markers before they are identified using a sequencing machine. For proteins, single amino acid residues can be removed from one end of the protein and identified one at a time using an automated system.
The terms “comprising”, “consisting of”, and “consisting essentially of” are defined according to their standard meaning and may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term.
The invention is further described in detail by reference to the following experimental examples. These examples are provided for the purpose of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
EXAMPLE 1
Amplification of cDNAs Encoding Secreted or Membrane-Bound Proteins Containing Specific Amino Acid Motifs
In order to select cDNAs that encode secreted or membrane-bound proteins, the cloning vector G7311 (referred to herein as an “Amptrap” vector) was designed by the present inventors, which allows selection for signal sequence-encoding regions by β-lactamase rescue (as shown in FIG. 2). The Amptrap vector shown contains a 5′-truncated β-lactamase gene driven by a Lac promoter. The product of the modified β-lactamase gene lacks the signal peptide present in the wild-type protein and is therefore unable to be exported into the bacterial periplasmic space. An asymmetric pair of SfiI sites for insertion of cDNA lies immediately 5′ to the β-lactamase open reading frame (ORF). A separate neomycin phosphotransferase marker was included to allow propagation of the vector without cDNA inserts. Because β-lactamase must be secreted into the periplasmic space to produce ampicillin resistance, bacterial cells bearing an unmodified Amptrap plasmid display a kanamycin-resistant, ampicillin-sensitive (KanR AmpS) antibiotic resistance phenotype. To select for signal peptide-encoding cDNAs, cDNA sequences can be cloned directionally into the Amptrap vector at its asymmetric SfiI sites, creating a fusion transcript between the inserted cDNA and the β-lactamase gene. If an inserted ORF contains a methionine start codon coupled to a signal peptide that is in frame with the β-lactamase ORF, secretion of β-lactamase is restored and transformed bacterial clones acquire a KanR AmPR doubly resistant phenotype, allowing their direct selection on Kan+Amp medium.
In order to increase specificity in cloning using the Amptrap vector, the inventors adopted a single short-primer PCR strategy for amplification of novel gene family members. In this technique, PCR amplification of cDNA is performed using a 5′-RACE primer coupled with a degenerate 3′ antisense primer, representing between three and five residues of a conserved amino acid motif in a given family of proteins. An anchor sequence containing an SfiI site is coupled to the 3′ degenerate sequence for subsequent cloning. The spacing between the degenerate codons and the SfiI site in the 3′ primer was designed so that rescue of β-lactamase secretion would require translation of the degenerate sequence in the desired frame upon cloning and expression in the Amptrap vector. Various primer sets were used to amplify subsets of cDNA sequences containing the 5′ regions of cDNAs plus potential coding sequence, all of which ended in the primer-encoded amino acid motifs.
EXAMPLE 2
Cloning of Expressed Sequence Tags (EST) Sequences Using the Amptrap Vector
cDNA was synthesized from Florida lancelet (Branchiostoma floridae) and sea lamprey (Petromyzon marinus) tissues and cloned into an Amptrap vector. 57 sequences were analyzed by BLASTX searching of the Genbank database. Although 17 sequences failed to match known sequences in Genbank, all of the remaining 40 sequences were found to encode proteins that are likely to be secreted or bound to membranes (Table 1).







TABLE 1







Beta-lactamase fusion (“amptrap”) EST sequences












G#
Tissue
Source
Stock?
Gel date
Comments





7024
PMP
20000827, clone 3
Y
20000928
no match*


7026
PMI
20000827, clone 5
Y
20000830
trypsinogen b1†


7027
PMI
20000827, clone 6
Y
20000830
no match*


7031
PMI
20000827, clone 10
Y
20000830
trypsinogen b1†


7033
PMI
20000827, clone 12
Y
20000830
trypsinogen b1†


7034
PMI
20000827, clone 13
Y
20000905
trypsinogen b1†


7035
PMI
20000827, clone 14
Y
20000905
chymotrypsinogen†


7037
PMI
20000827, clone 16
Y
20000905
trypsinogen†


7038
PMI
20000827, clone 17
Y
20000905
trypsinogen†


7039
PMI
20000827, clone 18
Y
20000928
no match*


7046
PMP
20000831, clone 3
Y
20000905
no match*


7047
PMI
20000831, clone 19
Y
20000905
trypsinogen†


7048
PMI
20000831, clone 21
Y
20000905
no match*


7049
PMI
20000831, clone 29
Y
20000905
chymotrypsinogen†


7212
PMI + PMP
20000915, clone 1
Y
20000928
elastase†


7213
PMI + PMP
20000915, clone 2
Y
20000928
chymotrypsin-like†


7214
PMI + PMP
20000915, clone 3
Y
20000928
no match*


7215
PMI + PMP
20000915, clone 4
Y
20000928
cytochrome C oxidase†


7216
PMI + PMP
20000915, clone 5
Y
20000928
defender against cell death-1 (DAD-1)†


7535
PMI + PMP
array, plate 3, C1
Y
20001120
no match*


7536
PMI + PMP
array, plate 3, D1
Y
20001120
trypsinogen†


7537
PMI + PMP
array, plate 3, E1
Y
20001120
no match*


7538
PMI + PMP
array, plate 3, F1
Y
20001120
no match*


7541
PMI + PMP
array, plate 3, A2
Y

trypsinogen†


7542
PMI + PMP
array, plate 3, B2
Y

procolipase†


7543
PMI + PMP
array, plate 3, C2
Y

trypsinogen†


7544
PMI + PMP
array, plate 3, D2
Y

trypsinogen†


7545
PMI + PMP
array, plate 3, E2
Y

trypsinogen†


7546
PMI + PMP
array, plate 3, F2
Y

trypsinogen†


7547
PMI + PMP
array, plate 3, G2
Y

trypsinogen†


7548
PMI + PMP
array, plate 3, H2
Y

trypsinogen†


7676
PMI + PMP
array, plate 3, F6
Y
20001213
trypsinogen a†


7677
PMI + PMP
array, plate 3, H6
Y
20001213
no match*


7679
PMI + PMP
array, plate 3, D8
Y
20001213
trypsinogen a†


7681
PMI + PMP
array, plate 3, H8
Y
20001213
trypsinogen b†


7682
PMI + PMP
array, plate 3, A9
Y
20001213
trypsinogen a†


7683
PMI + PMP
array, plate 3, B9
Y
20001213
trypsinogen a†


7684
PMI + PMP
array, plate 3, C9
Y
20001213
trypsinogen b†


7685
PMI + PMP
array, plate 3, G9
Y
20001213
trypsinogen b†


7686
PMI + PMP
array, plate 3, H9
Y
20001213
trypsinogen b†


7687
PMI + PMP
array, plate 3, A10
Y
20001213
trypsinogen b†


7688
PMI + PMP
array, plate 3, E10
Y
20001213
no match*


7689
PMI + PMP
array, plate 3, G10
Y
20001213
trypsinogen b†


7690
PMI + PMP
array, plate 3, A11
Y
20001213
trypsinogen b†


7691
PMI + PMP
array, plate 3, F11
Y
20001213
trypsinogen b†


7692
PMI + PMP
array, plate 3, G11
Y
20001213
no match*


7694
PMI + PMP
array, plate 3, C12
Y
20001213
trypsinogen b†


7695
PMI + PMP
array, plate 3, D12
Y
20001213
trypsinogen b†


7696
PMI + PMP
array, plate 3, F12
Y
20001213
trypsinogen b†


7748
BFD
pilot ligations, G7637
N
20001222
cytochrome C oxidase subunit III†


7750
BFD
pilot ligations, G7637
N
20001222
no match*


7752
BFD
pilot ligations, G7637
N
20001222
no match*


7754
BFD
pilot ligations, G7637
N
20001222
calsequestrin 1†


7756
BFD
pilot ligations, G7637
N
20001222
no match*


7758
BFD
pilot ligations, G7637
N
20001222
NADH dehydrogenase subunit 4L†


7760
BFD
pilot ligations, G7637
N
20001222
no match*


7762
BFD
pilot ligations, G7637
N
20001222
no match*





*No matches after BLASTX search of Genbank


†Membrane protein


‡Artifact (intracellular protein or 3′ UTR of cDNA))


BFD Branchiostoma floridae pooled dorsal regions


PMI Petromyzon marinus intestine


PMP Petromyzon marinus protovertebral arch






EXAMPLE 3
Amplification of Candidate Immune-Type Receptor Genes from Branchiostoma floridae, Raja eglanteria, and Petromyzon marinus 
In order to identify potential new members of the novel immune-type receptor (NITR) gene family previously described in teleost fish, cDNA sequences from Florida lancelet Branchiostoma floridae, clearnose skate Raja eglanteria, and sea lamprey Petromyzon marinus tissues were amplified by 5′-RACE PCR using various 3′ primers and the 5′-SMART oligonucleotide. These primers included:










nitrVYWFR-Sfi:
5′TGGCCGAGGCGGCCCNCGRAACCARTANAC-3′;
(SEQ ID NO. 8)



 


nitrVYWF-Sfi:
5′GACTGGCCGAGGCGGCCCRAACCARTANAC-3′;
(SEQ ID NO. 9)


 


nitrYWFR-Sfi:
5′-GACTGGCCGAGGCGGCCCNCGRAACCARTA-3′;
(SEQ ID NO. 10)


 


nitrYWFK-Sfi:
5′-GACTGGCCGAGGCGGCCCYTTRAACCARTA-3′;
(SEQ ID NO. 11)


 


nitrWFR1-Sfi:
5′-GACTGGCCGAGGCGGCCCNCGRAACCA-3′;
(SEQ ID NO. 12)


 


nitrWFR2-Sfi:
5′-GACTGGCCGAGGCGGCCCYCTRAACCA-3′; and
(SEQ ID NO. 13)


 


nitrWFK-Sfi:
5′GACTGGCCGAGGCGGCCCYTTRAACCA-3′.
(SEQ ID NO. 14)






The pool of amplicons were subsequently cloned into the Amptrap vector. After sequence analysis of 222 amplicons, 148 amplicons were found to encode secreted or membrane-bound proteins, 19 amplicons encoded artifactual sequences (ORFs for intracellular proteins or 3′ untranslated regions of cDNAs), and 55 amplicons failed to match any known sequences in Genbank (available at www.ncbi.nlm.nih.gov) after BLASTX searching. 41 of the 222 amplicons encoded candidate immunoglobulin-superfamily domains, which were the targets of the screen (Table 2).







TABLE 2







Beta-lactamase fusion (“Amptrap”) PCR-directed sequences











Target




G#
(primer)
Tissue
Comments





7950
NITR
OM
integral membrane protein 2B†



(pool of 7)


7951

OM
immunoglobulin superfamily molecule†


7952

OM
immunoglobulin superfamily molecule†


7953

OM
tetraspan protein family (TM4SF)†


7954

OM
immunoglobulin superfamily





molecule (2)†


7955

OM
immunoglobulin superfamily molecule†


7956

OM
immunoglobulin superfamily molecule†


7957

OM
immunoglobulin superfamily molecule†


7958

OM
immunoglobulin superfamily molecule†


7961

DR
No Match*


7962

DR
No Match*


7963

DR
claudin†


7964

DR
claudin†


7965

DR
claudin†


7966

DR
claudin†


7967

DR
claudin†


7968

DR
lipid kinase (?)†


7969

DR
claudin†


7971

DR
claudin†


7976

BFV
NADH dehydrogenase†


7977

BFV
lgSF domain†


8020
NITR
Reg
transmembrane protein, PIGPC1†



(pool of 7)


8021

Reg
transmembrane protein, PIGPC1†


8022

Reg
transmembrane protein, PIGPC1†


8023

Reg
transmembrane protein, PiGPC1†


8024

Reg
Immunoglobulin light chain II/III†


8025

Reg
transmembrane protein, PIGPC1†


8026

Reg
3′ UTR‡


8028

Reg
immunoglobulin superfamily molecule†


8031

Reg
Candidate immunoglobulin superfamily





molecule†


8032

Reg
transmembrane protein, PIGPC1†


8033

Reg
Candidate immunoglobulin superfamily





molecule†


8034

XL
Golgi membrane protein p18†


8036

XL
MHC Class II†


8037

XL
MHC Class II†


8038

XL
immunoglobulin light chain†


8039

XL
immunoglobulin light chain†


8052
CD3
BFV
α-amylase†



(YQPL)


8053

BFV
α-amylase†


8054

BFV
α-amylase†


8055

BFV
α-amylase†


8150
NITR
Reg
candidate lg domain (distinct from



(pool of 7)

G8152)†


8151

Reg
candidate lg domain (distinct from





G8152)†


8152

Reg
immunoglobulin superfamily molecule†


8153

Reg
candidate lg domain (distinct from





G8152)†


8227
MHC
BFV
amphi-lipase†



(CHVEH)


8293
NITR
BFV
No Match*



(pool of 5)


8294

BFV
No Match*


8295

BFV
No Match*


8296

BFV
No Match*


8297

BFV
immunoglobulin superfamily molecule†


8298

BFV
No Match*


8309
NITR
BFV
cytochrome C oxidase†



(WFR1,



WFR2)


8310

BFV
CD81/CD9-like†


8311

BFV
No Match*


8312

BFV
CD81/CD9-like†


8313

BFV
CD81/CD9-like†


8314

BFV
cytochrome B†


8315

BFV
No Match*


8316

BFV
CD81/CD9-like†


8317

BFV
No Match*


8318

BFV
CD81/CD9-like†


8358
MHC (CXV)
BFV
Cathepsin


8359

BFV
Fibropellin III, Notch (?)†


8360

BFV
Fibropellin III, Notch (?)†


8361

BFV
No Match*


8362

BFV
Fibropellin III, Notch (?)†


8363

BFV
Notch, SP1070 (D. melanogaster)†


8364

BFV
folate receptor†


8365
MHC
BFV
myosin heavy chain, 3′ end‡



(CXVXH2)


8366
MHC (CXV)
Reg
MHC Class II†


8367

Reg
MHC Class II†


8368

Reg
No Match*


8369

Reg
MHC Class II†


8370

Reg
MHC Class II†


8371

Reg
MHC Class II†


8372

Reg
connective tissue growth factor†


8373

Reg
connective tissue growth factor†


8382
NITR (1–5
BFV
No Match*



OR 6–7)


8383

BFV
CD81/CD9-like†


8384

BFV
cytochrome C oxidase†


8385

BFV
cytochrome C oxidase†


8386

BFV
CD81/CD9-like†


8387

BFV
cytochrome B†


8388

BFV
PDGF-b (?)A


8390

BFV
CD81/CD9-like†


8391

BFV
CD81/CD9-like†


8392

BFV
NADH dehydrogenase†


8393

BFV
cytochrome B†


8394
MHC (CXV)
BFV
fibropellin III†


8395

BFV
fibropellin III†


8396

BFV
No Match*


8397

BFV
Notch2†


8398

BFV
No Match*


8399

BFV
α2-macroglobulin receptor (LDL-related)†


8400

BFV
collagen (?)†


8401

BFV
asialoglycoprotein receptor†


8402

BFV
Cathepsin-like (?)†


8403

BFV
fibropellin III†


8404

BFV
No Match*


8405

BFV
Cathepsin-L-like†


8432
NITR (1–4
PMP
No Match*



OR 5–7)


8433

PMP
Transport protein (?)†


8435

PMP
No Match*


8436

PMP
similar to repeat-rich proteins‡


8437

PMP
Repetitive sequence‡


8438

PMP
Repetitive sequence‡


8439

PMP
Collagen†


8444

PMP
Repetitive sequence‡


8445

PMP
Repetitive sequence‡


8456
MHC (CXV)
PMP
No Match*


8457

PMP
No Match*


8458

PMP
β-actin‡


8459

PMP
β-actin‡


8460

PMP
lysosomal transporter protein†


8461

PMP
lysosomal transporter protein†


8462

PMP
lysosomal transporter protein†


8463

PMP
lysosomal transporter protein†


8488
“J” (FGXG)
BFV
short ORF with signal sequence; not





lg-like†


8489

BFV
short ORF with signal sequence; not





lg-like†


8490

BFV
short ORF with signal sequence; not





lg-like†


8491

BFV
short ORF with signal sequence; not





lg-like†


8492

BFV
short ORF with signal sequence; not





lg-like†


8493

BFV
short ORF with signal sequence; not





lg-like†


8495

BFV
possible immunoglobulin superfamily





molecule†


8496

BFV
short ORF with signal sequence; not





lg-like†


8497

BFV
No Match*


8498
“J” (FGXG)
Reg
ATP synthase F0, subunit 6†


8503

Reg
synaptophysin-like (short region of high





similarity)†


8505

Reg
ATP synthase F0, subunit 6†


8510
“J” (GXGT)
BFV
No Match*


8511

BFV
poly-A‡


8512

BFV
Repetitive sequence? ‡


8513

BFV
“barrier to autointegration” factor‡


8514

BFV
UCC1/ependymin (ECM protein)†


8517

BFV
No Match hypothetical H. sapiens gene,





F22162_1*


8519

BFV
PSSP-94 (secreted protein)†


8520

BFV
No Match C. elegans hypothetical protein;





“NOV”*


8521

BFV
NADH dehydrogenase†


8523

BFV
potassium channel†


8527

BFV
NADH dehydrogenase†


8528

BFV
NADH dehydrogenase†


8529

BFV
α-amylase†


8530

BFV
No Match*


8531

BFV
tetraspanin -- 29Fa; D1-7; CD63†


8532

BFV
No Match*


8533

BFV
Ca-binding protein†


8538
lgSF (YXC)
BFV
scavenger receptor†


8539

BFV
cytochrome C oxidase†


8540

BFV
No Match*


8541

BFV
scavenger receptor; zonadhesin†


8542

BFV
No Match -- possibly fibropellin*


8543

BFV
cytochrome C oxidase†


8544

BFV
cytochrome C oxidase†


8545

BFV
cytochrome C oxidase†


8546

BFV
scavenger receptor†


8547

BFV
No Match*


8548

BFV
cytochrome C oxidase†


8549

BFV
cytochrome C oxidase†


8550

BFV
poly-A?‡


8552
lgSF (YXC)
Reg
No Match*


8553

Reg
No Match*


8555
“J” GXGT)
Reg
ATP synthase subunit F0†


8557

Reg
cytochrome b558α†


8561
“J” GXGT)
Reg
No Match C. elegans hypothetical protein*


8562

Reg
immunoglobulin light chain†


8563

Reg
α-interferon-inducible protein - possible





signal peptide†


8565

Reg
α-interferon-inducible protein - possible





signal peptide†


8566

Reg
No Match*


8567

Reg
No Match*


8568
lgSF (YXC)
Reg
integrin (αE)†


8569

Reg
No Match*


8570

Reg
No Match*


8571

Reg
No Match*


8573

Reg
alcohol dehydrogenase†


8574

Reg
No Match*


8575

Reg
No Match*


8589
NITR
BFV
2 lgSF domains; distinct from G7977,



(pool of 7)

G8297a


8590

BFV
No Match*


8591

BFV
No Match*


8594

BFV
No Match*


8606
NITR
BFV
2 lgSF domains; distinct from G7977,



(pool of 7)

G8297A


8608

BFV
destabilase†


8609

BFV
serine protease (?)†


8610

BFV
No Match*


8622
NITR
BFV
immunoglobulin superfamily molecule†



(pool of 7)


8623

BFV
No Match*


8624

BFV
Immunoglobulin superfamily molecule†


8625

BFV
No Match*


8630
NITR
Reg
sorcin (Ca-binding protein)‡



(pool of 7)


8631

Reg
No Match Unknown human protein*


8632

Reg
sorcin (Ca-binding protein)‡


8633

Reg
sorcin (Ca-binding protein)‡


8634

Reg
sorcin (Ca-binding protein)‡


8635

Reg
sorcin (Ca-binding protein)‡


8636

Reg
No Match*


8637

Reg
folate receptor†


8658
NITR
BFV
kettin; G8589-like†



(WFK)


8659

BFV
immunoglobulin superfamily molecule†


8660

BFV
immunoglobulin superfamily molecule†


8661

BFV
immunoglobulin superfamily molecule†


8663

BFV
immunoglobulin superfamily molecule†


8664

BFV
immunoglobulin superfamily molecule†


8665

BFV
immunoglobulin superfamily molecule†


8666

BFV
RSV receptor - ?†


8667

BFV
No Match*


8668

BFV
No Match*


8669

BFV
immunoglobulin superfamily molecule†


8670
NITR
BFV
No Match*



(WFR1)


8671
NITR
BFV
No Match*



(WFR2)


8673

BFV
CD81/CD9†


8694
NITR?
BFV
Lysozyme†



(YWC)


8696

BFV
Lysozyme†


8697

BFV
Lysozyme†


8698

Reg
Repetitive sequence?‡


8700

Reg
No Match hypothetical protein Rv1796 -





Mycobacterium tuberculosis -?*


8701

Reg
immunoglobulin heavy chain†


8702

Reg
No Match*


8704

Reg
No Match*


8717

Reg
HMG-CoA reductase - ?†


8719

Reg
WD40-repeat type I transmembrane





protein A72.5†


8720

Reg
No Match hypothetical protein Rv1796 -





Mycobacterium tuberculosis - ?*





*No matches to proteins of known function after BLASTX search of Genbank


†Membrane or secreted protein


‡Artifactual sequence (intracellular protein or 3′ UTR of cDNA)


OM Onchorynchus mykiss, head kidney


DR Danio rerio, spleen


BFV Branchiostoma floridae, pooled ventral regions


Reg Raja eglanteria, spleen


XL Xenopus laevis, spleen


PMP Petromyzon marinus, protovertebral arch






EXAMPLE 4
Amplification of Candidate Major Histocompatibility Complex (MHC) Genes from Raja eglanteria: An Example of PCR Priming Using Only Two Known Amino Acids
A PCR primer corresponding to the amino acids cys-X-val (CXV), attached to an SfiI linker (PCR primer, CXV-Sfi: 5′-GACTGGCCGAGGCGGCCCNACNNNRCA-3′ (SEQ ID NO. 15)), was used to amplify sequences from Raja eglanteria spleen cDNA. Eight KanR AmpR Amptrap clones were sequenced and compared to the Genbank database using the BLASTX algorithm. Five of the eight clones were found to encode an MHC Class II protein (Table 2: 8366–8373).
The CXV amino acid sequence is conserved in the α3 domains of many major histocompatibility complex (MHC) class I proteins, as well as in the α2 domains of MHC class II proteins and β2-microglobin (β2m). A 3′ degenerate primer complementing the CXV motif (described above), in which the second codon position is degenerate (NNN), was used in directed Amptrap analysis of spleen cDNA from the clearnose skate (Raja eglanteria, a representative cartilaginous fish (FIG. 7A). The initial PCR reaction produced a broad ethidium bromide-staining band (FIG. 7B). Reaction products were digested with Sfi I and size-selected using a Chromaspin-1000 gel filtration column (CLONTECH) to remove unincorporated primers and very short amplicons before ligation to the Amptrap vector. After transformation and selection on ampicillin plates, eight colonies, containing inserts of at least  ̃600 bp; were sequenced (FIG. 7C). Five of these colonies were found to encode MHC class II (FIG. 7D). The failure to recover MHC I amplicons was likely due to both size selection bias and the need to change cycling conditions to favor the recovery of longer transcripts (unpublished observations). A similar experiment, in which the gel filtration step was omitted, yielded an amplicon homologous to β2m. The predicted coding region of a full-length cDNA encoding the skate homolog of β2m contains a 111 amino acid open reading frame that exhibits strong similarity to mammalian β2m protein (p=10−11–10−12). The identities between this gene and other β2 ms are shown in FIG. 7E, from which several conclusions can be drawn: 1) highly significant identities exist between skate β2m and the other members in this comparison set, 2) several regions of identity between all other β2 ms are not shared by skate β2m, and 3) several identities are shared by skate β2m and some but not all other β2 ms.
Therefore, as demonstrated in FIGS. 7A–7E, the methods of the subject invention are particularly useful in cloning divergent members of a gene family using three to five amino acid motifs. At times, the second amino acid can be completely divergent, allowing cloning based on knowledge of only two amino acids, such as described above with respect to the Raja eglanteria MHC Class II genes, although the primer should still contain sequences complementary to at least three codons (with the middle sequence being completely degenerate, “NNN”, in such a case).
EXAMPLE 5
Amplification of Candidate Genes from Amphioxus
Another example of cloning using the methods of the subject invention is presented in FIGS. 8A–8E. Seven unique primers were designed to complement three to five amino acid motifs surrounding a single conserved tryptophan (W) residue in the N-terminal Ig domains of novel immune-type receptors, which have been interpreted to possibly reflect a conserved feature of primordial immune receptors. These primers were:











(1)
nitrVYWFR-Sfi
(5′-TGGCCGAGGCGGCCCNCGRAACCARTANAC-3′;
(SEQ ID NO. 8))



 


(2)
nitrVYWF-Sfi
(5′-GACTGGCCGAGGCGGCCCRAACCARTANAC-3′;
(SEQ ID NO. 9))


 


(3)
nitrYWFR-Sfi
(5′-GACTGGCCGAGGCGGCCCNCGRAACCARTA-3′;
(SEQ ID NO. 10))


 


(4)
nitrYWFK-swfi
(5′-GACTGGCCGAGGCGGCCCYTTRAACCARTA-3′;
(SEQ ID NO. 11))


 


(5)
nitrWFR1-Sfi
(5′-GACTGGCCGAGGCGGCCCNCGRAACCA-3′;
(SEQ ID NO. 12))


 


(6)
nitrWFR2-Sfi
(5′-GACTGGCCGAGGCGGCCCYCTRAACCA-3′; and
(SEQ ID NO. 13))


 


(7)
nitrWFK-Sfi
(5′-GACTGGCCGAGGCGGCCCYTTRAACCA-3′.
(SEQ ID NO. 14))






These primers were used in individual reactions to amplify cDNA from amphioxus, as shown in FIG. 8A. The initial 5′ RACE PCR produced a 200 bp-2 kilobase (kb) polydisperse distribution of product without any prominent bands, as shown in FIG. 8B). The insert sizes of ampicillin resistant colonies were analyzed directly using PCR (FIG. 8C), and eight colonies containing inserts in the range of  ̃250–800 bp were selected for sequence analysis. Clone G7977 (FIG. 8D), which was amplified using a degenerate primer corresponding to the amino acid sequence Trp-Phe-Lys (WFK) (primer #7 above), encodes a 57 amino acid open reading frame with similarity to Ig V regions 5′ to the primer binding site. Using the G7977 amplicon as a hybridization probe, a full-length cDNA encoding a transmembrane protein bearing an IgSF domain at its N-terminus was isolated, followed by membrane-proximal extracellular domain of unknown function (FIG. 8E). Inspection of the full-length cDNA sequence, recovered separately from the Amptrap PCR, confirmed that the native sequence contains appropriately placed codons for the amino acid sequence WFK.
EXAMPLE 6
Identification of Domains Containing Genes Encoding Chitin Binding Proteins (CBPs)
The primers described in Example 5 also permitted the identification of three distinct families of amplicons with ORFs that encode IgSF proteins but do not match known molecules after standard BLASTP searches. These amplicons were labeled individually and used as probes to clone full-length cDNA sequences representing each of the three families. Sequencing of the full-length clones showed that all three families encode putative secreted proteins containing two Ig domains at their N-termini and single putative chitin binding domains at their C-termini, as shown in FIG. 9A. Because of their lengths and the presence of conserved “V” domain amino acids within each domain, the Ig domains of all of these proteins are best classified as “V” type; although similar in structure, the three families, designated V-region containing chitin binding proteins (V-CBPs) share only limited amino acid sequence identity (27–38%). The relationship of these genes to other Ig domain-encoding putative receptors that have been identified in invertebrates is unclear. FIG. 9B shows an amino acid alignment of the V-CBP Ig domains with V domains from mammalian immune receptors. Notably, the sequences exhibit V-type spacing of cysteines and share identity with the additional residues that are most conserved in Ig, TCR, NITRs and other V-type IgSF domains. Subsequent analyses have shown that the V-CBP multigene family is more extensively diversified (data not shown). An expressed recombinant V-CBP (G8297) binds chitin, and this binding is dependent on the presence of the predicted C-terminal chitin-binding domain (FIGS. 9C–9E). Finally, in situ hybridization to mRNA in transverse sections of adult B. floridae identified specific expression of G8297 in scattered cells in the intestine; identical hybridization patterns are seen with probes complementing the corresponding regions of the other two V-CBP genes.
Taken together, the above examples of Amptrap cloning demonstrate broad utility based on five successive levels of strong positive selection: 1) enrichment of 5′ ends of cDNAs using SMART technology (CLONTECH), 2) requirement for a methionine start codon in the inserted cDNA, 3) requirement for a signal peptide open reading frame downstream of the start codon, 4) requirement for conserved amino acid codons being in-frame with the start codon and open reading frame signal peptide, and 5) requirement for a specified distance between the 5′ end of the cDNA and the 3′ degenerate primer binding site, which defines a basis for size selection. By requiring a start codon in the cloned sequence, competing artifactual priming is reduced through minimization of introns, intergenic DNA regions and untranslated regions, all of which account for high levels of artifactual amplicons in other PCR-based cloning methods. In each experiment described herein, and numerous other applications (unpublished), relatively few clones are recovered but the frequency of significant targets is very high. In some cases, this integrated series of selection steps can result in the majority of sequenced clones containing inserts with the desired characteristics. In comparing Amptrap cloning to other systems, it is important to recognize that Amptrap is based on selection rather than simple screening; clones encoding irrelevant proteins are deleted from the experiment and do not appear in the pool of colonies for analysis. Amptrap selection for cDNAs allows cloning and selection to occur in bacteria, which are highly amenable to DNA transformation and propagation; clearly such an approach is preferable to yeast selection strategies, which have not received widespread application.
The preceding descriptions of the invention are merely illustrative and should not be considered as limiting the scope of the invention in any way. From the foregoing description, one of ordinary skill in the art can easily ascertain the essential characteristics of the instant invention, and without departing from the spirit and scope thereof, can make various changes and/or modifications of the inventions to adapt it to various usages and conditions. As such, these changes and/or modifications are properly, equitably, and intended to be, within the full range of equivalence of the following claims.
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

## Claims

1. A vector comprising the nucleic acid sequence of SEQ ID NO:1.

2. A method for selecting nucleic acid sequences encoding secreted or membrane-bound proteins which comprises:
(a) linearizing the vector of claim 1 with one or more restriction enzymes, wherein SEQ ID NO: 1 comprises a reporter gene, a selectable marker gene and a multiple cloning site, wherein the reporter gene encodes β-lactamase lacking a functional signal sequence, and wherein the reporter gene and selectable marker gene are operably linked to a promoter sequence;
(b) cutting a candidate nucleic acid sequence with the one or more restriction enzymes and ligating the candidate nucleic acid sequence to the linearized vector, thereby forming a ligation product, wherein the candidate nucleic acid sequence encodes a potential secreted or membrane-bound protein;
(c) transforming bacterial cells with the ligation product; and
(d) selecting for colonies based on export of β-lactamase to the periplasmic space of the bacterial cells.

3. A method for selecting nucleic acid sequences encoding secreted or membrane-bound proteins which comprises:
(a) providing the vector of claim 1, wherein the vector is linearized, wherein SEQ ID NO: 1 comprises a reporter gene, a selectable marker gene and a multiple cloning site, wherein the reporter gene encodes β-lactamase lacking a functional signal sequence, and wherein the reporter gene and selectable marker gene are operably linked to a promoter sequence;
(b) ligating a candidate nucleic acid sequence to the linearized vector, thereby forming a ligation product, wherein the candidate nucleic acid sequence encodes a potential secreted or membrane-bound protein;
(c) transforming bacterial cells with the ligation product; and
(d) selecting for colonies based on export of β-lactamase to the periplasmic space of the bacterial cells.

4. A vector comprising the nucleic acid sequence of SEQ ID NO. 2.

5. A method for selecting nucleic acid sequences encoding secreted or membrane-bound proteins which comprises:
(a) linearizing the vector of claim 4 with one or more restriction enzymes, wherein SEQ ID NO: 2 comprises a reporter gene, a selectable marker gene and a multiple cloning site, wherein the reporter gene encodes β-lactamase lacking a functional signal sequence, and wherein the reporter gene and selectable marker gene are operably linked to a promoter sequence;
(b) cutting a candidate nucleic acid sequence with the one or more restriction enzymes and ligating the candidate nucleic acid sequence to the linearized vector, thereby forming a ligation product, wherein the candidate nucleic acid sequence encodes a potential secreted or membrane-bound protein;
(c) transforming bacterial cells with the ligation product; and
(d) selecting for colonies based on export of β-lactamase to the periplasmic space of the bacterial cells.

6. A method for selecting nucleic acid sequences encoding secreted or membrane-bound proteins which comprises:
(a) providing the vector of claim 4, wherein the vector is linearized, wherein SEQ ID NO: 1 comprises a reporter gene, a selectable marker gene and a multiple cloning site, wherein the reporter gene encodes β-lactamase lacking a functional signal sequence, and wherein the reporter gene and selectable marker gene are operably linked to a promoter sequence;
(b) ligating a candidate nucleic acid sequence to the linearized vector, thereby forming a ligation product, wherein the candidate nucleic acid sequence encodes a potential secreted or membrane-bound protein;
(c) transforming bacterial cells with the ligation product; and
(d) selecting for colonies based on export of β-lactamase to the periplasmic space of the bacterial cells.