Shuttle vectors

The invention provides shuttle vectors, and methods of using shuttle vectors, capable of expression in, at least, a mammalian cell. Furthermore, the shuttle vectors are capable of replication in at least yeast, and optionally, bacterial cells. Also provided is a method wherein yeast are transformed with a shuttle vector as provided herein. Heterologous nucleic acids flanked by 5' and 3' ends identical to a homologous recombination site within the shuttle vector are introduced to the transformed yeast and allowed to homologously recombine with the shuttle vector such that they are inserted into the vector by the yeast organism. The shuttle vector is then recovered and transferred to a mammalian cell for expression.

FIELD OF THE INVENTION
 The invention relates to novel shuttle vectors, and more particularly,
 shuttle vectors capable of replication in at least yeast and capable of
 expression in at least a mammalian cell.
 BACKGROUND OF THE INVENTION
 The introduction of cloned nucleotide sequences into mammalian cells has
 greatly facilitated the study of the control and function of various
 eukaryotic genes. Mammalian cells provide an environment conducive to
 appropriate protein folding, post translational processing, feedback
 control, protein-protein interactions, and other eukaryotic protein
 modifications such as glycosylation and oligomerization. Thus, a number of
 expression vectors have been developed which allow the expression of a
 polypeptide in a mammalian cell.
 The typical mammalian expression vector will contain (1) regulatory
 elements, usually in the form of a viral promoter or enhancer sequences;
 (2) a multicloning site, usually having specific enzyme restriction sites
 to facilitate the insertion of a DNA fragment with the vector; and (3)
 sequences responsible for intron splicing and polyadenylation of mRNA
 transcripts. Generally, sequences facilitating the replication of the
 vector in both bacterial and mammalian hosts and a selection marker gene
 which allows selection of transformants in bacteria are also included. The
 bacterial elements, or in some cases phage elements, are included to
 provide the option of further analyzation of the nucleic acid inserts
 amplified and isolated from the bacteria or phage.
 In the past, the insertion of a heterologous nucleic acid (insert) into the
 multicloning site of a mammalian expression vector has generally been
 accomplished by one of two methods. In the first method, the insert is cut
 out of a bacterial expression vector and ligated into the mammalian
 expression vector. In the second method, often called "TA cloning",
 special ends are generated on the insert by PCR such that the modified
 insert can be put into the mammalian expression vector. Each of these
 methods requires a number of steps including enzymatic reactions which can
 be labor intensive and unreliable. Moreover, cloning efficiency drops
 significantly as the size of the insert increases.
 Another method used for inserting a heterologous nucleic acid (insert) into
 an expression vector takes advantage of yeast's high efficiency at
 homologous recombination in vivo. In this method, a nucleic acid fragment
 flanked by 5' and 3' homologous regions is co-introduced into a yeast with
 a vector which has regions identical to the 5' and 3' regions flanking the
 fragment. The yeast efficiently homologously recombines such that the
 fragment inserts into the region of the vector flanked by the
 before-mentioned 5' and 3' regions. H.a., et al., Plasmid, 38:91-96
 (1997), incorporated herein. Unfortunately, yeast are the only organisms
 able to efficiently recombine so as to insert heterologous nucleic acids
 into a vector. Therefore, to date, there is not an efficient method or
 means to transfer inserts into a specific region of a vector used for
 expression in mammalian cells.
 Accordingly, it is an object of the invention to provide compositions and
 methods useful in facilitating the insertion of a heterologous nucleic
 acid into a vector which can express the heterologous nucleic acid in at
 least a mammalian cell.
 Moreover, it is the object of this invention to provide a shuttle vector
 and methods of use which allow replication of the shuttle vector at least
 in yeast and which allow expression in at least a mammalian cell.
 SUMMARY OF THE INVENTION
 The invention provides shuttle vectors, and methods of using shuttle
 vectors, capable of expression in at least a mammalian cell. Furthermore,
 the shuttle vectors are capable of replication in at least yeast, and
 optionally, bacterial cells.
 In one aspect of the invention, the invention provides a shuttle vector
 comprising an origin of replication functional in yeast and preferably, a
 reporter gene functional in yeast. The shuttle vector further comprises a
 promoter functional in a mammalian cell, capable of directing
 transcription of a polypeptide coding sequence operably linked to said
 promoter.
 In another aspect of the invention, the shuttle vector comprises an
 insertion site operably linked to said promoter. The insertion site
 preferably allows for homologous recombination with a heterologous nucleic
 acid. In one embodiment, the insertion site has 5' and 3' regions
 identical to 5' and 3' regions flanking a nucleic acid to be inserted into
 the vector.
 Optionally, the shuttle vector comprises any one or more of the following:
 an internal ribosome entry sequence (IRES), a polyadenylation sequence and
 a splice sequence.
 In another aspect of the invention, the shuttle vector further comprises an
 origin of replication functional in a bacterial cell and preferably, a
 selectable gene functional in a bacterial cell. The shuttle vector may
 also comprise an origin of replication functional in a mammalian cell, and
 optionally, a selectable gene functional in a mammalian cell.
 The present invention also provides methods for using the shuttle vectors
 provided herein. In one embodiment, heterologous nucleic acids flanked by
 regions identical to flanking regions of the insertion site within a
 shuttle vector are co-introduced to yeast with the shuttle vector and
 allowed to homologously recombine such that the heterologous nucleic acids
 are inserted into the shuttle vector by the yeast organism. In preferred
 embodiments, the heterologous nucleic acids are introduced to the yeast in
 a linear nucleic acid. The shuttle vector is then recovered and
 transferred to a mammalian cell for expression.

DETAILED DESCRIPTION OF THE INVENTION
 The invention provides shuttle vectors, and methods of use, wherein the
 shuttle vectors are capable of expression in at least a mammalian cell and
 capable of replication in at least yeast. In the past, vectors have been
 constructed so as to be functional in certain aspects either in mammalian
 cells or yeast, but not both. As described herein, different hosts provide
 different advantages to an expression vector and in particular, to the
 expression product. By providing a vector which is functional as described
 herein in multiple hosts, the invention allows the advantages provided by
 varying hosts to be obtained by the use of a single tool.
 For example, a vector having the ability to replicate in yeast is useful
 for a variety of reasons. An advantage of the yeast system is its
 efficiency at homologous recombination. Orr-Weaver, et al., PNAS USA,
 80:4417-4421 (1983), incorporated herein by reference. By taking advantage
 of yeast's ability to insert heterologous nucleic acids into a vector,
 this eliminates the steps of manipulating the ends of the vector and the
 heterologous nucleic acid and ligating the two together. Another advantage
 of this system is that yeast can be transformed with large nucleic acids,
 i.e., up to at least 10 kilobases, which can then be inserted into the
 vector.
 Moreover, yeast is a well-studied organism which facilitates its use. In
 particular, yeast has been widely used to detect protein-protein
 interactions in the "two-hybrid system". The two-hybrid system is a method
 used to identify and clone genes for proteins that interact with a protein
 of interest. Briefly, the system indicates protein-protein interaction by
 the reconstitution of GAL4 function, which is detectable and only occurs
 when the proteins interact. This system and general methodologies
 concerning the transformation of yeast with expressible vectors are
 described in Cheng-Ting et al., PNAS USA, 88:9578-9582 (1991), Fields and
 Song, Nature, 340:245-246 (1989), and Chevray and Nathans, PNAS USA,
 89:5789-5793 (1992), each incorporated herein in their entirety.
 Regarding mammalian cells, these cells are preferred for the expression of
 eukaryotic proteins particularly when determining or studying the function
 of the protein. Mammalian cells are able to reproduce the protein's proper
 glycosylation and oligomerization, folding, post translational processing,
 feedback control, protein-protein interaction, etc., and thus are
 advantageous for expression of eukaryotic and particularly mammalian
 proteins.
 Regarding bacterial cells or phage, these systems are also very well
 studied and are therefore easily manipulated. In particular, bacteria and
 phage are useful for the rapid amplification of nucleic acids.
 Thus, while vectors which function in one of yeast, mammalian cells,
 bacteria or phage are useful, vectors which can successfully shuttle
 between these systems are particularly desirable. This invention provides
 such vectors. In a preferred embodiment, the shuttle vector functions in
 both yeast and mammalian cells. Such a shuttle vector can allow for the
 exploitation of the yeast two-hybrid system as well as yeast's ability to
 homologously recombine, as well as provide a convenient means for
 subsequent expression in mammalian cells to, for example, verify
 protein-protein interactions, study the protein's function, etc.
 In one embodiment, the invention provides a shuttle vector comprising an
 origin of replication functional in yeast and a promoter functional in a
 mammalian cell. Preferably, the shuttle vector also comprises a selectable
 gene functional in yeast.
 The origin of replication functional in yeast is any nucleic acid sequence
 which allows replication of the shuttle vector independently from the
 chromosome. Generally, the origin of replication is functional in at least
 one or more of the following: Saccharomyces cerevisiae, Candida albicans
 and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K.
 lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe,
 and Yarrowia lipolytica. Suitable origin of replication sites include, for
 example, ars 1, centromere ori, and 2.mu. ori. Yeast origin of replication
 sites can be used to increase the copy number and to retrieve the vector
 from yeast.
 The "promoter functional in a mammalian cell" or "mammalian promoter" is
 capable of directing transcription of a polypeptide coding sequence
 operably linked to said promoter. The choice of the promoter will depend
 in part on the mammalian cell into which the vector is put. Generally,
 this promoter is functional in at least one or more of the following:
 Chinese hamster ovary (CHO), BHK, 293, Hela, NH3T3 and COS cells. More
 specific examples include monkey kidney CV1 line; human embryonic kidney
 line (293 or 293 cells subcloned for growth in suspension culture, Graham
 et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR
 (CHO, Urlaub and Chasin, Proc Natl. Acad. Sci. USA, 77:4216 (1980)); mouse
 sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung
 cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse
 mammary tumor (MMT 060562, ATCC CCL51). The selection of the appropriate
 host cell is deemed to be within the skill in the art.
 Transcription from vectors in mammalian host cells is controlled, for
 example, by promoters obtained from the genomes of viruses such as
 adenoviruses, retroviruses, lentiviruses, herpes viruses, including but
 not limited to, polyoma virus, fowlpox virus (UK 2,211,504 published Jul.
 5, 1989), adenovirus 2, bovine papilloma virus, avian sarcoma virus,
 cytomegalovirus (CMV), hepatitis-B virus, Simian Virus 40 (SV40), Epstein
 Barr virus (EBV), feline immunedeficiency virus (FIV), and Sr.alpha., or
 are respiratory synsitial viral promoters (RSV) or long terminal repeats
 (LTRs) of a retrovirus, i.e., a Moloney Murine Leukemia Virus (MoMuLv)
 (Cepko et al. (1984) Cell 37:1053-1062). Moreover, the promoters can be
 selected from heterologous mammalian promoters, e.g., the actin promoter
 or an immunoglobulin promoter, and from heat-shock promoters, and
 functional derivatives thereof, provided such promoters are compatible
 with the host cell systems. The promoter functional in a mammalian cell
 can be inducible or constitutive.
 In an embodiment provided herein, the shuttle vector is double stranded and
 on a first strand, comprises a first promoter operably linked to either a
 coding sequence or a site for the insertion of a coding sequence of
 interest (i.e., a heterologous nucleic acid) followed by a polyadenylation
 site. On a second strand, the shuttle vector comprises two LTRs flanking
 said region comprising said first promoter and coding sequence or cloning
 site, wherein the LTRs operate in a direction opposite to said first
 promoter.
 "Operably linked" as used herein means that the transcriptional and
 translational regulatory nucleic acid is positioned relative to any coding
 sequences in such a manner that transcription is initiated. Generally,
 this will mean that the promoter and transcriptional initiation or start
 sequences are positioned 5' to the coding region. The transcriptional and
 translational regulatory nucleic acid will generally be appropriate to the
 host cell used, as will be appreciated by those in the art.
 By "vector" or "episome" herein is meant a nucleic acid replicon used for
 the transformation of host cells. The vectors may be either
 self-replicating extrachromosomal vectors or vectors which integrate into
 a mammalian host genome, such as a retroviral based vector. In a preferred
 embodiment, the shuttle vector remains as an extrachromosomal vector in
 bacteria and yeast, and is integrated into the genome of the mammalian
 cell.
 A preferred embodiment utilizes retroviral desired vectors. Currently, the
 most efficient gene transfer methodologies harness the capacity of
 engineered viruses, such as retroviruses, to bypass natural cellular
 barriers to exogenous nucleic acid uptake. The use of recombinant
 retroviruses was pioneered by Richard Mulligan and David Baltimore with
 the Psi-2 lines and analogous retrovirus packaging systems, based on NIH
 3T3 cells (see Mann et al., Cell 33:153-159 (1993), hereby incorporated by
 reference). Such helper-defective packaging lines are capable of producing
 all the necessary trans proteins--gag, pol, and env--that are required for
 packaging, processing, reverse transcription, and integration of
 recombinant genomes. Those RNA molecules that have in cis the .psi.
 packaging signal are packaged into maturing virions. In addition,
 transfection efficiencies of retroviruses can be extremely high, thus
 obviating the need for selection genes in some cases.
 Retroviral transfection systems are further described in Mann et al.,
 supra: Pear et al., PNAS USA 90(18):8392-6 (1993); Kitamura et al., PNAS
 USA 92:9146-9150 (1995); Kinsella et al., Human Gene Therapy 7:1405-1413;
 Hofmann et al., PNAS USA 3:5185-5190; Choate et al., Human Gene Therapy
 7:2247 (1996); WO 94/19478; PCT US97/01019, and references cited therein,
 all of which are incorporated by reference.
 Any number of suitable retroviral vectors may be used to construct the
 shuttle vectors of the invention. Preferred retroviral vectors include a
 vector based on the murine stem cell virus (MSCV) (see Hawley et al., Gene
 Therapy 1:136 (1994)) and a modified MFG virus (Rivere et al., Genetics
 92:6733 (1995)), and pBABE (see PCT US97/01019, incorporated by
 reference), and functional derivatives thereof.
 In addition, it is possible to configure a retroviral vector to allow
 expression of genes after integration in target cells. For example,
 Tet-inducible retroviruses can be used to express genes (Hoffman et al.,
 PNAS USA 93:5185 (1996)). Expression of this vector in cells is virtually
 undetectable in the presence of tetracycline or other active analogs.
 However, in the absence of Tet, expression is turned on to maximum within
 48 hours after induction, with uniform increased expression of the whole
 population of cells that harbor the inducible retrovirus, indicating that
 expression is regulated uniformly within the infected cell population.
 The shuttle vector can also be based on a non-retroviral vector. Any number
 of known vectors are suitable, including, but not limited to, pREP9,
 pCDNA, pCEP4 (Invitrogen), pCI and pCI-NEO (Promega). Basically, any
 vector can be reconstructed to contain the components as described herein.
 For example, construction of suitable vectors containing the components
 described herein can be achieved by employing standard ligation techniques
 which are known to the skilled artisan, using cloned or synthetic
 sequences.
 In a preferred embodiment, the shuttle vector includes a selectable gene
 functional in yeast (also referred to herein as a yeast reporter gene). By
 "selectable gene" or "reporter gene" herein is meant a gene that by its
 presence in a host cell, i.e. upon expression, can allow the host to be
 distinguished from a cell that does not contain the selectable gene.
 Selectable genes can be classified into several different types, including
 survival and detection genes. It may be the nucleic acid or the protein
 expression product that causes the effect. Additional components, such as
 substrates, ligands, etc., may be additionally added to allow selection or
 sorting on the basis of the selectable gene.
 In a preferred embodiment, the selectable gene is a survival gene that
 serves to provide a nucleic acid (or encode a protein) without which the
 cell cannot survive, such as a drug resistant gene, a growth regulatory
 gene, or a nutritional requirement. The selectable gene functional in
 yeast is preferably a survival gene. Wherein a selectable gene functional
 in bacteria is included in the shuttle vector, a survival gene is also
 preferred.
 Preferred survival genes functional in yeast are survival genes which
 include ADE2, HIS3, LEU2, TRP 1, URA3, and ALG7, which confer resistance
 to tunicamycin; the neomycin phosphotransferase gene, which confers
 resistance to G418; the CUP1 gene, which allows yeast to grow in the
 presence of copper ions; and an adenine producing gene, or the like, which
 may be used alone or in combinations of two or more thereof. In a
 preferred embodiment, the trp1 gene is utilized. Stinchcomb et al.,
 Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et
 al., Gene, 10:157 (1980). The trp1 gene provides a selection marker for a
 mutant strain of yeast lacking the ability to grow in tryptophan, for
 example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)]. The
 preferred selectable gene functional in bacteria is a drug resistant, such
 as an ampicillin resistant gene.
 In a preferred embodiment, the selectable gene is a detection gene. Wherein
 a selectable gene functional in mammalian cells is included in the vector,
 a detection gene is preferred. Detection genes encode a protein that can
 be used as a direct or indirect label, i.e., for sorting the cells, i.e.
 for cell enrichment by FACS. In this embodiment, the protein product of
 the selectable gene itself can serve to distinguish cells that are
 expressing the selectable gene. In this embodiment, suitable selectable
 genes include those encoding green fluorescent protein (GFP), blue
 fluorescent protein (BFP), yellow fluorescent protein (YFP), red
 fluorescent protein (RFP), luciferase, .beta.-galactosidase, all
 commercially available, i.e., Clontech, Inc.
 Alternatively, the selectable gene encodes a protein that will bind a label
 that can be used as the basis of selection; i.e. the selectable gene
 serves as an indirect label or detection gene. In this embodiment, the
 selectable gene should encode a cell-surface protein. For example, the
 selectable gene may be any cell-surface protein not normally expressed on
 the surface of the cell, such that secondary binding agents could serve to
 distinguish cells that contain the selectable gene from those that do not.
 Alternatively, albeit non-preferably, selectables comprising normally
 expressed cell-surface proteins could be used, and differences between
 cells containing the selectable construct and those without could be
 determined. Thus, secondary binding agents bind to the selectable protein.
 These secondary binding agents are preferably labeled, for example with
 fluors, and can be antibodies, haptens, etc. For example, fluorescently
 labeled antibodies to the selectable gene can be used as the label.
 Similarly, membrane-tethered streptavidin could serve as a selectable
 gene, and fluorescent biotin could be used as the label, i.e. the
 secondary binding agent. Alternatively, the secondary binding agents need
 not be labeled as long as the secondary binding agent can be used to
 distinguish the cells containing the construct; for example, the secondary
 binding agents may be used in a column, and the cells passed through, such
 that the expression of the selectable gene results in the cell being bound
 to the column, and a lack of the selectable gene (i.e. inhibition),
 results in the cells not being retained on the column. Other suitable
 selectable proteins/secondary labels include, but are not limited to,
 antigens and antibodies, enzymes and substrates (or inhibitors), etc.
 In one aspect of the invention, the shuttle vector includes an insertion
 site, which is used to insert a heterologous nucleic acid sequence of
 choice, for ultimate expression in mammalian cells. The insertion site can
 be either be a cloning site, preferably a multicloning site (MCS), or a
 site suitable for homologous recombination, (referred to herein as a
 homologous recombination site). The vector can include multiple insertion
 sites, including both cloning sites and at least one homologous
 recombination site.
 In a preferred embodiment, the insertion site is a cloning site. A cloning
 site as used herein is a known sequence, preferably the only one on the
 vector, (i.e., it is a unique sequence on the vector) upon which a
 restriction enzyme operates to linearize or cut the vector. A multicloning
 site, also sometimes referred to as a multiple cloning site, polylinker,
 or polycloning site, is a cluster of cloning sites such that many
 restriction enzymes operate thereon. A wide variety of these sites are
 known in the art.
 In a preferred embodiment, the insertion site is a site that allows the
 introduction of the heterologous nucleic acid into the shuttle vector by
 homologous recombination. Homologous recombination is, briefly, the
 process of strand exchange that can occur spontaneously with the alignment
 of homologous sequences (i.e. sets of complementary strands). As is known
 in the art, yeast are efficient at homologous recombination. Orr-Weaver,
 et al, supra; H.a., et al., supra; Ma, et al., Gene, 58:201-216 (1987);
 Petermann, Nucleic Acids Res., 26(9):2252-2253 (1998); each incorporated
 herein by reference.
 Thus, in general, the homologous recombination site contains two distinct,
 but generally contiguous, regions. The first region, referred to herein as
 the 5' region, is generally identical to the 5' region flanking the
 heterologous nucleic acid to be inserted into the vector. The second
 region, referred to herein as the 3' region, is generally identical to the
 3' region flanking the heterologous nucleic acid to be inserted into the
 vector. Preferably, the 5' and 3' regions are each at least 12 or 15
 nucleic acids long. More preferably, the 5' and 3' regions are each at
 least about 20 or 30 nucleic acids long, and more preferably at least
 about 50 nucleic acids long, and most preferably about 60 nucleic acids
 long. These regions are preferably less than about 100 nucleic acids long.
 Preferably, the homologous recombination site sequence is unique to the
 vector in that the vector does not comprise another sequence corresponding
 to the sequence of the homologous recombination site.
 The insertion site is used to insert a heterologous nucleic acid. A
 "heterologous nucleic acid" as used herein refers to any nucleic acid
 inserted into the shuttle vector at a site operably linked to the
 promoter. Various embodiments of heterologous nucleic acids are further
 defined below. In a preferred embodiment, the heterologous nucleic acid is
 flanked by 5' and 3' regions identical to the 5' and 3' regions of a
 homologous recombination site on the shuttle vector provided herein. Thus,
 when the heterologous nucleic acid is inserted into the vector, the 5' and
 3' regions flanking the heterologous nucleic acid replace the 5' and 3'
 regions of the homologous recombination site during homologous
 recombination.
 In a further aspect, the shuttle vector further comprises an origin of
 replication functional in a bacterial cell. The bacterial cell is
 generally any bacterial cell which can be used to amplify the shuttle
 vector. Examples include Gram-negative or Gram-positive organisms, for
 example, Enterobacteriaceae such as E. coli, Bacillus subtilis,
 Streptococcus cremoris, Streptococcus lividans. Various E. coli strains
 are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E.
 coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772
 (ATCC 53,635). Origin of replication sites are known in the art and are
 further described in Sambrook, et al., Molecular Cloning, 2nd Ed., Vol. 3,
 Chapter 1, particularly sections 12-20 (1989), Promega, 1998 catalog
 number E1841 (pCI-neo).
 In one embodiment, the shuttle vector also comprises an origin of
 replication functional in mammalian cells. As is known in the art, the
 only extrachromosomal vectors which replicate in mammalian cells are
 virally derived. A number of viral origin of replications require the
 binding of a specific viral replication protein to effect replication.
 Suitable origin of replication/viral replication protein pairs include,
 but are not limited to, the Epstein Barr origin of replication and the
 Epstein Barr nuclear antigen (see Sugden et al., Mole. Cell. Biol.
 5(2):410-413 (1985)); the SV40 origin of replication and the SV40 T
 antigen (see Margolskee et al., Mole. Cell. Biol. 8(7):2837 (1988)). The
 coding sequence for the viral replication protein can be on the shuttle
 vector provided herein, or on a separate vector.
 In an additional aspect of this invention, the shuttle vector comprises
 additional sequences, including but not limited to at least one or all of
 the following: an internal ribosome entry sequence (IRES), an RNA splice
 site (also called a splice signal or sequence herein) and a
 polyadenylation site (also called a polyadenylation signal or sequence
 herein).
 IRES elements function as initiators of the efficient translation of
 reading frames. In particular, IRES allows for the translation of two
 different genes on a single transcript. IRES thus greatly facilitates the
 selection of cells expressing peptides at uniformly high levels. IRES
 elements are known in the art and are further characterized in Kim, et
 al., Molecular and Cellular Biology 12(8):3636-3643 (August 1992) and
 McBratney, et al., Current Opinion in Cell Biology 5:961-965 (1993).
 All of those sequences of viral, cellular, or synthetic origin which
 mediate an internal binding of the ribosomes can be used as an IRES.
 Examples include those IRES elements from poliovirus Type I, the 5'UTR of
 encephalomyocarditis virus (EMV), of "Thelier's murine encephalomyelitis
 virus (TMEV) of "foot and mouth disease virus" (FMDV) of "bovine
 enterovirus (BEV), of "coxsackie B virus" (CBV), or of "human rhinovirus"
 (HRV), or the "human immunoglobulin heavy chain binding protein" (BIP)
 5'UTR, the Drosophila antennapediae 5'UTR or the Drosophila ultrabithorax
 5'UTR, or genetic hybrids or fragments from the above-listed sequences.
 The shuttle vectors provided herein may include a splice donor and acceptor
 site (splicing signals or splice sites) within the transcription unit.
 Splicing signals are known to increase mRNA stability and protein
 expression levels. Splicing signals are known in the art and are further
 described in Sambrook, et al., Molecular Cloning, 2nd Ed., Vol. 3, Chapter
 16, particularly section 7 (1989).
 A polyadenylation site or signal refers to sequences necessary for the
 termination of transcription and for stabilizing the mRNA of eukaryotes.
 Such sequences are commonly available and are further described in
 Sambrook, et al., Molecular Cloning, 2nd Ed., Vol. 3, Chapter 16,
 particularly sections 6-7 (1989).
 Optionally, the shuttle vector may further comprise transcription
 enhancers. Enhancers are cis-acting elements of DNA, usually about from 10
 to 300 bp, that act on a promoter to increase its transcription. Many
 enhancer sequences are now known from mammalian genes including for
 example, globin, elastase, albumin, .alpha.-fetoprotein, and insulin.
 Typically, however, one will use an enhancer from a eukaryotic cell virus.
 Examples include the SV40 enhancer on the late side of the replication
 origin (bp 100-270), the cytomegalovirus early promoter enhancer, the
 polyoma enhancer on the late side of the replication origin, and
 adenovirus enhancers. The enhancer may be spliced into the vector at a
 position 5' or 3' to a coding sequence, but is preferably located at a
 site 5' from the promoter.
 Optionally, the vector can be constructed so as to allow of the
 heterologous nucleic acid expression in yeast and/or bacterial cells. In
 this embodiment, the vector would further include a promoter functional in
 yeast and/or bacterial cells. Examples of suitable promoting sequences for
 use with yeast hosts include the promoters for 3-phosphoglycerate kinase
 [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic
 enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland,
 Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate
 dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,
 glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,
 triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
 Other yeast promoters, which are inducible promoters having the additional
 advantage of transcription controlled by growth conditions, include the
 promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid
 phosphatase, degradative enzymes associated with nitrogen metabolism,
 metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes
 responsible for maltose and galactose utilization.
 Promoters for bacterial cells are known in the art and further described
 i.e., in Sambrook, et al., Molecular Cloning, 2nd Ed., Vol. 3, Chapter 17,
 particularly sections 11-17 (1989). Generally, promoters suitable for use
 with prokaryotic hosts include the .beta.-lactamase and lactose promoter
 systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature,
 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system
 [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid
 promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci.
 USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will
 contain a Shine-Dalgarno (S.D.) sequence.
 In an embodiment provided herein, the insertion site is linked to a
 selection system, i.e., a detection gene. In a preferred embodiment, from
 the 5' to 3' direction the construct comprises the mammalian promoter, the
 heterologous nucleic acid, the IRES site, and the selectable gene.
 In a preferred embodiment, the vectors are used to screen heterologous
 nucleic acids. "Heterologous nucleic acids" as used herein refers to
 naturally occurring nucleic acids, random nucleic acids, or "biased"
 random nucleic acids, e.g. in nucleotide/residue frequency generally or
 per position. By "randomized" or grammatical equivalents herein is meant
 that each nucleic acid consists of essentially random nucleotides. For
 example, digests of procaryotic or eukaryotic genomes may be used, or cDNA
 fragments. They are heterologous in that they are inserted into the
 shuttle vector.
 In a preferred embodiment, the heterologous nucleic acids are presented to
 the shuttle vector in the form of a cloning vector wherein the
 heterologous nucleic acid is flanked by 5' and 3' regions identical to 5'
 and 3' regions of an insertion site (i.e., a homologous recombination
 site) on the shuttle vector. That is, heterologous nucleic acids are
 recombined into cloning vectors containing homologous recombination
 flanking regions. The cloning vectors and the shuttle vectors are
 introduced into yeast, where recombination takes place. In a preferred
 embodiment, the cloning vectors are linear when introduced to the yeast.
 In one aspect of the invention, the shuttle vectors provided herein are
 used to transform yeast. Heterologous nucleic acids are then, or
 simultaneously introduced to the yeast, and in a preferred embodiment,
 homologous recombination takes place such that the yeast inserts the
 heterologous nucleic acid into the shuttle vector at a specific insertion
 site, i.e., a homologous recombination site.
 Transformations into yeast are typically carried out according to the
 method of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al.,
 Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). The shuttle vectors are then
 isolated from the yeast and used to transform mammalian cells for
 expression of the heterologous nucleic acid.
 For transforming mammalian cells, the calcium phosphate precipitation
 method of Graham and van der Eb, Virology, 52:456-457 (1978) can be
 employed. General aspects of mammalian cell host system transformations
 have been described in U.S. Pat. No. 4,399,216. However, other methods for
 introducing DNA into cells, such as by nuclear microinjection, biolistics,
 electroporation, bacterial protoplast fusion with intact cells, or
 polycations, e.g., polybrene, polyornithine, may also be used. For various
 techniques for transforming mammalian cells, see Keown et al., Methods in
 Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352
 (1988). Expression in mammalian cells is also described in Sambrook, et
 al., Molecular Cloning, 2nd Ed., Vol. 3, Chapter 16, particularly sections
 68-72 (1989).
 Isolation of the shuttle vectors is performed by standard techniques known
 in the art. Generally, the shuttle vectors can be isolated by breaking the
 cell open and separating the vector nucleic acid based on weight, i.e.,
 centrifugation, or size, i.e. gel permeability. The vectors need only be
 isolated to the extent required to perform transformation.
 In one aspect of this invention, the invention involves expression of
 heterologous nucleic acid inserts in a mammalian cell population. The
 expression of heterologous nucleic acids is identified by the production
 of a label or tag. Thus, when the shuttle vector expresses a heterologous
 nucleic acid, a selectable gene will also be expressed thereby verifying
 the presence of an expressed heterologous nucleic acid.
 In another aspect of the present invention, expressed heterologous nucleic
 acids are selected on the basis of activity or phenotype. For example, the
 expressed insert or the cell type expressing that particular insert can be
 screened for its ability to interact with an antibody or ligand, capable
 of specific binding to the encoded product of that insert, which has been
 previously bound to a solid support such as a petri dish. Positive cDNA
 inserts (those expressed in cell types binding to the solid support) are
 recovered, transformed into a convenient host (E. coli) and characterized
 by known recombinant DNA techniques. This procedure is also referred to as
 panning, and is further described in Wysocki and Sata, 1979 PNAS
 75:2844-2848 and Seen and Aruffo, 1987 PNAS 84:3365-3369.
 Thus, in one embodiment, the present invention allows for creating shuttle
 vectors with inserts therein, without necessarily requiring the skilled
 artisan to insert the heterologous nucleic acid into the shuttle vector.
 Rather, the invention herein provides for the yeast organism to perform
 this step in a preferred embodiment. Moreover, the present invention also
 allows for expression in mammalian cells, which provides for a native
 environment for expressing mammalian genes.
 Additionally, the invention provides for a variety of options, such as
 replication in bacteria for amplification of shuttle vectors containing
 selected heterologous nucleic acids. Moreover, the shuttle vectors
 provided herein can perform the traditional aspects of expression vectors,
 whether or not "shuttling" is desired.
 Furthermore, the present invention provides for screening for heterologous
 nucleic acids which encode candidate agents. "Candidate agents" as used
 herein are peptides which may have a desired effect on the phenotype or
 genotype of a cell. Heterologous nucleic acids expressing a candidate
 agent can be designed in a number of ways so as to facilitate their
 identification. Generally, this is achieved by the use of fusion partners,
 or combinations of fusion partners. Examples include presentation
 structures, targeting sequences, rescue sequences, and stability
 sequences, all of which can be used independently or in combination, with
 or without linker sequences.
 By "fusion partner" or "functional group" herein is meant a sequence that
 is associated with the heterologous nucleic acid expressing a candidate
 agent, that confers upon all members of the library in that class a common
 function or ability. Fusion partners can be heterologous (i.e. not native
 to the host cell), or synthetic (not native to any cell). Suitable fusion
 partners include, but are not limited to: a) presentation structures, as
 defined below, which provide the candidate bioactive agents in a
 conformationally restricted or stable form; b) targeting sequences,
 defined below, which allow the localization of the candidate bioactive
 agent into a subcellular or extracellular compartment; c) rescue sequences
 as defined below, which allow the purification or isolation of either the
 candidate bioactive agents or the nucleic acids encoding them; d)
 stability sequences, which confer stability or protection from degradation
 to the candidate bioactive agent or the nucleic acid encoding it, for
 example resistance to proteolytic degradation; e) dimerization sequences,
 to allow for peptide dimerization; or f) any combination of a), b), c),
 d), and e), as well as linker sequences as needed.
 In a preferred embodiment, the fusion partner is a presentation structure.
 By "presentation structure" or grammatical equivalents herein is meant a
 sequence, which, when fused to a heterologous nucleic acid expressing a
 candidate agent, causes the candidate agents to assume a conformationally
 restricted form. Proteins interact with each other largely through
 conformationally constrained domains. Although small peptides with freely
 rotating amino and carboxyl termini can have potent functions as is known
 in the art, the conversion of such peptide structures into pharmacologic
 agents is difficult due to the inability to predict side-chain positions
 for peptidomimetic synthesis. Therefore the presentation of peptides in
 conformationally constrained structures will benefit both the later
 generation of pharmaceuticals and will also likely lead to higher affinity
 interactions of the peptide with the target protein. This fact has been
 recognized in the combinatorial library generation systems using
 biologically generated short peptides in bacterial phage systems. A number
 of workers have constructed small domain molecules in which one might
 present randomized peptide structures.
 Suitable presentation structures include, but are not limited to, minibody
 structures, loops on beta-sheet turns and coiled-coil stem structures in
 which residues not critical to structure are randomized, zinc-finger
 domains, cysteine-linked (disulfide) structures, transglutaminase linked
 structures, cyclic peptides, B-loop structures, helical barrels or
 bundles, leucine zipper motifs, etc.
 In a preferred embodiment, the presentation structure is a coiled-coil
 structure, allowing the presentation of the randomized peptide on an
 exterior loop. See, for example, Myszka et al., Biochem. 33:2362-2373
 (1994), hereby incorporated by reference). Using this system investigators
 have isolated peptides capable of high affinity interaction with the
 appropriate target. In general, coiled-coil structures allow for between 6
 to 20 randomized positions.
 A preferred coiled-coil presentation structure is as follows:
 MGCAALESEVSALESEVASLESEVAALGRGDMPLAAVKSKLSAVKSKLASVKSKLAACGPP (SEQ ID
 NO:5). The underlined regions represent a coiled-coil leucine zipper
 region defined previously (see Martin et al., EMBO J. 13(22):5303-5309
 (1994), incorporated by reference). The bolded GRGDMP region represents
 the loop structure and when appropriately replaced with randomized
 peptides (i.e. candidate bioactive agents, generally depicted herein as
 (X).sub.n, where X is an amino acid residue and n is an integer of at
 least 5 or 6) can be of variable length. The replacement of the bolded
 region is facilitated by encoding restriction endonuclease sites in the
 underlined regions, which allows the direct incorporation of randomized
 oligonucleotides at these positions. For example, a preferred embodiment
 generates a XhoI site at the double underlined LE site and a HindIII site
 at the double-underlined KL site.
 In a preferred embodiment, the presentation structure is a minibody
 structure. A "minibody" is essentially composed of a minimal antibody
 complementarity region. The minibody presentation structure generally
 provides two randomizing regions that in the folded protein are presented
 along a single face of the tertiary structure. See for example Bianchi et
 al., J. Mol. Biol. 236(2):649-59 (1994), and references cited therein, all
 of which are incorporated by reference). Investigators have shown this
 minimal domain is stable in solution and have used phage selection systems
 in combinatorial libraries to select minibodies with peptide regions
 exhibiting high affinity, Kd=10.sup.-7, for the pro-inflammatory cytokine
 IL-6.
 A preferred minibody presentation structure is as follows:
 MGRNSQATSGFTFSHFYMEWVRGGEYIAASRHKHNKYTTEYSASVKGRYIVSRDTSQSILYLQKKKGPP (SEQ
 ID NO:6). The bold, underline regions are the regions which may be
 randomized. The italized phenylalanine must be invariant in the first
 randomizing region. The entire peptide is cloned in a
 three-oligonucleotide variation of the coiled-coil embodiment, thus
 allowing two different randomizing regions to be incorporated
 simultaneously. This embodiment utilizes non-palindromic BstXI sites on
 the termini.
 In a preferred embodiment, the presentation structure is a sequence that
 contains generally two cysteine residues, such that a disulfide bond may
 be formed, resulting in a conformationally constrained sequence. This
 embodiment is particularly preferred when secretory targeting sequences
 are used. As will be appreciated by those in the art, any number of random
 sequences, with or without spacer or linking sequences, may be flanked
 with cysteine residues. In other embodiments, effective presentation
 structures may be generated by the random regions themselves. For example,
 the random regions may be "doped" with cysteine residues which, under the
 appropriate redox conditions, may result in highly crosslinked structured
 conformations, similar to a presentation structure. Similarly, the
 randomization regions may be controlled to contain a certain number of
 residues to confer .beta.-sheet or .alpha.-helical structures.
 In a preferred embodiment, the fusion partner is a targeting sequence. As
 will be appreciated by those in the art, the localization of proteins
 within a cell is a simple method for increasing effective concentration
 and determining function. For example, RAF1 when localized to the
 mitochondrial membrane can inhibit the anti-apoptotic effect of BCL-2.
 Similarly, membrane bound Sos induces Ras mediated signaling in
 T-lymphocytes. These mechanisms are thought to rely on the principle of
 limiting the search space for ligands, that is to say, the localization of
 a protein to the plasma membrane limits the search for its ligand to that
 limited dimensional space near the membrane as opposed to the three
 dimensional space of the cytoplasm. Alternatively, the concentration of a
 protein can also be simply increased by nature of the localization.
 Shuttling the proteins into the nucleus confines them to a smaller space
 thereby increasing concentration. Finally, the ligand or target may simply
 be localized to a specific compartment, and inhibitors must be localized
 appropriately.
 Thus, suitable targeting sequences include, but are not limited to, binding
 sequences capable of causing binding of the expression product to a
 predetermined molecule or class of molecules while retaining bioactivity
 of the expression product, (for example by using enzyme inhibitor or
 substrate sequences to target a class of relevant enzymes); sequences
 signalling selective degradation, of itself or co-bound proteins; and
 signal sequences capable of constitutively localizing the candidate
 expression products to a predetermined cellular locale, including a)
 subcellular locations such as the Golgi, endoplasmic reticulum, nucleus,
 nucleoli, nuclear membrane, mitochondria, chloroplast, secretory vesicles,
 lysosome, and cellular membrane; and b) extracellular locations via a
 secretory signal. Particularly preferred is localization to either
 subcellular locations or to the outside of the cell via secretion.
 In a preferred embodiment, the targeting sequence is a nuclear localization
 signal (NLS). NLSs are generally short, positively charged (basic) domains
 that serve to direct the entire protein in which they occur to the cell's
 nucleus. Numerous NLS amino acid sequences have been reported including
 single basic NLS's such as that of the SV40 (monkey virus) large T Antigen
 (Pro Lys Lys Lys Arg Lys Val), (SEQ ID NO:7) Kalderon (1984), et al.,
 Cell, 39:499-509; the human retinoic acid receptor-.beta. nuclear
 localization signal (ARRRRP (SEQ ID NO:8)); NF.kappa.B p50 (EEVQRKRQKL
 (SEQ ID NO:9); Ghosh et al., Cell 62:1019 (1990); NF.kappa.B p65
 (EEKRKRTYE (SEQ ID NO:10); Nolan et al., Cell 64:961 (1991); and others
 (see for example Boulikas, J. Cell. Biochem. 55(1):32-58 (1994), hereby
 incorporated by reference) and double basic NLS's exemplified by that of
 the Xenopus (African clawed toad) protein, nucleoplasmin (Ala Val Lys Arg
 Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Leu Asp (SEQ ID
 NO:11)), Dingwall, et al., Cell, 30:449-458, 1982 and Dingwall, et al., J.
 Cell Biol., 107:641-849; 1988). Numerous localization studies have
 demonstrated that NLSs incorporated in synthetic peptides or grafted onto
 selectable proteins not normally targeted to the cell nucleus cause these
 peptides and selectable proteins to be concentrated in the nucleus. See,
 for example, Dingwall, and Laskey, Ann, Rev. Cell Biol., 2:367-390, 1986;
 Bonnerot, et al., Proc. Natl. Acad. Sci. USA, 84:6795-6799, 1987; Galileo,
 et al., Proc. Natl. Acad. Sci. USA, 87:458-462, 1990.
 In a preferred embodiment, the targeting sequence is a membrane anchoring
 signal sequence. This is particularly useful since many parasites and
 pathogens bind to the membrane, in addition to the fact that many
 intracellular events originate at the plasma membrane. Thus,
 membrane-bound peptide libraries are useful for both the identification of
 important elements in these processes as well as for the discovery of
 effective inhibitors. The invention provides methods for presenting the
 randomized expression product extracellularly or in the cytoplasmic space.
 For extracellular presentation, a membrane anchoring region is provided at
 the carboxyl terminus of the peptide presentation structure. The
 randomized epression product region is expressed on the cell surface and
 presented to the extracellular space, such that it can bind to other
 surface molecules (affecting their function) or molecules present in the
 extracellular medium. The binding of such molecules could confer function
 on the cells expressing a peptide that binds the molecule. The cytoplasmic
 region could be neutral or could contain a domain that, when the
 extracellular randomized expression product region is bound, confers a
 function on the cells (activation of a kinase, phosphatase, binding of
 other cellular components to effect function). Similarly, the randomized
 expression product-containing region could be contained within a
 cytoplasmic region, and the transmembrane region and extracellular region
 remain constant or have a defined function.
 Membrane-anchoring sequences are well known in the art and are based on the
 genetic geometry of mammalian transmembrane molecules. Peptides are
 inserted into the membrane based on a signal sequence (designated herein
 as ssTM) and require a hydrophobic transmembrane domain (herein TM). The
 transmembrane proteins are inserted into the membrane such that the
 regions encoded 5' of the transmembrane domain are extracellular and the
 sequences 3' become intracellular. Of course, if these transmembrane
 domains are placed 5' of the variable region, they will serve to anchor it
 as an intracellular domain, which may be desirable in some embodiments.
 ssTMs and TMs are known for a wide variety of membrane bound proteins, and
 these sequences may be used accordingly, either as pairs from a particular
 protein or with each component being taken from a different protein, or
 alternatively, the sequences may be synthetic, and derived entirely from
 consensus as artificial delivery domains.
 As will be appreciated by those in the art, membrane-anchoring sequences,
 including both ssTM and TM, are known for a wide variety of proteins and
 any of these may be used. Particularly preferred membrane-anchoring
 sequences include, but are not limited to, those derived from CD8, ICAM-2,
 IL-8R, CD4 and LFA-1.
 Useful sequences include sequences from: 1) class I integral membrane
 proteins such as IL-2 receptor beta-chain (residues 1-26 are the signal
 sequence, 241-265 are the transmembrane residues; see Hatakeyama et al.,
 Science 244:551 (1989) and von Heijne et al, Eur. J. Biochem. 174:671
 (1988)) and insulin receptor beta chain (residues 1-27 are the signal,
 957-959 are the transmembrane domain and 960-1382 are the cytoplasmic
 domain; see Hatakeyama, supra, and Ebina et al., Cell 40:747 (1985)); 2)
 class II integral membrane proteins such as neutral endopeptidase
 (residues 29-51 are the transmembrane domain, 2-28 are the cytoplasmic
 domain; see Malfroy et al., Biochem. Biophys. Res. Commun. 144:59 (1987));
 3) type III proteins such as human cytochrome P450 NF25 (Hatakeyama,
 supra); and 4) type IV proteins such as human P-glycoprotein (Hatakeyama,
 supra). Particularly preferred are CD8 and ICAM-2. For example, the signal
 sequences from CD8 and ICAM-2 lie at the extreme 5' end of the transcript.
 These consist of the amino acids 1-32 in the case of CD8
 (MASPLTRFLSLNLLLLGESILGSGEAKPQAP (SEQ ID NO:12); Nakauchi et al., PNAS USA
 82:5126 (1985) and 1-21 in the case of ICAM-2 (MSSFGYRTLTVALFTLICCPG (SEQ
 ID NO:13); Staunton et al., Nature (London) 339:61 (1989)). These leader
 sequences deliver the construct to the membrane while the hydrophobic
 transmembrane domains, placed 3' of the random candidate region, serve to
 anchor the construct in the membrane. These transmembrane domains are
 encompassed by amino acids 145-195 from CD8
 (PQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSR (SEQ ID NO:14);
 Nakauchi, supra) and 224-256 from ICAM-2
 (MVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQR (SEQ ID NO:15); Staunton, supra).
 Alternatively, membrane anchoring sequences include the GPI anchor, which
 results in a covalent bond between the molecule and the lipid bilayer via
 a glycosyl-phosphatidylinositol bond for example in DAF
 (PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO:16), with the bolded
 serine the site of the anchor; see Homans et al., Nature 333(6170):269-72
 (1988), and Moran et al., J. Biol. Chem. 266:1250 (1991)). In order to do
 this, the GPI sequence from Thy-1 can be cassetted 3' of the variable
 region in place of a transmembrane sequence.
 Similarly, myristylation sequences can serve as membrane anchoring
 sequences. It is known that the myristylation of c-src recruits it to the
 plasma membrane. This is a simple and effective method of membrane
 localization, given that the first 14 amino acids of the protein are
 solely responsible for this function: MGSSKSKPKDPSQR (SEQ ID NO:17) (see
 Cross et al., Mol. Cell. Biol. 4(9):1834 (1984); Spencer et al., Science
 262:1019-1024 (1993), both of which are hereby incorporated by reference).
 This motif has already been shown to be effective in the localization of
 selectable genes and can be used to anchor the zeta chain of the TCR. This
 motif is placed 5' of the variable region in order to localize the
 construct to the plasma membrane. Other modifications such as
 palmitoylation can be used to anchor constructs in the plasma membrane;
 for example, palmitoylation sequences from the G protein-coupled receptor
 kinase GRK6 sequence (LLQRLFSRQDCCGNCSDSEEELPTRL (SEQ ID NO:18), with the
 bold cysteines being palmitolyated; Stoffel et al., J. Biol. Chem
 269:27791 (1994)); from rhodopsin (KQFRNCMLTSLCCGKNPLGD (SEQ ID NO:19);
 Barnstable et al., J. Mol. Neurosci. 5(3):207 (1994)); and the p21 H-ras 1
 protein (LNPPDESGPGCMSCKCVLS; Capon et al., Nature 302:33 (1983)).
 In a preferred embodiment, the targeting sequence is a lysozomal targeting
 sequence, including, for example, a lysosomal degradation sequence such as
 Lamp-2 (KFERQ (SEQ ID NO:20); Dice, Ann. New York Acad. Sci. 674:58
 (1992); or lysosomal membrane sequences from Lamp-1
 (MLIPIAGFFALAGLVLIVLIAYLIGRKRSHAGYQTI (SEQ ID NO:21), Uthayakumar et al.,
 Cell. Mol. Biol. Res. 41:405 (1995)) or Lamp-2
 (LVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF (SEQ ID NO:22), Konecki et la.,
 Biochem. Biophys. Res. Comm. 205:1-5 (1994), both of which show the
 transmembrane domains in italics and the cytoplasmic targeting signal
 underlined).
 Alternatively, the targeting sequence may be a mitrochondrial localization
 sequence, including mitochondrial matrix sequences (e.g. yeast alcohol
 dehydrogenase III; MLRTSSLFTRRVQPSLFSRNILRLQST (SEQ ID NO:23); Schatz,
 Eur. J. Biochem. 165:1-6 (1987)); mitochondrial inner membrane sequences
 (yeast cytochrome c oxidase subunit IV; MLSLRQSIRFFKPATRTLCSSRYLL (SEQ ID
 NO:24); Schatz, supra); mitochondrial intermembrane space sequences (yeast
 cytochrome c1;
 MFSMLSKRWAQRTLSKSFYSTATGAASKSGKLTQKLVTAGVAAAGITASTLLYADSLTAEAMTA (SEQ ID
 NO:25); Schatz, supra) or mitochondrial outer membrane sequences (yeast 70
 kD outer membrane protein; MKSFITRNKTAILATVAATGTAIGAYYYYNQLQQQQQRGKK (SEQ
 ID NO:26); Schatz, supra).
 The target sequences may also be endoplasmic reticulum sequences, including
 the sequences from calreticulin (KDEL (SEQ ID NO:27); Pelham, Royal
 Society London Transactions B; 1-10 (1992)) or adenovirus E3/19K protein
 (LYLSRRSFIDEKKMP (SEQ ID NO:28); Jackson et al., EMBO J. 9:3153 (1990).
 Furthermore, targeting sequences also include peroxisome sequences (for
 example, the peroxisome matrix sequence from Luciferase; SKL; Keller et
 al., PNAS USA 4:3264 (1987)); farnesylation sequences (for example, P21
 H-ras 1; LNPPDESGPGCMSCKCVLS (SEQ ID NO:29), with the bold cysteine
 farnesylated; Capon, supra); geranylgeranylation sequences (for example,
 protein rab-5A; LTEPTQPTRNQCCSN (SEQ ID NO:30), with the bold cysteines
 geranylgeranylated; Farnsworth, PNAS USA 91:11963 (1994)); or destruction
 sequences (cyclin B1; RTALGDIGN (SEQ ID NO:31); Klotzbucher et al., EMBO
 J. 1:3053 (1996)).
 In a preferred embodiment, the targeting sequence is a secretory signal
 sequence capable of effecting the secretion of the candidate agent. There
 are a large number of known secretory signal sequences which are placed 5'
 to the variable peptide region, and are cleaved from the peptide region to
 effect secretion into the extracellular space. Secretory signal sequences
 and their transferability to unrelated proteins are well known, e.g.,
 Silhavy, et al. (1985) Microbiol. Rev. 49, 398-418. This is particularly
 useful to generate a peptide capable of binding to the surface of, or
 affecting the physiology of, a target cell that is other than the host
 cell, e.g., the cell infected with the retrovirus. In a preferred
 approach, a fusion product is configured to contain, in series, secretion
 signal peptide-presentation structure-randomized expression product
 region-presentation structure. In this manner, target cells grown in the
 vicinity of cells caused to express the library of peptides, are bathed in
 secreted peptide. Target cells exhibiting a physiological change in
 response to the presence of a peptide, e.g., by the peptide binding to a
 surface receptor or by being internalized and binding to intracellular
 targets, and the secreting cells are localized by any of a variety of
 selection schemes and the peptide causing the effect determined. Exemplary
 effects include variously that of a designer cytokine (i.e., a stem cell
 factor capable of causing hematopoietic stem cells to divide and maintain
 their totipotential), a factor causing cancer cells to undergo spontaneous
 apoptosis, a factor that binds to the cell surface of target cells and
 labels them specifically, etc.
 Suitable secretory sequences are known, including signals from IL-2
 (MYRMQLLSCIALSLALVTNS (SEQ ID NO:32); Villinger et al., J. Immunol.
 155:3946 (1995)), growth hormone (MATGSRTSLLLAFGLLCLPWLQEGSAFPT (SEQ ID
 NO:33); Roskam et al., Nucleic Acids Res. 7:30 (1979)); preproinsulin
 (MALWMRLLPLLALLALWGPDPAAAFVN (SEQ ID NO:34); Bell et al., Nature 284:26
 (1980)); and influenza HA protein (MKAKLLVLLYAFVAGDQI (SEQ ID NO:35);
 Sekiwawa et al., PNAS 80:3563)), with cleavage between the
 non-underlined-underlined junction. A particularly preferred secretory
 signal sequence is the signal leader sequence from the secreted cytokine
 IL-4, which comprises the first 24 amino acids of IL-4 as follows:
 MGLTSQLLPPLFFLLACAGNFVHG (SEQ ID NO:36).
 In a preferred embodiment, the fusion partner is a rescue sequence. A
 rescue sequence is a sequence which may be used to purify or isolate
 either the candidate agent or the heterologous nucleic acid encoding it.
 Thus, for example, peptide rescue sequences include purification sequences
 such as the His.sub.6 tag for use with Ni affinity columns and epitope
 tags for detection, immunoprecipitation or FACS (fluoroscence-activated
 cell sorting). Suitable epitope tags include myc (for use with the
 commercially available 9E10 antibody), the BSP biotinylation target
 sequence of the bacterial enzyme BirA, flu tags, lacZ, and GST.
 Alternatively, the rescue sequence may be a unique oligonucleotide sequence
 which serves as a probe target site to allow the quick and easy isolation
 of the retroviral construct, via PCR, related techniques, or
 hybridization.
 In a preferred embodiment, the fusion partner is a stability sequence to
 confer stability to the candidate bioactive agent or the heterologous
 nucleic acid encoding it. Thus, for example, peptides may be stabilized by
 the incorporation of glycines after the initiation methionine (MG or
 MGG0), for protection of the peptide to ubiquitination as per Varshavsky's
 N-End Rule, thus conferring long half-life in the cytoplasm. Similarly,
 two prolines at the C-terminus impart peptides that are largely resistant
 to carboxypeptidase action. The presence of two glycines prior to the
 prolines impart both flexibility and prevent structure initiating events
 in the di-proline to be propagated into the candidate peptide structure.
 Thus, preferred stability sequences are as follows: MG(X).sub.n GGPP (SEQ
 ID NO:37), where X is any amino acid and n is an integer of at least four.
 In one embodiment, the fusion partner is a dimerization sequence. A
 dimerization sequence allows the non-covalent association of one random
 peptide to another random peptide, with sufficient affinity to remain
 associated under normal physiological conditions. This effectively allows
 small libraries of random peptides (for example, 10.sup.4) to become large
 libraries if two peptides per cell are generated which then dimerize, to
 form an effective library of 10.sup.8 (10.sup.4.times.10.sup.4). It also
 allows the formation of longer random peptides, if needed, or more
 structurally complex random peptide molecules. The dimers may be homo- or
 heterodimers.
 Dimerization sequences may be a single sequence that self-aggregates, or
 two sequences, each of which is generated in a different retroviral
 construct. That is, nucleic acids encoding both a first random peptide
 with dimerization sequence 1, and a second random peptide with
 dimerization sequence 2, such that upon introduction into a cell and
 expression of the nucleic acid, dimerization sequence 1 associates with
 dimerization sequence 2 to form a new random peptide structure.
 Suitable dimerization sequences will encompass a wide variety of sequences.
 Any number of protein-protein interaction sites are known. In addition,
 dimerization sequences may also be elucidated using standard methods such
 as the yeast two hybrid system, traditional biochemical affinity binding
 studies, or even using the present methods.
 The fusion partners may be placed anywhere (i.e. N-terminal, C-terminal,
 internal) in the structure as the biology and activity permits.
 In a preferred embodiment, the fusion partner includes a linker or
 tethering sequence, as generally described in PCT US 97/01019, that can
 allow the candidate agents to interact with potential targets unhindered.
 For example, when the candidate bioactive agent is a peptide, useful
 linkers include glycine-serine polymers (including, for example,
 (GS).sub.n (GSGGS).sub.n (SEQ ID NO:38) and (GGGS).sub.n (SEQ ID NO:39),
 where n is an integer of at least one), glycine-alanine polymers,
 alanine-serine polymers, and other flexible linkers such as the tether for
 the shaker potassium channel, and a large variety of other flexible
 linkers, as will be appreciated by those in the art. Glycine-serine
 polymers are preferred since both of these amino acids are relatively
 unstructured, and therefore may be able to serve as a neutral tether
 between components. Secondly, serine is hydrophilic and therefore able to
 solubilize what could be a globular glycine chain. Third, similar chains
 have been shown to be effective in joining subunits of recombinant
 proteins such as single chain antibodies.
 In addition, the fusion partners, including presentation structures, may be
 modified, randomized, and/or matured to alter the presentation orientation
 of the randomized expression product. For example, determinants at the
 base of the loop may be modified to slightly modify the internal loop
 peptide tertiary structure, which maintaining the randomized amino acid
 sequence.
 Thus, heterologous nucleic acids can be sequences which have not been
 manipulated in any way, or alternatively, they can be constructed to have
 fusion partners. In either case, they can be inserted into the shuttle
 vectors by conventional methods such as enzymatic manipulation and
 ligation, or preferably, are inserted into the shuttle vector by
 homologous recombination as described herein.
 It is understood by the skilled artisan that while various options (of
 compounds, properties selected or order of steps) are provided herein, the
 options are also each provided individually, and can each be individually
 segregated from the other options provided herein. Moreover, steps which
 are obvious and known in the art are intended to be within the scope of
 this invention. For example, there may be additionally washing steps,
 segregation, or isolation steps. Moreover, additional components to
 vectors, particularly regulatory elements, cells, cell media, etc., which
 are routine and known in the art can be incorporated herein without
 deviating from the spirit and scope of the invention.
 The following examples serve to more fully describe the manner of using the
 above-described invention, as well as to set forth the best modes
 contemplated for carrying out various aspects of the invention. It is
 understood that these examples in no way serve to limit the true scope of
 this invention, but rather are presented for illustrative purposes. All
 references cited herein are expressly incorporated by reference in their
 entirety.
 EXAMPLES
 Example 1
 Mammalian Cells Transfected with a Shuttle Vector Show Expression
 A shuttle vector (pPYC-R) was constructed in accordance with the schematic
 shown in FIG. 1. The sequence is provided in FIG. 2. The vector has an
 IRES at positions 6001-6505, a GFP at 6506-7258, Amp.sup.R at 9888-655, an
 E. colireplication origin at 656-1456, a yeast 2.mu. replication origin at
 1461-2808, Trp at 3344-4018 and a CMV promoter at 4853-5614 of SEQ ID NO:1
 shown in FIG. 2.
 1 .mu.g of pPYC-R plasmid was transfected into 30% confluent 293 (Phoenix)
 cells by a standard Ca.sup.2+ Phosphate transfection method known in the
 art to test expression of GFP. After incubation for 48 hours in 37.degree.
 C. CO.sub.2 incubator, cells transfected by pPYC-R show green fluorescence
 color under UV microscope as depicted in FIG. 3.
 Example 2
 Use of Yeast to Construct Shuttle Vector with Insert, Expression of Insert
 This example demonstrates the in-frame fusion of Rip cDNA, an
 apoptosis-inducing gene when over-expressed in mammalian cells, to a
 hemagglutinin (HA) tag in pPYC by recombination with non-virus based
 vector. Rip is further described in Hsu, et al., Immunity, 4:387-396
 (1996), incorporated herein by reference.
 1 .mu.g of pPYC plasmid (FIGS. 4 and 5) was cut by EcoRI to linearize and
 was purified from agarose gel. Rip cDNA was amplified by PCR and was
 purified from agarose gel. The oligo-nucleotide sequences used to amplify
 Rip were
 ACGACTCACTATAGGCTAGCCGCCACCATGGCTTACCCATACGATGTTCCAGATTACGCTGGGCAACCAGACATG
 TCCTTGAA (SEQ ID NO:3)
 and
 TTGCCAAAAGACGGCAATATGGTGGAAAATAACGTGTCGACTCTAGAGGTACCACGCGTGTTAGTTCTGGCTGAC
 GTAAA (SEQ ID NO:4).
 Flanking sequences required for homologous recombination between PCR
 fragment and vector are underlined. The purified vector and PCR fragment
 was co-transfected into yeast by a standard Li/PEG method known in the
 art. Transformants were plated on SD-W selection plate and were incubated
 in 30.degree. C. incubator for 4 days. Colonies were harvested and pooled
 together for plasmid mini-preparation to recover recombinant plasmid from
 yeast. The plasmid from mini-preparation was transformed into E. coli to
 isolate single colony on LB plus 50 .mu.g/ml ampicilin. Five colonies were
 picked to grow up for plasmid mini-preparation and subsequent restriction
 enzyme digestion and sequencing verification.
 Clones with Rip cDNA inserted in-frame downstream of the tag (HA) were
 co-transfected with pGDB, an apoptosis reporter vector, into 30% confluent
 mammalian 293 (Phoenix) cells by Ca.sup.2+ phosphate transfection method
 to test expression of Rip. FIG. 6 shows the results, extensive cell death
 due to the expression of HA tagged Rip.