WO1992000385A1 - Universal mapping probes for identifying and mapping conserved dna sequences - Google Patents

Abstract

The present invention provides a method for preparing Universal Mapping Probes for identifying and locating DNA sequences which are conserved in genomes of many different species, and which are likely to be contained within or near expressed genes. Chromosomal DNA is digested with an infrequently-cutting site-specific cleaving agent and a second site-specific cleaving agent, and the cut segments are inserted into vectors for cloning. Clones containing non-repetitive chromosomal DNA segments are selected and tested for cross-hybridization with DNA from other species. Selected clones are then assigned positions on the chromosomes from which they were derived, using known mapping techniques.

Description

UNIVERSAL MAPPING PROBES FOR IDENTIFYING AND MAPPING CONSERVED DNA SEQUENCES

BACKGROUND

This invention relates to the field of genetic mapping. In particular, it provides a method for preparing Universal Mapping Probes, used to identify and locate DNA sequences which are conserved in genomes of many different species, and which are likely to be contained within or near expressed genes.

The eucaryotic genome consists of millions of base pairs of DNA compacted into several chromosomes. A large fraction of chromosomal DNA consists of short, repeated nucleotide sequences (highly repetitive DNA) , with longer repeated sequences also interspersed throughout. The function of these sequences is largely unknown. Repetitive DNA sequences tend to diverge a great deal over the course of evolution, presumably because their function is not sequence- dependent.

In contrast, the eucaryotic genome also contains non-repetitive or "unique" sequence DNA, which comprises genes. Because the nucleotide sequence within a gene is the "code" which determines the ultimate structure and function of a protein, gene sequences change slowly over the course of evolution, and remain relatively similar in genomes of different species. This "conservation" of genetic information is proportional to the elapsed time since the species diverged. More conservation exists among mammalian species, for example, than between a mammal and an insect. I Environmental considerations aside, the set of visible traits or "phenotype" belonging to an individual is an expression of its genetic makeup. For example, in economically important plants and animals, desirable genetic traits are selected through years of breeding. The less that is known about the genes specifying a certain trait, the more of a long-term "trial and error" procedure the breeding program becomes. To shorten breeding programs and increase the efficiency by which desirable traits can be selected in these economically important, often essential species, an understanding of their genetic organization is fundamental.

In humans many diseases and disorders are genetically inherited. These include such well-studied disorders as thalasemia, sickle-cell anemia, hemophelia and Tay-Sachs disease. In addition, it is becoming increasingly more apparent that the pre-disposition to certain diseases is genetically inherited. This has been shown to be the case for several types of human cancer, including retinoblastoma and renal carcinoma. Diagnosis, treatment or elimination of these diseases requires an understanding of their genetic basis, but contemporary knowledge of the genes involved is still very limited. Traditionally, genes have been "found" within a genome through classic genetic analysis, by following the inheritance patterns of particular traits through several generations of offspring. Recent breakthroughs have come about through the use of molecular genetic analysis, in which genes can be physically located and analyzed. The most striking advances have occurred, however, in organisms that can be studied by both classical and molecular genetic analysis.

The result of classical genetic analysis is the genetic linkage map, which describes the relationship among genes and DNA markers based on the inheritance patterns of the expressed phenotype. Closely linked genes will be inherited together, while unlinked genes (genes on separate chromosomes being the most extreme example) will be inherited independently. A recent extension of such an analysis is the RFLP (restriction fragment length polymorphism) map, which tracks the inheritance patterns of individual variations in DNA sequence which do not necessarily result in an altered phenotype. These slight variations, then, become "markers" which can be physically assigned to positions on the genome.

Both linkage maps and RFLP maps depend on following inheritance patterns. For this reason, the most complete maps exist for organisms which possess a short generation time, relatively simple genomes, and can be easily manipulated in the laboratory. These include the mouse, frog and fruit fly. In addition, such organisms can be manipulated to produce particularly useful genetic lines, which are of critical importance in studying the inheritance pattern of a particular phenotype, such as the predisposition to a disease.

Though humans don't fall into the category of "convenient lab animals", the obvious importance of the human genome has prompted an intensive effort toward mapping and sequencing it. To supply the necessary genetic material to study inheritance patterns in humans, "genetic banks" have been formed through collaborative efforts of scientists worldwide. One such repository is the Centre d¹ Etude Polymorphisme Humain (CEPH) in Paris, which contains cultured cells of members of 40 large families, comprising at least eight children and both sets of grandparents.

Three technological advances in the field of chromosome mapping have revealed aspects of genome organization that will certainly facilitate localizing and characterizing eucaryotic genes. These are: (1) the realization that restriction fragment length polymorphisms can be used as markers; (2) the development of electrophoretic methods for separating very large DNA fragments; and (3) the discovery of site specific restriction endonucleases that cleave infrequently in the mammalian genome. Such endonucleases possess the common property of cleaving DNA less frequently than predicted by the length of the recognition sequence.

Many infrequently-cutting restriction endonucleases possess the feature that the sequence they recognize for cleavage contains the dinucleotide C-G. The cytosine of this dinucleotide is frequently methylated in mammalian DNA, rendering it resistant to cleavage by restriction enzymes. However, these rare-cutting restriction enzymes are able to cleave DNA in "islands" of GC-rich sequence dispersed along the chromosome. Bird et al., Cell 4_0: 91-99 (1985); Brown and Bird, Nature 322: 477-81 (1986). The C-G's in these "islands" (termed "Hpall tiny fragment (HTF) islands" due to the abundance of Hpall restriction sites) are not methylated, and therefore can be cleaved by restriction enzymes specific for this dinucleotide. These islands were have been found to contain an abundance of rare restriction sites clustered within them. More importantly, these clusters of GC-rich rare restriction sites have now been found in association with many genes. Bird et al., Cell 40: 91-99 (1985); Brown and Bird, Nature 322: 477-81 (1986). In one study, three of four HTF islands analyzed contained transcribed DNA. Lindsay and Bird, Nature 327: 336-38 (1987). Thus, HTF islands are diagnostic for genes, and occur on the average of once in 100 kb in the mammalian genome.

The enzyme Notl recognizes the 8-base pair sequence GCGGCCGC, and cuts least frequently of all the C-G rare-cutting restriction enzymes recognizing the C-G element. . Notl sites have also been found to be clustered in islands of GC rich DNA (termed "NTF islands"), but with only one tenth the frequency of HTF islands (Hpall recognizes a 4 bp sequence while Notl recognizes an 8 bp sequence). Thus, Notl clusters occur on the average of once per 1,000 kb in the mammalian genome, and contain an average of 5 Notl sites. Smith et al., Nuc. Acids Res. 15: 1173-84 (1987) . Notl clusters are also indicative of transcribed regions, and because of their rarity and placement near genes, tend to be evolutionarily conserved among different species. For example, Smith et al., Am. J. Hum. Genet. 45: 443-47, (1989) isolated two cosmids containing human clusters of rare restriction sites, including Notl, and found that portions of them, corresponding to unique sequence DNA, cross-hybridized with mouse and hamster unique sequence DNA. This and the other above mentioned features of Notl have made it useful in the development of mapping strategies for both procaryotic and eucaryotic genomes. Poustka et al., Nature 325: 353-55 (1987; Smith et al., Am. J. Hum. Genet. 4_5: 443-47 (1989); Tartof et al., Gene. 67: 169-82 (1988); Daniels et al., Nature 3_25_: 831-32 (1987).

Molecular genetic techniques have yielded many advances in the search for genes in the eucaryotic genome; however, the greatest advances have been made in organisms which can also be easily studied using classical genetic techniques. It is often the case that linkage maps and known patterns of inheritance provide the first clues regarding the location of an unknown gene. Then, after a gene has been isolated and characterized by molecular methods, inheritance patterns provide critical verification of the gene's functional characteristics. In species whose patterns of inheritance cannot easily be observed, this critical verification may not be accomplished. Such species therefore remain less well-characterized genetically. Unfortunately, the species which are often difficult to characterize are the economically important ones, such as large mammals and perennial crop plants. It would be of great utility, therefore, to be able to transfer map information from well-characterized species to species which are not so well-characterized due to limitations of classical genetic analysis.

Current technology allows cloned genes or cDNA from one species to be used to locate that same gene in the chromosomes of another species. But, because of the large average size of the advanced encaryotic genome, it would be so time-consuming and laborious to locate each gene in this manner, one by one, as to be impracticable. Additionally, the fact that not all genes are represented in a cDNA library exacerbates the inherent difficulty of such an undertaking. What is needed is a way to transfer map information from large regions of a well-characterized chromosome to that of an uncharacterized one. This could be accomplished upon satisfying two prerequisites: (1) large contiguous segments of chromosomal DNA sequence must be conserved from one species to the next; and (2) the conserved regions must bear convenient "landmarks", which are also conserved.

The mean length of conserved segments between the human and mouse genome has been estimated, and it has been found that large, contiguous sequences are indeed conserved between these mammals. Nadeau and Taylor, Proc. Nat. Acad. Sci. USA 81: 814-18 (1984) found that, on the average, segments of approximately 8 million base-pairs in length showed conservation with regard to the structure and organization of genes within such segments. Furthermore, this conservation reaches to the actual DNA sequence level, where sequence homology between human, mouse and hamster DNA was found to exist in the rare restriction site clusters discussed earlier. Smith et al.. Am. J. Hum. Genet. 45: 443-47 (1989). Clearly, these rare - i -

restriction site clusters would be ideal "landmarks" to ai in transferring map information from one species to another. They are spaced at large intervals along chromosomes (100 kbp for HTF islands, 1,000 kbp for NTF islands), are gene-associated and cross-hybridize with homologous regions in genomes of other species.

Though the above-mentioned findings resulted from studies conducted for different purposes, they provide the necessary prerequisite for developing a system by which mapping information from one species may be used to construct a genetic map in related species. This invention is directed toward utilizing these conserved, potentially gene-associated DNA sequences for the purpose of transferring genetic map information from well-characterized species to species where such genetic information is needed.

SUMMARY OF THE INVENTION This invention is directed toward generating an effective means of identifying and locating gene-associated DNA sequences that are conserved in genomes of different species. Such a means, presently unavailable, will facilitate the transfer of genetic map information from genetically well-characterized species to species of economic or other importance, which are less well-characterized.

One feature of the invention is a method of preparing a set of Universal Mapping Probes (hereinafter sometimes referred to as UMPs) to function as an ordered library of probe/markers, spaced at predictable intervals along a selected chromosome. Such libraries offer a significant advantage over the current technology, where often only a few, sporadically-positioned cloned markers are available for genomes of important species. More importantly, Universal Mapping Probes are constructed in such a way that they are likely to cross-hybridize with homologous sequences in other genomes. This is accomplished by utilizing certain site-specific cleaving agents, such as restriction endonucleases which cleave infrequently, in potentially conserved sequences likely to be transcribed. Recognition sequences for these endonucleases are often clustered in, but not restricted to, highly GC-rich regions, in which the normally methylated element C-G remains unmethylated, enabling the endonucleases to cleave.

Universal mapping probes can be constructed from individual chromosomes or entire genomes. Chromosomal DNA is digested with a selected infrequently-cutting cleaving agent and a second selected cleaving agent, generating appropriately-sized cut segments with convenient termini for cloning. These segments will contain DNA sequences from one or both of two classes found in eucaryotic genomes: (1) repeated sequences that are not usually associated with genes; and (2) unique, or non-repetitive, sequences that constitute potentially gene-associated sequences. The segments are inserted into vectors for cloning, after which clones containing the unique-sequence chromosomal DNA may be selected. Once cloned, these unique sequences are mapped to their corresponding chromosomal positions, using mapping techniques such as restriction fragment length polymorphism (RFLP) mapping, linkage mapping and in situ hybridization. According to another aspect of this invention, the method summarized above will produce a set of cloned markers, wherein each member of the set contains a different segment of chromosomal DNA, but possesses several common features. Each clone will contain a segment of chromosomal DNA possessing non-repetitive sequences likely to be gene-associated and conserved among species. These sequences will be assigned corresponding positions on the chromosome from which the Universal Mapping Probe set was derived. One terminus of each segment will comprise a recognition sequence for the selected infrequently-cutting site-specific cleaving agent, while the other terminus will comprise a recognition sequence for the second cleaving agent. Each segment will be inserted into a vector which has been modified to comprise the appropriate site-specific cleaving agent recognition sequences. Further aspects of this invention relate to methods for utilizing Universal Mapping Probes to transfer genetic map information among different species. In one aspect, a selected member of a Universal Mapping Probe set is used to identify and locate homologous sequences in chromosomes from related species. Chromosomal DNA from the species of interest is digested with one or more site-specific restriction endonucleases, and the cut fragments are separated electrophoretically. The selected Universal Mapping Probe is labelled by one of various means and used as a hybridization probe in Southern blot analysis of the separated chromosomal fragments to reveal restriction fragment length polymorphisms. Such RFLPs allow one to genetically map the probes in question. Used in this manner, a Universal Mapping Probe positioned in or near a known gene (a gene responsible for a known function) in one species can pinpoint the location of that gene in the genome of another species. Thus, a gene encoding a desirable trait in mice, for instance, can be identified and located in the genome of cattle. This type of molecular genetic "short-cut" could obviate traditional breeding programs, designed to yield the same information. According to another aspect of this invention, there is provided a method for physically determining the location of a human disease gene in an experimental organism where independently derived mutations of this locus more frequently occur. In this case, the UMP is used to clone and restriction map considerable extents (megabase lengths) of the homologous normal and mutant chromosome segments from the second species. By comparing the maps of normal and mutant individuals it should be possible to reveal structural aberrations such as deletions, translocations, duplications etc. that cause dysfunction of the gene and thereby identify those portions of the DNA that constitute the locus itself. Either^' strategy for the use of Universal Mapping Probes will facilitate the transfer of genetic map information from one species to another. Each of the various aspects of this invention is set forth more fully in the detailed description of the invention provided below. DESCRIPTION OF THE DRAWINGS

Figure 1A represents a base pair sequence (RN-1; top strand=SEQUENCE ID.l, bottom strand=SEQUENCE ID.2) comprising a Notl linker construct for insertion into an EcoRI restriction endonuclease site; the locations of restriction sites and linker region are indicated for this sequence (R = EcoRI; R^~ = defective EcoRI; TGA = a termination codon) ; ***

Figure IB represents a base pair sequence (HN-1; top strand=SEQUENCE ID.3, -bottom strand=SEQUENCE ID.4) comprising a Notl linker construct for insertion into a Hindlll restriction endonuclease site; the Notl and linker segments are as shown in Fig. 1A; the locations of restriction sites and linker region are indicated for this sequence (H = Hindlll; H^~~ = defective

Hindi!I) ;

Figure 1C represents a base pair sequence (HN-2; top strand=SEQUENCE ID.5, bottom strand= SEQUENCE

ID.6) comprising a Notl linker construct for insertion into a Hindlll restriction endonuclease site; the

SUBSTITUTE ISA/US construct having a linker segment different from that shown in Figs. 1A and IB; the locations of restriction sites and linker region are indicated for this sequence (H and H^~* are as defined in IB; T *A*G* = a termination codon) ;

Figure ID represents a base pair sequence (RN-2; top strand=SEQUENCE ID.7, bottom strand=SEQUENCE ID.8) comprising a NotI-T7 promoter construct for insertion into an EcoRI restriction endonuclease site. The locations of restriction sites and T7 promoter region are indicated for this sequence (R = EcoRI; R^— = defective EcoRI; T *G*A* = a termination codon) .

Figure 2A illustrates a vector constructed in accordance with the invention, derived from the plasmids pUC18 or pUC19 and including a Notl linker construct, with the location of its insertion shown adjoining the multiple cloning site (MCS) of the vector (R, R^—, H, and H^— are as defined in Figure 1) ; Figure 2B depicts vectors constructed in accordance with the invention, derived from lambda phage EMBL4 and including either a Notl linker construct (EMBL4N) or a NotI-T7 promoter construct (KT4) , with the location of insertion shown at the EcoRI site on the left arm of EMBL4; the "Stuffer" segment represents a DNA fragment necessary for achieving a phage DNA size adequate for packaging (R = EcoRI; B = Ba HI; S = Sail) . DETAILED DESCRIPTION OF THE INVENTION

Sections I-IV below detail steps in the construction and use of an ordered set of cloned markers, referred to herein as Universal Mapping Probes. The object of this invention is to provide a means by which genetic map information may be transferred from one species to construct a genetic map in another. Furthermore, all genetic maps so constructed from a species to which UMPs are mapped can also be related to one another, thus providing a

SUBSTITUTE ISA/US "network" of map information among related species. The invention relies on the inclusion in each cloned marker of a "rare" cleavage site which has a high probability of occurring within or near a transcribed gene, and which, as a result, is likely to be conserved among genomes of different species.

A rare cleavage site is defined, for purposes of this invention, as one which is cleaved less frequently by a site-specific cleaving agent than would be mathematically predicted by the length of the recognition sequence. For example, in a preferred embodiment of the invention, a cleavage site, comprised of the recognition sequence for the endonuclease Notl is included in each member of the Universal Mapping Probe set. This recognition sequence comprises eight base pairs, which should occur once in approximately 65,000 base pairs (4®) . However, it has been empirically determined that Notl cleaves only about once in 200,000 base pairs in the human genome: less than half the frequency predicted in a genome comprised of 50% GC. Thus, the Notl recognition sequence satisfies the definition of a rare cleavage site. Cleaving agents, such as Notl, which are site-specific to rare cleavage sites, are referred to herein as infrequently-cutting site-specific cleaving agents. Additionally, a collection of cloned, ordered Universal Mapping Probes from a selected chromosome or genome is referred to herein as a Universal Mapping Probe (UMP) library or set.

The rationale for constructing ordered libraries of DNA segments whose members contain a rare cleavage site has both a theoretical and an empirical basis. Theoretically, if a specific agent, such as a restriction endonuclease, cleaves less frequently than mathematically predicted, it is likely that its recognition site has been selected against in evolution. Sequences subjected to selective pressure are usually maintained in a genome because they serve some function (for example, they are associated with expressed genes) . For this reason also, they are often conserved among species as the species diverge evolutionarily. Thus, this invention is not limited to the particular rare restriction sites discussed below, but is intended to encompass the use of any rare cleavage site as included by the above-stated general definition.

A preferred embodiment of the invention incorporates the empirical finding that certain rare cleavage sites occur in GC-rich clusters in the human genome, which are often associated with the 5' ends of genes. As previously stated, GC-rich regions of DNA are often associated with genes. Moreover, in mammals, the dinucleotide element C-G is usually methylated, and cannot be cleaved by restriction endonucleases. In these GC-rich "islands" containing clusters of rare cleavage sites, however, the C-G element is not methylated, and can, therefore, be cleaved. Though the exact function of these cleavage site clusters is not known, their rarity provides is evidence that they have been subjected to some selective pressures. Additional supporting evidence comes from the finding that these restriction site clusters have been found to be conserved among various species. These gene-associated, conserved cleavage sites are preferred for use in the practice of this invention. Examples of such sites, and the corresponding retriction endonucleases that cleave them, are listed in Table I below. TABLE I. Restriction endonuclease cleavage sites containing the C-G element:

SITE ENDONUCLEASE

GCGGCCGC Notl

CGGCCG Eagl

GCGCGC BssHII

ACGCGT Mlul

CCGCGG SacII

CGATCG Pvul

CCCGGG Smal

GCCGGC Nael 0 GGCGCC Na l (Nunl)

CCGG Hpall

GCGC Hhal

The use of the shorter cleavage sites is contemplated in the practice of this invention even though they may

-,,- occur more frequently than is desirable. Such restriction sites may nevertheless be of use in restriction endonuclease digestions that are not carried to completion. As mentioned earlier, the present invention is not limited to the use of the

2_Q restriction sites listed above. For example, the infrequently-cutting restriction endonuclease Sfil, whose restriction site (GGCCNNNNNGGCC) does not fall into the above-mentioned class, is also contemplated for use in the practice of this invention.

_2c Although restriction endonucleases are presently the most widely-used means of site-specific cleavage of double stranded DNA, this invention also contemplates the use of alternative methods of site-specific cleavage. For example, it may be

2_Q possible to achieve greater specificity than that provided by infrequently-cutting restriction enzymes, such as Notl, using the sequence-specific cleavage reaction described by Moser and Dervan, Science, 238: 645-50 (1987) . In carrying out this non-enzymatic gc reaction, a 20-bp homopyrimidine sequence would replace the restriction sites. When a complementary homopyrimidine oligodeoxynucleotide containing an EDTA-Fe II complex at its 5' end is hybridized to the corresponding sequence in a vector, a triplex structure is created. Upon addition of dithiothreitol, a double-strand break is produced at the site of hybridization.

Another example of alternatives to the use of naturally-occurring restriction enzymes is the use of synthetic DNA binding and cleaving proteins that are specifically designed to recognize a selected oligonucleotide sequence. Such "designer" proteins are likely to become increasingly important as tools in recombinant DNA technology.

Cleaving the geno ic DNA with a second site-specific cleaving agent, in accordance with the present invention, accomplishes three purposes: first, it reduces the segments to a preferred size range of 1 to 15 kb; second, it introduces a convenient cohesive cloning terminus on the cut segments; and third, it generates segments with non-identical termini, which facilitates cloning only in the desired orientation.

The description which follows sets forth the general procedures involved in practicing the present invention. All temperatures are given in degrees Centigrade unless otherwise indicated. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention.

I. SOURCES AND PREPARATION OF CHROMOSOMAL DNA. In general, this invention is applicable to eucaryotic chromosomal DNA which contains rare cleavage sites. It is contemplated that a Universal Mapping Probe library will be most useful in the species range where large contiguous portions of genomes are expected to be conserved. Some possibilities for useful UMP libraries include, but are not limited to the following: (1) mouse and human UMP libraries for use with mammals; (2) Arabidopsis thaliana UMP library for use with higher plants; (3) Drosophila melanogaster UMP library for use with insects; and (4) Neurospora or Saccharomyces UMP libraries for use with fungi. Mammalian UMP libraries are preferred because mammalian DNA has been shown to contain gene-associated rare restriction site clusters, which are likely to be conserved among mammalian species. While such clusters are also likely to exist in other eucaryotic genomes, this has not yet been conclusively demonstrated.

One specifically preferred embodiment utilizes human chromosomes as the starting material for an UMP library. Human chromosome 3 in particular is preferred. Human chromosome UMP libraries should enable the elucidation of the genetic basis for disease, as well as aiding in the worldwide effort to map and sequence the human genome. Universal Mapping Probes may be constructed from single chromosomes or entire genomes. Total genomic DNA is prepared from cells and tissues according to standard methods known to those skilled in the art. A typical example of such a preparative method is given in Example 4 below. To reduce ambiguity, it is often preferable to construct Universal Mapping Probes from single chromosomes. Sufficient quantities of a particular chromosome may be obtained by isolating condensed metaphase chromosomes and separating them on the basis of size by flow cytometry. Alternatively, a heterologous chromosome may be introduced into a cell line of another species, using techniques known in the ^art* See Patterson et al., Somatic Cell Genetics 9_: 359-74 (1983). For example, in a preferred embodiment, human chromosome 3 is introduced into a cell line of Chinese hamster ovarian tissue. A genomic library is then prepared from DNA isolated from the cell line containing the extra chromosome. Clones containing human chromosomal DNA fragments are selected by colony or plaque hybridization using labelled total human DNA as a probe. Because 5 mammalian DNA is comprised largely of highly repetitive sequences which are not conserved among species, total human DNA will serve as a probe to easily identify clones containing human, rather than hamster, DNA fragments. In this way, large amounts of 10 chromosome-specific DNA clones may be prepared and identified by standard techniques. From these, UMPs can be obtained as described below.

II. PREPARATION AND MODIFICATION OF CLONING VECTORS

15 FOR UMP LIBRARIES

Vector modification may be accomplished by any one of several methods known in the art. A preferred method is the preparation of synthetic oligo- nucleotides incorporating the specific cleavage sites

20 of interest, as well as any other useful sequences, such as transcription promotors or universal primer targets for DNA restriction mapping and sequence analysis. Such synthetic oligonucleotides are referred to herein as "linkers" or "linker constructs"

-,. Synthetic linker constructs may be prepared by the phosphoramadite method employed in the Applied Biosystems 380A DNA synthesizer or similar devices. The resultant constructs may be purified according to procedures well known in the art, e.g. by

_₀ electrophoresis on a 10% polyacrylamide gel.

Different cloning vectors may be modified for use in the present invention. Indeed, most of the more commonly-used vectors may be successfully applied in constructing UMP libraries according to this invention.

₃₅ Suitable vectors include the pUC plasmid vectors, such as pUC18 or pUC19, both of which possess the lacZ complementation system. These vectors are used to particular advantage in that colonies of bacteria successfully transformed therewith are readily identifiable using known techniques. Other suitable vectors include lambda phage vectors, such as lambda EMBL4, and cosmid vectors such as cos4.

The plasmids pUClδ and pUC19, described in Perron et al., supra 33: 103 (1985), may be routinely propagated in E. coli strain DH5°<, available from Bethesda Research Laboratories. The pUC plasmids, as well as their derivatives decribed hereinafter, are grown in LB medium or "T-broth" (TB) medium. Preparation of LB medium is described in Maniatis et al. , Molecular Cloning, Cold Spring Harbor Laboratory (1989) (hereinafter "Maniatis"). One liter of TB is made by adding 100 ml of a sterile solution of 0.17 M KH2PO4 to a separately sterilized solution containing 12 g bacto-tryptone, 24 g bacto-yeast extract, 4 ml glycerol and water to a final volume of 900 ml.

In order to prepare plasmid DNA, plasmid-bearing bacteria are grown to saturation overnight with vigorous shaking at 37°. Plasmid DNA is readily obtained by the alkaline lysis procedure described in Maniatis, supra. It has been found preferable to carry out an additional phenol extraction step after precipitation of the DNA from the CsCl gradient. The additional extraction step eliminates trace amounts of exonuclease that frequently contaminate plasmid DNA preparations. The presence of such contaminants can result in a slightly smeared appearance to the bands produced after restriction enzyme digestion.

Liquid cultures of lambda phage EMBL4, as described in Frischauf et al., J. Mol Bio. , 170: 827 (1983), and related phage, are grown on E. coli strain BHB2600 in NZYM medium, as disclosed in Maniatis, supra. Phage stocks and phage derived by in vitro packaging are titered or amplified using E. coli strain LE392, and plated on soft agar. Quantities of lambda phage are prepared by adding 1.3 x lθ8 phage to 6.7 ml. of a saturated culture of E. coli strain BHB2600 grown in NZY medium and incubating at 37° for 20 minutes. The infected culture is then transferred to 330ml of NZYM broth in a 2 liter flask and vigorously shaken at 37° for about 6 hours, at which time complete lysis is apparent. The lysate is adjusted to 0.5 M NaCl and 0.02 M MgCl2 and cell debris is removed by centrifugation, and the phage precipitated with polyethylene glycol(PEG 8000). Lambda phage DNA is extracted by the method of Thomas et al., J. Mol. Biol., 91: 315 (1975).

A preferred cosmid vector is cos4, constructed as disclosed in Steller et al., EMBO J. , 4:167(1985). The cos4 vector, and its derivatives, are propagated in E. coli strain DH1 or DH5o< and cultured in TB medium. Xgal and a picillin are added to agar plates or broth as necessary. It is noted that when DH1 or DH5c are grown in LB broth, a typical yield of cosmid DNA is about 100 ug. per 330 ml. of culture. With the use of TB medium, however, it has been found that about 2 mg. of cosmid DNA per 330 ml. of culture can be routinely obtained. Therefore, by growing bacteria in TB medium, it is possible to process many 50 to 100 ml. cultures, and to obtain approximately 250 ug. of cosmid DNA. TB has also been found to increase the yield of pUC plasmids.

To prepare vector DNA for modification by insertion of the linker constructs described above, plasmid or lambda DNA is digested at 37° for 60 minutes with a fourfold excess of restriction endonuclease in 20 to 50 ul of "Universal Restriction Buffer" (URB). This buffer is a modification of the Tris-acetate buffer described by O'Farrell, Focus 3: 1 (1980), and contains 33 mM Tris-acetate, pH 7.9, 66 mM potassium acetate, 10 mM magnesium acetate, 100 ug/ml bovine serum albumin (BSA) , 0.5 mM dithiothreitol (DTT) and 4 mM spermidine. All of the restriction enzymes tested possess the same activity in URB as in the manufacturers' recommended buffer.

To insert a linker construct into a vector of choice, 5 ug of the complementary strands of the appropriate unphosphorylated synthetic oligonucleotide are annealed in 10 ul of ligation buffer (22 mM Tris acetate, pH 7.4, 7.5 mM MgCl₂, 0.1 mM EDTA, 0.5 mM adenosine triposphate (ATP), 1.0 mM DTT) at 37° for 60 minutes to produce the desired double stranded segments. One microgram of this DNA is ligated to a similar amount of restriction enzyme-digested plasmid or lambda DNA in 10 ul of ligation buffer containing 3-10 units of T4 DNA ligase and 50 ug/ml BSA, then incubated at 14° for 12 to 24 hours. Cells competent for transformation by plasmid DNA are prepared as described in Maniatis, supra, and plated on LB-agar supplemented with ampicillin and/or Xgal as necessary. Ligated lambda DNA is packaged into phage and plated on E. coli strain LE392.

III. CONSTRUCTION OF UNIVERSAL MAPPING PROBE LIBRARIES (a) . Insertion of Chromosomal DNA fragments into modified vectors. Recombinant DNA libraries may be prepared with the above-described modified cloning vectors by digesting both vector and genomic DNA with the specific cleaving agent of choice, ligating with T4 DNA ligase, and transforming or transfecting host cells. Each of these recombination steps may be performed according to standard procedures disclosed in Maniatis, supra.

More specifically, genomic DNA segments are generated by cleaving, either partially or completely, with a selected infrequently-cutting restriction endonuclease and a second restriction endonuclease of choice, generating segments terminated at one end with a rare restriction site, and at the other end with some other restriction site convenient for cloning purposes. The order and extent of digestion with the two restriction endonucleases will vary, depdnding on the enzymes used, the source of chromosomal DNA anc the desired size of the resulting segments. In a preferred embodiment, the genomic DNA of choice is first partially digested with Mbol, and fractions containing the desired segment size range are selected. Those segments are then digested to completion with the infrequently-cutting restriction enzyme Notl.

Genomic DNA segments are dephosphorylated, then inserted into modified cloning vectors which have been previously digested with appropriate restriction enzymes. In the aforementioned embodiment, modified EMBL4 vectors are digested with Notl and BamHI (BamHI and Mbol termini are compatible) , then incubated with the chromosomal DNA segments and T4 DNA ligase. The products of this reaction are packaged in vitro into lambda phage and propagated in E. coli strain LE392 by plating on soft agar.

(b) . Selection of clones containing non- repetitive DNA sequences.

Clones containing non-repetitive DNA sequences may be easily selected using total chromosomal DNA as a probe. Chromosomal DNA segments are excised from vector DNA by cleavage with the appropriate cleaving agents, the segments are separated by agarose gel electrophoresis, then transferred and immobilized upon a solid support for Southern hybridization analysis. Total chromosomal DNA from the chromosomes from which the UMP library was derived is labelled and used as a hybridization probe. This type of probe contains a high percentage of repetitive DNA sequences, as previously discussed, and will hybridize strongly with excised segments which contain repetitive sequences. Non-repetitive DNA sequences, on the other hand, will not be detected by the total chromosomal DNA probe. Thus, non-hybridizing fragments will be those which contain non-repetitive (unique-sequence) DNA, and may be selected accordingly. (c) . Assigning UMPs to their corresponding position on the chromosomes.

Once cloned, unique chomosomal DNA segments may be assigned to a position on the chromosome from which they were derived using one or several techniques known in the art. One method that is of particular utility is restriction fragment length polymorphism (RFLP) mapping. This method has widespread applicability because restriction fragment length polymorphisms can usually be found in any fragment of genomic DNA, providing there is a suitably large population of familialally-related DNA to analyze, and provided that appropriate restriction endonucleases are used in the analysis. Restriction fragment length polymorphisms are often prevalent in flanking regions of polymorphic alleles or members of a gene family. Polymorphic banding patterns are detected in restriction endonuclease-cleaved DNA by Southern blot analysis of total genomic DNA from two parents and two generations of offspring, using the UMP as a radiolabelled probe. Restriction fragment polymorphisms behave as co-dominant mendelian elements, so that the first offspring (F_j generation) shows the sum of the polymorphisms of the two parents. The second offspring (F₂ generation), however, shows classical mendelian segregation of the polymorphisms. By correlating the segregation of the polymorphisms with the segregation of known genetic markers in a particular breeding cross, the newly-identified locus can be placed on a chromosome linkage map. Because polymorphisms for each UMP marker can be obtained, they too may be localized to positions on a chromosome map by RFLP mapping. In addition, UMPs may be located on the physical map by classical restriction endonuclease mapping.

Another method contemplated for localizing a Universal Mapping Probe to a particular chromosomal region is in situ hybridization of a labelled UMP to condensed metaphase chromosomes. This method, originally applied to polytene chromosomes of Drosophila, has recently been improved for utility with other chromosomes. Lawrence et al., Cell 52: 51-61 (1988). Individual DNA sequences are localized on chromosomes using fluorescence detection of biotinated probes hybridized in situ. The use of fluorescence detection provides low background and high hybridization efficiency, allowing high resolution localization of single sequences on chromosomes within the interphase nucleus.

Thus, a Universal Mapping Probe library consists of an ordered set of cloned markers, each member corresponding to a known position on the chromosome or genome from which it is derived. Physically, each Universal Mapping Probe in the library contains a segment of genomic (chromosomal) DNA, terminated at one end by a rare cleavage site and at the other end by a second cleavage site to facilitate cloning, and comprising sequences of unique (non-repetitive) DNA. The genomic DNA fragment is inserted into one of several common cloning vectors, which has been modified to contain the appropriate cleavage sites. Because of the way they are constructed. Universal Mapping Probes are likely to contain DNA sequences comprising genes, and are conserved among genomes of different species. Therefore, they can be powerful tools for the transfer of genetic mapping information from one species to another. IV. UTILIZATION OF UNIVERSAL MAPPING PROBES

A Universal Mapping Probe located in or near a known gene can be used to identify and locate homologous sequences in chromosomes from another species. Samples of chromosomal DNA from the species of interest are each digested with a different selected site-specific cleaving agent, and the cut fragments separated eleσtrophoretically, as described in Maniatis, supra. After the separated chromosomal fragments have been immobilized on a suitable membrane support, the selected Universal Mapping Probe is labelled and used as a hybridization probe in Southern blot analysis. The labelled UMP will identify fragments containing homologous sequences that exist in the target chromosomes of interest.

Positively-identified sequences may themselves then be isolated, cloned and assigned a position in the genome from which they were derived, using methods described previously for assigning UMPS to corresponding positions on chromosomes. Alternatively, if the

Universal Mapping Probe cross-hybridizes strongly with the homologous sequences in the target chromosome of interest, the additional cloning step may not be needed. The Universal Mapping Probe may itself be used to determine the position of the homologous sequence in the other genome.

In another application of this invention, an UMP library may be "screened" to determine if it contains sequences homologous to those of a known gene from another species. DNA samples from each member of the UMP library are immobilized on a solid support. The gene of interest is then labelled and used to probe the ordered library. UMP clones containing homologous sequences are then referenced back to their assigned positions on the chromosome from which they were derived. In a preferred embodiment, Universal Mapping Probe libraries from human chromosomes are used to screen animal model systems in which genes encoding diseases have already been identified and located. For example, UMPs from human chromosome 3 are examined for linkage to the putative renal carcinoma (RC) gene of the rat. By testing progeny from the appropriate backcrosses of two rat breeding lines, a collection of UMPs displaying tight linkage to the RC gene is identified. Probes tightly linked to the rat RC locus are then used to screen DNA obtained from normal and tumor tissue of renal carcinoma patients. It is anticipated that some tumor DNAs will be deleted for at least a portion of the RC locus, a phenomenon which will aid in defining the boundaries of the gene and ultimately faciliating its cloning.

Clearly, the present invention will be of great utility in identifying and locating potentially gene-associated DNA sequences that are conserved in genomes of different species. This will greatly facilitate the transfer of genetic map information from genetically well-characterized species to species of economic or other importance where such information is needed. This invention can be used to particular advantage in plant and animal breeding programs, where it can provide a faster, more efficient means of genetic mapping than tradition breeding techniques. Ongoing efforts to treat or cure genetically-inherited human diseases will also benefit from the present invention, as it will enable transfer of genetic information from animal model systems to their human DNA counterparts. This will facilitate the elucidation of the genetic basis for such diseases. The following examples are provided to describe the invention in further detail. These examples are intended merely to illustrate and not to limit the invention. Example 1.

Preparation of Notl linker constructs for vector modification.

Four separate Notl linker constructs, each 30 base pairs in length, were synthesized by the phosphoramadite method. The resultant constructs are shown in Figs. 1A-1D of the drawing. Each construct possesses ligatable, cohesive termini to facilitate insertion of the construct into the various vectors. The Notl linker construct depicted in Fig. 1A was synthesized such that the left terminus contains a ligatable, but not subsequently digestible, defective EcoRI site (R~) , followed by the 8-base pair Notl recognition sequence, followed by a 17-base pair linker segment. The right terminus is a cohesive

EcoRI site (R) which, when ligated, can subsequently be cut with EcoRI. Thus, the Notl linker construct of Fig. 1A (designated "RN-1"), when inserted in the desired orientation into the multiple cloning site of the lacZ gene of various vectors, will be in the proper reading frame to maintain full lacZ function. However, if inserted in the opposite orientation, the termination codon TGA will be read, therby preventing expression of the LacZ gene. Other variations of the same Notl linker construct may be prepared for insertion into vectors. The Notl linker construct shown in Fig. IB, for example, contains the identical 17 base pair linker segment as that shown in Fig. 1A, as well as the 8-base pair Notl recognition sequence. However, the sequence shown in Fig. IB (designated "HN-1") is constructed with one Hindlll" (defective Hindlll) cohesive terminus and one Hindlll cohesive terminus. As in the previously-described sequence, the reading frame is positioned to maintain lacZ function when the construct is inserted in the proper orientation. Fig. 1C shows an alternative construct (designated "HN-2"), containing Hindlll and Hindlll^" cohesive termini, but with a different linker segment. Fig. ID also shows an alternative construct (designated RN-2) , containing EcoRI and EcoRI" cohesive termini, but with a linker segment containing a promotor sequence for T7 RNA polymerase.

Example 2. Insertion of the Notl linker construct into pUC vectors.

The RN-1 and HN-1 constructs prepared as described in Example 1, were inserted at the EcoRI and Hindlll sites of pUC19 and pUC18, respectively, to form pUC19N and pUC18N, as illustrated in Fig. 2A. As seen in Fig. 2A, the construct is inserted adjacent to the multiple cloning sites of the pUC plasmids. Note that since the lacZ function has been preserved in pUC19N and pUC18N, appropriate host cells, for example DH5°(, containing the plasmids give rise to blue-colored colonies when grown on Xgal agar plates. When chromosomal DNA is inserted into the multiple cloning sites of these vectors, white-colored colonies are produced. Transformation was accomplished by standard procedures, as disclosed in Maniatis, supra.

Example 3.

Insertion of the Notl linker construct into the EMBL4 lambda vector. The RN-1 construct described in Example 1 above was inserted into the EcoRI site on the left arm of EMBL4 as illustrated in Fig. 2B. The resulting genome is referred to as EMBL4N, and has a cloning capacity of 9-23 kilobase pairs. The orientation of the "stuffer" fragment in EMBL4N is identical to that in EMBL4. See Frischauf^' et al., J. Mol Biol. , 170: 827 (1983). The RN-2 construct described in Example 1 above was inserted into the EcoRI site on the left arm of EMBL4 as depicted also in Fig 2B. The resulting genome is referred to as KT4, and contains the T7 transcription promotor, in addition to possessing the characteristics of EMBL4N.

Example 4.

Preparation of chromosomal DNA from cells or tissues.

Tissue was homogenized (or cells were resuspended) in a buffer containing 0.02 M Tris acetate (pH 7.5), 0.1 M NaCl, 1 mM EDTA. SDS was added to a final concentration of 1%, and Proteinase K added to a final concentration of 200-500 ug/ml.

Following an incubation, overnight at 37°C, solutions were extracted 2-3 times with buffered phenol. DNA was precipitated by adding 2 volumes ethanol and chilling to -70°C for 20 minutes. Precipitated DNA was spooled onto a pasteur pippette and transferred into 1-2 ml water, to which NaCl was added to a final concentration of 100 mM. DNA was precipitated, again by addition of 2 volumes ethanol, for 20 min at -70°C. Precipitated DNA was transferred to a fresh tube and resuspended in TE buffer (lOmM Tris acetate, 0.1 mM EDTA) .

Example 5.

Restriction endonuclease digestion of genomic DNA to yield fragments punctuated by a Notl and an Mbol restriction site.

Typically,- 5-10 ug of genomic DNA was digested with 40-80 units of the appropriate restriction enzyme in "Universal Restriction Buffer" (URB) : 33 mM Tris-acetate, pH7.9, 77 mM KOAc, 10 mM MgOAc, 100 ug/ml bovine serum albumin (BSA) and 5 mM dithiothreitol (DTT), for 60 min at 37°C. For a digestion with Mbol, dilutions of Mbol were made in Storage Buffer, containing 50 M KC1, 10 mM Tris-acetate (pH 7.4), 10 mM EDTA, 0.1 M DTT, 100 ug/ml BSA and 50% glycerol. Five to 10 ug genomic DNA was incubated with various dilutions of Mbol in 50 ul final volume URB at 37°C for 60 min. Enzyme reactions were inactivated by heating at 65°C for 5 min. Samples (5 ul) were removed from each reaction and examined for size by agarose gel electrophoresis. Digests were selected in which most of the DNA fragments had been reduced to a size range of 15-20 kb. To those digests was added 40-80 units of Notl, which was a sufficient amount to result in complete Notl digestion in 60 minutes at 37°C. This procedure produced a collection of DNA fragments of approximately 15 kb having a Notl site at one end and an Mbol site at the other.

Example 6. Ligation of genomic DNA into phage vectors and transfection of host strains.

To prevent self-ligation, genomic DNA fragments were dephosphorylated by the addition of approximately 20 units (1 ul) calf intestinal alkaline phosphatase to each digest described in Example 5. Following incubation at 37°C for 1 hr, samples were heated to 65°C for 15 min to deactivate the alkaline phosphatase. Samples were extracted once with phenol/chloroform (1:1), once with chloroform only, once with ether, followed by a flush of the ether with nitrogen gas. Fragments were precipitated by the addition of 0.1 volume of 3 M NaOAc (pH 7.4) and 2 volumes ethanol, followed by chilling at -70°C for 20 min (alternatively, samples were chilled to -20°C for 2 or more hours). Samples were centrifuged at 4°C for 10 min to collect the precipitate, which was then washed with 70% ethanol, dried and resuspended in 5 ul of TE buffer.

The alkaline phosphatase-treated NotI-Mbol genomic segments were then ligated to EMBL4N or KT4 lambda phage vectors described in Example 3. After digestion to completion with Notl and BamHI, approximately 1 ug of vector and 0.5 ug genomic DNA fragments were combined in a 10 ul reaction consisting of ligation bufer (22 mM Tris acetate, pH 7.4, 7.5 M MgCl₂, 0.1 mM EDTA, 0.5 mM adenosine triphosphate

(ATP) and 1.0 mM DTT) and 10 units of T4 DNA ligase. The reaction was incubated overnight at 14°C

The resulting ligation products were packaged into mature lambda phage using the "Gigapack" packaging extract obtained from Stratagene Cloning

Systems, Inc., and associated procedures. Recombinant phage derived by this in vitro packaging method were amplified and titered using E. coli strain LE392, and plated on soft agar in the customary manner. Phage clones containing unique-sequence chromosomal DNA were selected, mapped and their species cross-hybridization potential determined by methods described in the upcoming Examples.

Example 7.

Preparing Universal mapping probes from a single human chromosome.

(a). Source of human chromosome 3. A NotI-Mbol genomic library was constructed in lambda KT4 using DNA from a Chinese hamster cell line into which human chromosome 3 had been introduced. The cell line (designated CHO-HU3 (314-2)) was generated according to known methods. See Drabkin et al., Proc. Nat'l. Acad. Sci. 2: 6890-94 (1985); Patterson et al. , Somatic Cell Genet. 9: 359-74 (1983).

Lambda phage clones that hybridized specifically to human DNA sequences were identified by plaque hybridization, using 32p_ι_abeled total human DNA as a probe,- and following standard techniques. Such clones could contain only repetitive sequences, or might be composed of interspersed unique (non-repetitive) sequences that are characteristic of most mammalian DNA. In either case, the cloned fragments were necessarily of human chromosome 3 origin.

(b) . Selecting clones containing unique DNA sequences and determining their species cross-hybridization potential. The library described in part (a) above yielded 413 lambda NotI-Mbol Hu3 clones hybridizing to human DNA. DNA was extracted from each and then characterized by Southern blot analysis. Each clone was digested with Notl and EcoRI, subjected to gel electrophoresis, and the resulting fragments transferred to a nylon membrane. The membrane was then probed with ³2p-_ι_ab_eχ_e(3 total human DNA. Of the 413 clones analyzed in this way, 164 (40%) contained unique sequence fragments whose average size was approximately 3.2 kb (ranging from 1.5 to 17 kb) . From these, 17 unique sequence fragments were subcloned and used as 32p_ι_ak_eι_e(3 probes to assess their ability to cross hybridize to rat, mouse, cow and horse DNA under stringent hybridization conditions of 65°C, 2X SSC (0.3 M NaCl, 0.03 M Na Citrate, pH 7.0). Of the 17, 9 (53%) were found to cross hybridize to DNA of each of the aforementioned species.

(c) . Assigning cloned unique sequence fragments to positions of Human chromosome 3. Five of the nine species cross-hybridizing probes also displayed restriction length polymorphisms with various restriction enzymes. Such polymorphisms can be exploited for constructing meiotic genetic maps with these probes, as well as establishing their syntenic (linkage) relationships in other mammalian genomes. Example 8 .

Locating the human renal carcinoma gene using human chromosome 3 UMPS in conjunction with an animal model system. A breeding colony of rats bearing a single dominantly inherited Eker mutation that predisposes individuals to renal carcinoma (Eker et al. 1981) has been established. The phenotype is highly penetrant so that 50% of the progeny of a mating of RC/RC+ heterozygotes to normal RC+/RC+ animals develop renal tumors. Because the predisposition to renal carcinoma in humans has been localized to chromosome 3, Universal Mapping Probes described in Example 7 can be analyzed for linkage to the rat RC gene utilizing a very sensitive method for detecting polymorphisms between two different strains of rat, one bearing the RC mutation (Long Evans) and the other being normal (Brown Norway) . Denaturing gradient gels (DGG) offer the ability to detect single base differences in fragments as large as 1500 bp. Myers et al., Meth. Enz. 155: 501 (1987). Panels of 20 rat backcross DNAs, derived from mating RC/RC+ heterozygotes of the Long Evans strain to normal RC+/RC+ Brown Norway individuals, are submitted to DGG elctrophoresis and probed with each UMP. Those probes showing linkage with the RC gene are analyzed further using a larger collection of backcross animals to establish precise genetic distances.

UMP probes can also be analyzed by in situ hybridization to human metaphase chromosomes. See Lawrence et al. , Cell 5_2: 51-61 (1988). UMPs localizing at or distal to 3pl4 (the approximate location of the human putative RC gene) can then be used to screen for deletions in tumor DNA in or near the human RC gene as follows. Normal and tumor tissue samples from renal cell carcinoma patients have been collected from patients undergoing treatment at American Oncologic Hospital of the Fox Chase Cancer Center. Cell cultures from tumors have been initiated, and some are propagating as established cell lines. In addition, normal lymphocytes from each patient are immortalized with Epstein-Barr virus.

Epstein-Barr virus-immortalized lymphocytes (control) and RC cell lines (experimental) can be used as the source of high molecular weight DNA. Samples are digested with Notl and the resulting fragments separated by pulsed field electrophoresis. By comparing band sizes from immortalized lymphocyte and RC tumor cell DNA, deficiencies in or near the RC gene can be identified. UMPS that detect smaller bands in tumor compared to normal DNA can be used to test for linkage to the rat RC gene. Although karyotyping solid tumors is difficult, it should be possible to examine chromosome 3 from established RC cell lines during early passages and scrutinize them for abberrations such as deletions, translocations or inversions as a means of defining further the location of the human RC locus.

Once close linkage to the rat and/or human RC gene is obtained a substantial portion of the region can be cloned, and it can be determined which segments code for transcripts that are present in normal kidney cells but are missing or are aberrant in the tumor. Definitive proof that the relevant gene has been cloned can be obtained by sequence comparisons of normal and tumor DNA from RC patients and from similar comparisons between homologous regions in normal and mutant rats.

While certain aspects of the present invention have been described and exemplified above as preferred embodiments, various other embodiments should be apparent to those skilled in the art from the foregoing disclosure. Thus, while the cleavage steps of the above-described technique have been exemplified by use of restriction endonucleases, other site specific cleaving agents may be advantageously used. These include, but are not limited to, the previously-described non-enzymatic cleavage method of Moser and Dervan, supra, as well as synthetic DNA-cleaving proteins and other agents.

In addition, though this invention may have the greatest utility in eucaryotic chromosomal DNA, it may also be applicable to procaryotic DNA in some instances. Therefore, the use of procaryotic DNA in the practice of this invention is contemplated.

Furthermore, the above-described amplification of genomic DNA segments by cloning, using the modified vectors of the invention, may be carried out by alternative means. For example, genomic DNA segments may be amplified by the polymerase chain reaction using methods known to those skilled in the art.

The present invention is, therefore, not limited to the embodiments specifically described and exemplified above, but is capable of variation and modification without departure from the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing a set of Universal Mapping Probes for identifying and locating DNA sequences which are conserved among genomes of different species, comprising the steps of: a) isolating chromosomal DNA from a selected source; b) cutting said chromosomal DNA into fragments with an infrequently-cutting site specific cleaving agent; c) cutting said chromosomal DNA fragments with a second site-specific cleaving agent to provide segments possessing DNA sequences from at least one of the group comprising repetitive DNA and non-repetitive DNA; d) inserting the cut chromosomal DNA segments into clonable vectors; e) producing clones of said insert-containing vectors; f) selecting clones containing said non-repetitive DNA sequences; g) assigning each selected clone to a corresponding position on the chromosome from which said DNA segment comprising that clone was derived.

2. A method as claimed in claim 1, wherein the chromosomal DNA undergoes complete digestion by the infrequently-cutting site-specific cleaving agent.

3. A method as claimed in claim 1, wherein the chromosomal DNA undergoes partial digestion by the infrequently-cutting site-specific cleaving agent.

4. A method as claimed in claim 1 wherein the infrequently-cutting site-specific cleaving agent is a restriction endonuclease.

5. A method as claimed in claim 4, wherein the infrequently-cutting site-specific restriction endonuclease is Notl.

6. A method as claimed in claim 1, wherein the cut chromosomal DNA fragments undergo complete digestion by the second site-specific cleaving agent.

7. A method as claimed in claim 1, wherein the cut chromosomal DNA fragments undergo partial digestion by the second site-specific cleaving agent.

8. A method as claimed in claim 1, wherein the second site-specific cleaving agent is a restriction endonuclease.

9. A method as claimed in claim 8, wherein the second site-specific restriction endonuclease is selected from the group consisting of BamHI and Mbol.

10. A method as claimed in claim 1, wherein the method for selecting the clones having said non-repetitive DNA sequences comprises the steps of: a) excising the chromosomal segments from the vectors; b) separating the chromosomal segments from the vectors by electrophoresis; c) transferring the electrophoresed segments to a solid support; d) Southern blot analysis, using the chromosome from which the UMP library was derived as a labelled hybridization probe; e) selecting clones which contain segments that exhibit no detectable hybridization.

11. A method as claimed in claim 1, wherein the method for assigning the selected clones to a corresponding position on the source chromosome is restriction fragment length polymorphism mapping.

12. A method as claimed in claim 1, wherein the method for assigning the selected clones to a corresponding position on the source chromosome is linkage mapping.

13. A method as claimed in claim 1, wherein the method for assigning the selected clones to a corresponding position on the source chromosome is in situ hybridization.

14. A set of Universal Mapping Probes for identifying and locating DNA sequences which are conserved among genomes of different species, prepared by the method of claim 1.

15. A set of Universal Mapping Probes as claimed in claim 14, wherein the recognition sequence for the infrequently-cutting site-specific cleaving agent is GGCCNNNNNGGCC, N being any nucleotide.

16. A set of Universal Mapping Probes as claimed in claim 14, wherein the recognition sequence for the infrequently-cutting site-specific cleaving agent is comprised of at least 50% (G + C) .

17. A set of Universal Mapping Probes as claimed in claim 16, wherein the recognition sequence for the infrequently-cutting site-specific cleaving agent comprises the dinucleotide sequence C-G.

18. A set of Universal Mapping Probes as claimed in claim 17, wherein the recognition sequence for the infrequently-cutting site-specific cleaving agent is GCGGCCGC.

19. A set of Universal Mapping Probes as claimed in claim 14, wherein the recognition sequence for the second site-specific cleaving agent is GGATCC.

20. A set of Universal Mapping Probes as claimed in claim 14, wherein the recognition sequence for the second^' site-specific cleaving agent is contained within a multiple cloning site.

21. A set of Universal Mapping Probes as claimed in claim 14, wherein the vector is modified to comprise recognition sequences for site-specific cleaving agents, whereby the cut source chromosomal DNA segments may be inserted.

22. A set of Universal Mapping Probes as claimed in claim 21, wherein the vector to be modified is selected from the group consisting of a plasmid, a lambda phage and a cosmid.

23. A set of Universal Mapping Probes as claimed in claim 22, wherein the plasmid is selected from the group consisting of pUC 18 and pUC 19.

24. A set of Universal Mapping Probes as claimed in claim 22, wherein the lambda phage is EMBL4.

25. A set of Universal Mapping Probes as claimed in claim 22 wherein the cosmid is cos4.

26. A set of Universal Mapping Probes as claimed in claim 14, wherein the source of the chromosomal DNA is eucaryotic DNA.

27. A set of Universal Mapping Probes as claimed in claim 26, wherein the source of the chromosomal DNA is mammalian DNA.

28. A set of Universal Mapping Probes as claimed in claim 27, wherein the source of the chromosomal DNA is human DNA.

29. A set: of Universal Mapping Probes as claimed in claim 28, wherein the source of chromosomal DNA is human chromosome 3 DNA.

30. A set of Universal Mapping Probes, comprising a multiplicity of members, each member of said set comprising: a) a segment of chromosomal DNA, wherein one terminus comprises a recognition sequence for an infrequently-cutting site-specific cleaving agent and the other terminus comprises a recognition sequence for a second site-specific cleaving agent; said segment further comprising a non-repetitive DNA sequence which corresponds to a known position on the chromosome from which the segment was derived; b) a modified vector comprising recognition sequences for said infrequently-cutting and said second site-specific cleaving agents whereby said source chromosomal DNA segment is capable of insertion into said modified vector.

31. A method of using Universal Mapping Probes to identify and locate DNA sequences in chromosomal DNA other than that from which the Universal Mapping Probes were derived, said sequences being homologous to the non-repetitive source chromosomal DNA sequences of a selected member of the Universal Mapping Probe set, comprising the steps of: a) cutting said other chromosomal DNA with site-specific cleaving agents; b) separating the cut segments by electrophoresis; c) Southern blot analysis of the separated segments, using said selected member of the Universal Mapping Probe set as a labelled hybridization probe; d) cloning the DNA segments which hybridize with said selected member of the Universal Mapping Probe set; e) assigning a position in said other chromosomal DNA for said cloned DNA segments hybridizing with the selected member of the Universal Mapping Probe set.

32. A method of identifying and locating homologous DNA sequences as claimed in claim 31, wherein the selected member of the universal mapping probe set comprises DNA sequences of a known gene.

33. A method of identifying and locating homologous DNA sequences as claimed in claim 31, wherein the method for assigning a position in the other genome for said cloned DNA segment is restriction fragment length polymorphism mapping.

34. A method of identifying and locating homologous DNA sequences as claimed in claim 31, wherein the method for assigning a position in the other genome for said cloned DNA segment is linkage mapping.

35. A method of identifying and locating homologous DNA sequences as claimed in claim 31, wherein the method for assigning a position in the other genome for said cloned DNA segment is in situ hybridization.

36. A method of determining whether or not members of a Universal Mapping Probe set contain chromosomal DNA sequences homologous to a selected sequence of DNA from a chromosomal source other than that from which the Universal Mapping Probe set was derived, comprising the steps of:

a) cloning and labelling said selected other DNA sequence; b) immobilizing on a solid support a sample of each member of the Universal Mapping Probe set; c) Southern blot analysis of said immobilized Universal^' Mapping Probes, using the cloned, labelled other DNA sequence as the hybridization probe; d) identifying members of the Universal Mapping Probe set which hybridize with said selected, labelled other DNA sequence; e) correlating said identified members with their corresponding positions on the chromosome from which the Universal Mapping Probe set was derived.

37. A method as claimed in claim 36, wherein said selected DNA sequence from a chromosomal source other than that from which the Universal Mapping Probe set was derived comprises DNA sequences from a known gene.

38. A method as claimed in claim 36, wherein said selected DNA sequence comprises DNA sequences closely linked to the rat renal carcinoma gene.