WO1992007092A1

WO1992007092A1 - Method of obtaining a physical map of a genome

Info

Publication number: WO1992007092A1
Application number: PCT/US1991/007651
Authority: WO
Inventors: Michael J. Lane; Brian Faldasz
Original assignee: Genmap, Inc.
Priority date: 1990-10-18
Filing date: 1991-10-18
Publication date: 1992-04-30
Also published as: AU8934291A

Abstract

The invention is directed to a method for obtaining a physical map of a eukaryotic genome or portion thereof, or in a specific embodiment, a chromosome or portion thereof and the map obtained therefrom by detecting repetitive DNA sequences on DNA fragments generated by digestion with a rare restriction enzyme. Such a map would provide immediate access to any segment of the chromosome defined genetically and would provide a starting point for studying the large scale organization of the chromosome.

Description

MKTHOD OF OBTAINING A PHYSICAL MAP OF A GENOME

1. FIELD OF THE INVENTION

The invention is directed to a method for rapidly obtaining a physical map of a genome or large portion thereof or a physical map of a chromosome or portion thereof and the map obtained therefrom. Such a map would provide immediate access to any segment of the genome or chromosome defined genetically and physically and would allow the study of the large scale organization of the entire genome.

2. BACKG OTΓND OF THE INVENTION The successful mapping of the genome of a number of lower organisms has been reported. Examples include the nematode (Coulson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:7821), yeast (Olson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:7826), and E_. coli (Smith et al., 1987, Science 236: 1448) . There has recently been a great deal of interest in mapping the entire human genome. (McKusic and Ruddle, 1987, Genomics, 1:103). Knowledge of the location of human genes and corresponding genetic traits that are produced would lead to major human health benefits. Specifically, identification of genes or regions of DNA involved in several diseases such as Alzheimer's, hereditary forms of cancer, Huntington's Chorea, and manic-depressive illness would lead to new methods of diagnosis and treatment of such disease. Additionally, such studies would provide knowledge of patterns of genomic organization with important functional consequences.

There are two main types of genome maps, genetic linkage maps and physical maps. Genetic linkage maps are formulated primarily by studying families and measuring the frequency with which two different traits are inherited together or linked. Physical maps which may include restriction maps and ordered DNA clone collections, as well as lower resolution maps of expressed genes or anonymous DNA fragments that are mapped by somatic cell hybridization or by in situ chromosome hybridization, are derived from chemical measurements made on the DNA molecules that form the human genome. The present invention is related to methods for obtaining a framework physical map of a chromosome and genome.

2.1. CHROMOSOME MAPPING STRATEGIES

Chromosome mapping strategies have heretofore involved bottom-up strategies, specifically the alignment of smaller DNA segments (cloned into existent vehicles to create a larger map) .

Leach et al., (1989, Genomics 5:167) generated hybrid cell lines by microcell-mediated transfer of human chromosome 17 into rat recipient cells. A partial map of human chromosome 17 was generated by detecting segregation of markers in reduced chromosomes using single copy chromosome 17 probes. Such cell lines may be generated by lethal ionizing radiation of the donor (Rose et al., 1990, Cell 60:495) .

Gardiner et al., (1990, EMBO J 9:25 and 1988, Som. Cell Mol. Genet. 12:185), using a similar procedure as that employed by Leach et al., (1989, Genomics 5:167) generated hybrid cell lines containing portions of chromosome 21. Chromosomal DNA sequences were cleaved with NotI and probed with chromosome 21 specific DNA sequences. The location of translocation breakpoints were also detected by detecting CpG islands.

Fountain et al., (1989, Am. J. Hum. Genet. 44:58) discloses the isolation of probe, 17L1 from an NotI linking library made from flow-sorted chromosome 17 material. Fountain et al. further discloses the mapping of the neurofibromatosis locus on chromosome 17 by probing with chromosome 17 specific probes, specifically DNA isolated from somatic cell hybrids obtained from flow sorting or microcell fusion techniques.

Pohl et al., (1988, Nucl. Acids Res. 16:9185), discloses the generation of an ^"~± linking clone library representing unmethylated ^~~~j ^~ sites from HHW693 DNA, a hamster hybrid cell line containing 4pl5-4pter and a fragment of 5p as its only human chromosome contribution.

Human clones were identified by hybridization with a cloned human Alu repeat sequence, and subsequently localized further to subregions of human chromosome 4pl5-4pter using a panel of additional hybrids.

2.2. REPETITIVE DNA SEQUENCES The chromosomes of eukaryotes are organized into distinct banding patterns when stained by a variety of procedures (reviewed in Holmquist, DNA Sequences in G Bands and R Bands In: Chromosomes and Chromatin. Adolph, ed. 1988. CRC Press, Boca Raton, FL.). Considerable evidence has been accumulated which suggests that these staining patterns reflect underlying non-uniform sequence representation (reviewed in Bernardi, 1989, Annual Rev. Genet. 23:637). Dark staining bands have been found to be relatively rich in long interspersed repeats (Korenberg and Rykowski et al., 1989, Cell 53:391) and light staining were relatively rich in short interspersed repeats, especially those human sequences released with cleavage by the uI restriction enzyme, hereinafter referred to as Alu sequences (Moyzis et al., 1989, Genomics 4:273 and Korenberg and Rykowski et al., 1989, Cell 53:391). Recently, this organizational feature has been detected by Southern blotting of pulsed field (see for example Schwartz and Cantor, 1984, Cell 37:67) gels when human monochromosome (Avdolovic and Furst, 1989, Am. Biotechnol. Lab 7:26) and trichromosome (Chen and Manuelidis, 1989,

Chromosoma 98:309) rodent hybrid dell DNAs are cleaved with enzymes that cut mammalian genomes infrequently. Alu sequence exclusion from, or preference for, regions of the human genome has been suggested as the basis of the bimodal distribution of Alu sequences found in the Genbank database

(Moyzis et al., 1989, Genomics 4:273) and in biochemical studies (Houck et al., 1979, J. Mol. Biol. 132:289) .

In a specific example, giant DNA fragments containing chromosome 21 or chromosome 22 were generated by sorting chromosomes 21 and 22, digesting the chromosomal

DNA with NotI, and analyzing the fragments generated by pulsed field gel electrophoresis (Minoshima et al., 1990,

Cytometry 11:539) . The separated fragments were subsequently probed with the Alu repetitive sequence using

Southern hybridization. However, these authors are not aware of what they are seeing. Furthermore, distinct human specific restriction fragments can be visualized by hybridization with an Alu sequence even when the total amount of human DNA present represents in excess of twenty percent of the human genome (Chen and Manuelidis, 1989,

Chromosoma 98:309) .

Alu repetitive DNA sequences have in some limited instances been used to map small portions of chromosomes.

For example, Alu repetitive DNA sequences have been used to identify chromosomal locations of large fragments of the human X chromosome cloned in a yeast artificial chromosome

(Nelson et al. , 1989, Proc. Natl. Acad. Sci. U.S.A.

86:6686) through amplification of such sequences by PCR.

Additionally, a portion of chromosome 11 was mapped by probing somatic cell mutants containing deletions in chromosome 11 with a hybridization probe consisting of an individual member copy of a repetitive human DNA family

(Gusella et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79:7804) . It is however noted in Moyzis et al. (1989, Genomics 4:273) that problems may be encountered in generating physical maps of chromosomes or significant portions thereof due to these repetitive sequence. Specifically, since repetitive sequences are in such high abundance, there may be a danger of cross-hybridization. . .

Secondly, since repetitive sequences tend to be clustered,- linkage by comparison to higher-order maps obtained by alternative methods may be required.

3. SUMMARY PF THE INVENTION The invention is directed to a method for obtaining a physical map of a genome or significant portion thereof from a eukaryotic organism comprising the steps of:

(a) providing a plurality of hybrid cells in which each hybrid cell is obtained from the fusion of a first progenitor cell from a first species and a second progenitor cell from a second species and in which each hybrid cell comprises no more than one piece of the genome from the first species, which genome or portion thereof is the genome to be mapped, and in which the sum of the pieces in the plurality of hybrid cells represent the total genome or portion to be mapped;

(b) isolating DNA from the hybrid cells of step (a) to isolated DNA;

(c) cutting the isolated DNA of step (b) with a rare restriction enzyme to obtain DNA fragments;

(d) separating the DNA fragments of step (c) to obtain separated DNA fragments;

(e) detecting the presence of at least one type of repetitive DNA sequence at high density in which the repetitive DNA sequence is specific to the first species on the separated DNA fragments of step (d) ;

(f) determining the relative location of the repetitive DNA detected in step (e) on the separated DNA fragments; (g) ordering the separated DNA fragments in which the relative location of the repetitive DNA has been determined; and

(h) generating a physical map of the genome or portion thereof therefrom.

The method of the present invention may be used to obtain a physical map of the genome from any eukaryotic organism. In one embodiment, step (a) of the method of the invention may further comprise providing a hybrid cell comprising the genome or portion thereof to be mapped in which the hybrid cells obtained from the fusion of a first progenitor cell from a first species comprising the genome or portion thereof to be mapped and a second progenitor cell from a second species.

The invention is further directed to a method for obtaining a physical map of a chromosome or portion thereof from a eukaryotic organism comprising the steps of:

(a) providing a plurality of hybrid cells in which each hybrid cell is obtained from the fusion of a first progenitor cell from a first species and a second progenitor cell from a second species and in which each hybrid cell comprises no more than one piece of one chromosome or portion thereof from the first species, which chromosome or portion thereof is the chromosome to be mapped, and in which the sum of the pieces in the plurality of hybrid cells represent the total chromosome or portion to be mapped;

(b) isolating DNA from the hybrid cells of step (a) to isolated DNA;

(d) separating the DNA fragments of step (c) to obtain separated DNA fragments;

(f) determining the relative location of the repetitive DNA detected in step (e) on the separated DNA fragments;

(g) ordering the separated DNA fragments in which the relative location of the repetitive DNA has been determined; and

(h) generating a physical map of the chromosome or portion thereof therefrom.

In a specific embodiment, the invention is directed to a method for obtaining a physical map of a human chromosome or portion thereof comprising the steps of:

(a) providing a plurality of hybrid cells in which each hybrid cell is obtained from the fusion of a human cell and a non-human cell, and in which each hybrid cell comprises no more than one piece of the human chromosome or portion thereof to be mapped, and in which the sum of the pieces in the plurality of hybrid cells represent the total chromosome or portion to be mapped;

(b) isolating DNA from the hybrid cells of step (a) to isolated DNA;

(d) separating the DNA fragments of step (c) to obtain separated DNA fragments;

(e) detecting the presence of at least one type of human repetitive DNA sequence at high density on the separated DNA fragments of step (d) ;

(f) determining the relative location of the repetitive DNA detected in step (e) on the separated DNA fragments; (g) ordering the separated DNA fragments in which the relative location of the human repetitive DNA has been determined; and

(h) generating a physical map of the human chromosome or portion thereof therefrom. . .

In another specific embodiment, the invention is directed to a method for obtaining a physical map of a human chromosome or portion thereof comprising the steps of:

(a) providing (i) a hybrid cell which comprises no more than one human chromosome or portion thereof, the human chromosome to be mapped and (ii) a plurality of hybrid cells in which each hybrid cell is obtained from the fusion of a human cell and a no -human cell and in which each hybrid cell comprises no more than one piece of the human chromosome or portion thereof to be mapped, and in which the sum of the pieces in the plurality of hybrid cells represent the total chromosome or portion to be mapped;

(b) isolating DNA from the hybrid cells of step (a) to isolated DNA;

(d) separating the DNA fragments of step (c) to obtain separated DNA fragments;

(g) ordering the separated DNA fragments in which the relative location of the human repetitive DNA has been determined; and (h) generating a physical map of the human chromosome or portion thereof therefrom.

In a most specific embodiment which is disclosed in Section 6, the method of the present invention may be used to map human chromosome 17 or portion thereof.

Chromosome 17 is thought to contain for example the gene encoding the HTLV-I receptor and the gene encoding the neurofibromatosis locus and breast cancer. In another embodiment, the method of the present invention may be used to generate a physical map of chromosome 21. Chromosome 21 is thought to contain the gene which may be responsible for Familial Alzheimer's Disease (FAD). The map generated using the method of the present invention could be a first step in determining the locus of the FAD gene, which further reduces the locus to a DNA fragment which is then obtainable by genetic mapping.

4. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the Alu hybridization pattern observed after the probing of a ^~~~£ ^~ digest of DNA from the human: mouse (LMTK-) hybrid cell line, LI. Lane 1: DNA from the LMTK- mouse cell line cleaved with NotI and pulse electrophoresed. Lane 2: lambda phage ladder. Lane 3: DNA from the LI hybrid cell line treated identically to lane 1.

Figure 2 shows the cytogenetic examination of human DNA in the parent LI cell line and the microcell derived daughter line, 9spl8. Figure 2a shows metaphase chromosomes from parental LI cell line "painted" with human Cot 1 DNA. A small acrocentric chromosome is decorated. Figure 2b shows human metaphase preparation "painted" with inter-alu PCR product obtained by amplification of LI DNA. Two small submetacentric chromosomes are stained, primarily on the long arm. Figure 2c shows Giemsa banding of metaphase preparation shown in Figure 2b demonstrating the stained chromosomes are 17. Figure 2d shows a metaphase chromosome preparation prepared from microcell fusion

"daughter" cell line 9spl8 - methods as in 2a above. A short acrocentric chromosome is decorated at its distal end. Figure 2e shows inter-Alu product from 9sp-18 DNA employed to paint human metaphase chromosomes as in b above. Two metacentric chromosomes are stained but only at the extreme distal ends. Figure 2f shows Gie sa banding of chromosomes shown in Figure 2e demonstrating that band

17q25 is the decorated portion of chromosome 17.

Figure 3 shows Alu hybridization patterns exhibited by probing DNA obtained from 17q daughter clones after NotI cleavage and resolution of restriction fragments by pulsed electrophoresis. Figure 3a shows a composite autoradiogram showing Alu detectable NotI fragments from nine microcell daughter clones derived from Ll: LMTK- microcell fusions (see Section 6) . Lanes 1-3, lanes 4-6 and lanes 7-9 are microcell hybrid clones designated, in order; 9sp-2, 9sp-6 and 9sp-7, 9sp-10, 6sp-23 and 6sp-l and 9sp-19, 9sp-18 and 9sp-15. Lanes labelled L are lambda concatemer size markers with sizes indicated in kilobases. Figure 3b shows an autoradiogram of a pulse gel run at a lower pulse time than a resolving lower molecular weight alu detected Not I fragments from several representative daughter hybrids. Lanes 1-4 are clones 9sp-18, 9sp-19, 6sp- 1 and 6sp-23, respectively. Lanes labelled L are lambda concatemer size markers.

Figure 4 shows consensus NotI restriction fragment information obtained from all hybrid cell lines. Figure 4a shows Alu detected NotI fragment lists, arranged in descending size, for Ll and all Ll derived cell lines . Inclusion of a fragment in this table indicates observation of that fragment in three independent experiments employing different fragment separation parameters. Clones which displayed the same alu fragment pattern (for example 9sp-2, 9sp-6 and 9sp-7) were tested for independence of isolation by digestion of DNA from all three with Ba Hl and/or Hindlll, electrophoresed on a standard gel, blotted and the blot probed with probe homologous to the retrovirally encoded neomycin gene. This revealed unique size fragments in all cases indicating that all clones are independently derived. Figure 4b shows an illustration of "bucket" map construction procedure and summary bucket map.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. HYBRIP CELLS The hybrid cells used in the method of the present invention may be obtained using the following procedures. A first progenitor cell from a first species which comprises the genome or portion thereof to be mapped, or in another embodiment, a chromosome or portion thereof to be mapped is fused to a second progenitor cell from a second species. The progenitor cells may be any eukaryotic cell which may for example include but is not limited to a plant, animal, or insect cell. In a specific embodiment, the first progenitor cell is a human cell and the second progenitor cell is a non-human mammalian cell. The non- human mammalian cell may for example be a rodent cell which may include but is not limited to a rat, mouse or hamster cell. The human cell may be fused to the non-human mammalian cell using procedure known in the art, e.g. treatment with polyethylene glycol. Hybrid cells may contain chromosomes from both parents, but in the case of human:animal cell hybrids, human chromosomes rapidly segregate out most of their human chromosome complement in a random fashion after passaging the cells. Hybrid cells comprising no more than one chromosome from the first progenitor species, the chromosome to be mapped may be obtained by procedures known in the art, for example, selecting for a selectable marker. In a preferred embodiment, a cell comprising the human chromosome may be obtained by microcell-mediated transfer (see for example, Saxon and Stanbridge, 1987,

Methods in Enzymology 151:313) . A human cell(s) is transfected with a plasmid vector and/or infected with a retrovirus which contains a dominant selectable marker

(e.g. pSV2-gpt or pSV2-neo) . Transfected clones may be screened for those containing only single integrations of the plasmid per cell by detecting the relative amount of plasmid DNA present in a sample using a probe containing plasmid specific sequences using techniques known in the art such as Southern Blot hybridization.

The transfected clones may be micronucleated using procedures known in the art, for example prolonged colcemid treatment followed by enucleation, which may be accomplished by centrifugation of flasks containing monolayers of micronucleate cells in medium containing cytochalasin B. The microcells are fused to non-human mammalian recipient cells, e.g. mouse cells, and the resulting hybrids comprising the single marked human chromosome, are isolated by growth in appropriate selective medium. The resulting hybrid cell which comprises the chromosome to be mapped or portion thereof may be detected by procedures known in the art, such as chromosome staining or hybridizing to a probe specific for a DNA sequence (s) on the chromosome using the hybridization techniques known in the art such as in situ hybridization. The hybrid may also be probed with a sequence specific to a human cell, such as a human repetitive DNA sequence(s) .

Hybrid cells which comprise a piece of the chromosome to be mapped may also be obtained by treating a hybrid cell comprising no more than one human chromosome with ionizing radiation (see for example Benham et al.,

1989, Genomics 4:509) . In another embodiment, such cells may be obtained by microcell mediated transfer of pieces of the chromosome to be mapped from a hybrid cell comprising no more than one chromosome from the first progenitor species of the hybrid cell.

5.2. PNA ISOLATION

The hybrid cells may be lysed using procedures known in the art. Such techniques include, but are not limited to prior suspension of cells in agarose, e.g., agarose microbead technique (Cook, 1984, EMBO J. 3:1837) and agarose block technique (Schwartz and Cantor, 1984,

Cell 37:67 and U.S. Patent No. 4,473,452. These techniques allow in situ cell lysis by enzymes, detergents and proteins diffused into the agarose while maintaining DNA integrity. Any method of DNA isolation which leaves the

DNA in a state available for subsequent treatment is acceptable for obtaining chromosomal DNA (see e.g.

Maniatis, 1989, Molecular Cloning: A laboratory Manual,

Cold Spring Harbor Laboratory, Cold Spring Harbor, New

York) .

5.3. RARE RESTRICTION ENZYME DIGESTION The isolated DNA is subsequently digested with a rare restriction enzyme. Examples of restriction enzymes which cut human DNA infrequently include but are not limited to MΩi.I, SacII. Miu.1, and Bs≤HII.

The term "rare restriction enzyme" may be defined operationally. In the method of the present invention, it is an enzyme which generates fragments of sufficient size to approach the average size of a domain on the fragment carrying the repetitive DNA sequence detected in step (e) of the method of the present invention.

Once a general determination is made as to a fragment size which would be acceptable for the purposes of the genome under consideration, the choice of a rare cutter can be made in a number of ways. One approach is to simply treat the DNA with an appropriate enzyme (appropriate to be defined below) , and observe the size of the fragments produced. If the fragment size is acceptable in accordance with the guidelines noted above, then a useful sequence has been chosen, and may be used in the present procedure.

On the other hand, a more systematic approach to the selection of a sequence may be desired. In such a case, an appropriate sequence may be predicted empirically by reference to the overall nucleotide composition of the genome to be mapped. For example, a general knowledge of the approximate GC content of the genome provides a convenient means by which the expected average fragment size, in base pairs, generated by cleavage at any given restriction site can be predicted. Information relating to GC content of various organisms is readily available in the literature (see for example, Hill, 1966, J. Gen. Microbiol. 44:419) or is readily determinable by known techniques (Owen and Pitcher, 1985, in "Chemical Methods in Bacterial Systematics" pp. 1-15, M. Goodfellow and D.E. Minnikin, Academic Press, London) . Given the fraction of total DNA which is GC, AT content can also be determined (fraction GC + fraction AT = 1) . Assuming random order of dinucleotides/trinucleotides, then average fragment size (AFS) generable by cleavage of a given recognition sequence can be calculated by the following formula

2 2

AFS = ( ri)* (l-rχ)^b

where ri = fractional GC content

1-rχ = fractional AT content a = # G + C in recognition sequence b = # A + T in recognition sequence.

The above schemes are not the only methods by which an appropriate restriction sequence can be chosen. but modifications thereof will be readily apparent to those skilled in the art. Similar equations have been previously described (e.g. Nei and Li, 1979, Proc. Natl. Acad. Sci. U.S.A. 76:5269). Also, summaries of rare v. common sequences are available in the literature (McClelland et al., 1987, in Gene Ampli.fi.cati.on and Analysis, Chirikjian

(ed.), Elsevier Science Publishing Co., pp. 258-282 and references cited therein) . Thus, the skilled artisan can routinely make an appropriate selection of a rare restriction site, and thus a rare restriction enzyme for the genome in which he is interested.

5.4. DETECTION OF REPETITIVE DNA SEQUENCES ON THE CHROMOSOMAL DNA RESTRICTION FRAGMENTS Following restriction enzyme digestion, the chromosomal DNA restriction fragments are separated either according to size or molecular weight. This separation may be achieved by any method which is capable of resolving fragments of the size produced. A majority of the current techniques rely on electrophoretic separation. The choice of technique will to a large extent govern the ultimate resolution that can be obtained in the mapping procedure. For example, HPLC methods are not particularly suited to separation of large fragments. In a preferred embodiment of the present method, fragments are separated via pulsed field electrophoresis, as described, for example in Schwartz and Cantor, 1984, Cell 37:67 and U.S. Patent No. 4,473,452. This technique is particularly well suited to separation of the large fragments. In this technique, the genomic fragments are contained in a suitable medium, preferably a gel medium and the DNA fragments subjected to pulsing electric fields.

Once DNA fragments have been separated, fragments which comprise at least one high density region of at least one type of repetitive DNA sequence are identified. "High density region" may be defined operationally.

Specifically, a high density region of at least one type of repetitive DNA on a DNA fragment from the genome or chromosome to be mapped is a region on the DNA fragment which contains a large amount of at least one type of repetitive DNA sequence relative to other regions of the genome on the DNA fragment. A convenient method for specifically identifying a high density region of at least one type of repetitive DNA sequence is by hybridizing each fragment generated to a labelled probe(s) comprising a known repetitive DNA sequence specific to the species of the genome or chromosome to be mapped using procedures known in the art (see for example Maniatis et al., 1989,

Molecular Cloning: A laboratory Manual, Cold Spring Harbor

Laboratory, Cold Spring Harbor, New York) using a standard hybridization buffer (for example see Church and Gilbert,

1984, Proc. Natl. Acac. Sci. U.S.A. 81:1991). Low density sequences will have less intensity on an autoradiogram. If the chromosome is a human chromosome, the probe will comprise a human repetitive DNA sequence. The human repetitive DNA sequence may be a short interspersed repeats

(e.g. Alu sequences) or long interspersed repeats (e.g. Ll sequences) .

Hybridization may be detected by detecting the labeled probe. In one embodiment the nucleotide sequence may be labeled with a radioisotopic label (e.g. 32p_f 3H,

^~~S, or 1251) and may be detected by, for example, scintillation counting, Cerenkov counting, and autoradiography. In another embodiment, the second nucleotide sequence is labeled with a fluorescer and can be detected by fluoremetry. In yet another embodiment, the second nucleotide sequence is labeled with biotin and may be detected by means of horseradish peroxidase linked streptavidi . A further embodiment of the invention involves using a label that is a saccharide and which may be selected from the group consisting of a monosaccharide and a polysaccharide, and is detected by means of a lectin which is selected from the group including but not limited to concanavalin A, soybean lectin, wheat germ lectin, and lotus seed lectin. Another embodiment of the invention involves using a label that is a hapten or antigen that is detected by means of an enzyme-linked immunoadsorption assay.

5.5. MAPPING PROCEDURES The fragments comprising repetitive DNA sequences may be ordered by inspecting hybridization patterns and identifying contiguous or overlapping sequences using procedures known in the art (see for example Staden, 1980, Nucl. Acids Res. 8:3673-3694). Alternatively, the order of fragments relative to one another may be determined by comparing the hybridization patterns of DNA fragments obtained by restriction digestion of DNA obtained from cells comprising the genome or chromosome to be mapped with the hybridization patterns of DNA fragments obtained by restriction digestion of DNA obtained from cells comprising no more than one piece of the genome or chromosome to be mapped. Hybridization patterns of genomic or chromosomal DNA from the hybrid cells are compared to one another to ascertain overlapping portions. By aligning these overlapping portions, a complete physical map of the chromosome or portion thereof can be generated.

5.6. APPLICATIONS AND METHODS OF USE

In a specific embodiment, the method of the present invention may be used to obtain a map of a human chromosome or portion thereof. Knowledge of the location of human genes and corresponding genetic traits that are produced would lead to major human health benefit. Specifically, such a map would provide immediate access to any segment of the chromosome and could ultimately be used to generate multipoint linkage maps.

The method of the present invention may also be used to obtain a physical map of a non-human chromosome or portion thereof. Such information would ultimately allow the counterparts of important human genes to be readily identified in organisms where their functional roles are easier to study. Additionally, chromosome maps of animals which are of use agriculturally would be particularly valuable.

6. EXAMPLE: CONSTRUCTION OF A FRAMEWORK PHYSICAL MAP OF HUMAN CHROMOSOME BASED ON ALU REPEAT SEQUESTRATION

Human restriction fragments containing relatively high Alu repeat density can be preferentially detected in the of other human restriction fragments in DNA from human: rodent somatic cell hybrids when the DNA is fragmented with enzymes that cleave human DNA infrequently (Chen, and Manuelidis, 1989, Chromosoma 98: 309) . In the Example disclosed herein, a new physical mapping approach capable of rapidly ordering Alu-rich restriction fragments spanning chromosome size (100,000 kb) human genomic domains is described. The feasibility of the approach is demonstrated by ordering some of the alu-rich No I fragments retained in a set of mouse: human 17q somatic hybrid cell lines.

The hybridization pattern exhibited when NotI cleaved genomic DNA from the somatic cell hybrid line Ll (Sanford, and Stubblefield, 1987, Som. Cell Mol. Gen. 13: 279) is pulse electrophoresed under conditions yielding resolution to 1000 kb, blotted to nylon membrane and the membrane subsequently probed with an alu repeat probe, is shown in Figure 1. DNA was prepared in agarose plugs by adding 1.5% low melting point agarose (F.M.C.) to an equal volume of cells suspended in phosphate buffered saline at a concentration of 2xl0⁷ cells/ml. This suspension was then pippeted into 100 μl plug molds and the molds were placed at -20°C to solidify the agarose. The solidified plugs were removed from the molds and placed in ESP (Schwartz and

Cantor, 1984, Cell 37:67; Carle et al., 1986, Science

232:65; Patterson, et al., 1987, Som. Cell Gen. 9:359-372) lysis solution (0.5 M EDTA, pH 9.5, 1% sodium laurylsarcosine, 2 mg/ml proteinase K [Boehringer

Mannheim] ) , and incubated with gentle shaking in a 50°C water bath for 24 hours. Plugs were then transferred to fresh ESP and stored at 4°C until use. In preparation for restriction enzyme digestion, agarose plugs were washed twice at room temperature for 20 minutes in sterile TE (10 mM Tris [pH 7.5], 1 mM EDTA) . Plugs were then washed twice for 20 minutes in TE containing 1 mM PMSF

(phenylmethylsulfonyl fluoride) . Two final washes in TE were then employed to remove the PMSF. At this point a 15-

20 μl slice was removed from the 100 ul plug and incubated at room temperature for 30 minutes in NotI buffer.

Digestions were carried out in 100 μl containing 50 units of restriction enzyme for 8-12 hours at 37°C. Prior to electrophoresis the reactions were terminated by a 30 minute incubation of the plug slice at 50° C. If not immediately loaded onto a gel the sample was stored at 4°C in ESP until needed. Pulsed electrophoresis was carried out using an LKB Pulsaphor apparatus on a 1.5% agarose gel and was run for 48 hours at 275 volts with a 100 second pulse time. After electrophoresis, the gel was Southern blotted onto nylon membrane (Hybond-N, Amersham) using capillary transfer following depurination. The nylon membrane was washed for 5 min. following depurination. The nylon membrane was washed for 5 min. in 6X SSC (900 mM sodium chloride, 90 mM sodium citrate) , air dried and baked for 1 hour at 65°C. DNA was then crosslinked by U.V. at 0.3 Joules/cm2 (Bioslink 312T, BIOS Corp.) . Alu probe was labelled using the random prime method (Feinberg and Vogelstein, 1983, Anal. Biochem. 132:6) and hybridization was carried out by published protocol (Church and Gilbert, 1984, Proc. Natl. Acad. Sci. USA 81: 1991) except that 100 μg/ml sheared salmon tests DNA was included in the hybridization mixture. Non-specific probe was removed by three five minute washes in 2X SSC at room temperature and two 20 minute washes in 2X SSC at 55° C prior to autoradiography. Examination of Figure 1 reveals a set of Alu hybridizing fragments present in the Ll cell line which are not present in the mouse (LMTK-) host cell line. From this and other blotting experiments we have identified fifteen No I restriction fragments in this size range reproducibly found in NotI digests of DNA from this cell line. An additional fragment of 4300 kb can be resolved using a hexagonal (Chu et al., 1986, Science 237: 1582) gel configuration and S. pombe chromosome size markers. A summary of the Alu identified Not I fragments detected in digests of DNA from the Ll cell line is shown in column 1 of Figure 4a. The total Alu detectable DNA length (sum of detected NotI restriction fragments) in this hybrid cell line is 9,360 kb.

To confirm that the entire long arm of the chromosome was indeed present in the hybrid metaphase chromosomes from this cell line was next examined. DNA probes, either human Cot 1 DNA (a generous gift of P. Watkins, Life Technologies, Inc.) or inter-Alu PCR products from hybrid cell lines Ll and 9sp-18 (using primer 559 ( Ledbetter et al., 1990, Genomics 5:475 and Nelson et al., 1989, Proc. Natl. Acad. Sci. USA 86:6686) were biotinylated using the random primer approach (Feinberg and Vogelstein, 1983, Anal. Biochem. 132:6). Hybridizations were performed essentially as described by Lichter et al., 1990, Science 247: 64; unlabeled competitor human Cot 1 DNA was included in the PCR hybridizations to suppress non specific hybridization (Lichter et al., 1988, Human Genetics 80: 224; Kievis et al., 1990, Cytometry 11: 109; Fan et al., 1990, Proc. Natl. Acad. Sci. USA 87:6223). In brief, hybridization was performed on standard acetic acid/methanol treated chromosomes. Metaphase chromosomes on aged slides (either baked at 80 C overnight n a vacuum oven or kept at room temperature for at least one week) were treated with RNase A in 2X SSC for 1 hour at 37°C. The slides were dehydrated in an increasing ethanol series followed by denaturation in 50% formamide; 2X SSC (pH 7.0) . The chromosomes were immersed in a cold ethanol series and then incubated with Proteinase K. The slides were dehydrated in an increasing ethanol series then dried. Competitor Cot 1 DNA at 200 μg/ml and carrier DNA (non- biotinylated sheared salmon sperm DNA) at 10 μg/ml were added to 8-10 μg/ml of probe DNA solution and denatured at 75°C for 5 minutes. Chilled hybridization cocktail (stock solution consisting of 100% formamide, 20X SSC, 50% dextran sulfate, 20x Denhardt's solution in 20X SSC, 1 M phosphate buffer, 10% SDS) was added to the probe DNAs and the resulting hybridization mix was kept on ice until time of application to the slides. Each slide received 50 μl of mix followed by a coverslip and was incubated at 37°C overnight in a moist chamber. Slides were then washed twice in 50% formamide; 2X SSC at 42°C, then washed twice in 2X SSC followed by a ten min. wash in IX B buffer (0.1 M NaH₂P0₄; 0.05% NP40) . The slides were incubated with blocking solution (IX BN buffer containing 0.02% sodium azide, 10% non-fat dry milk) for five minutes then incubated with FITC conjugated avidin DCS (5 ug/ml in blocking solution) . The slides were rinsed in 1XBN buffer at 42°C and amplified with anti-avidin DCS solution (12 μg/ml in IX BN buffer and 6.25% goat serum) for 20 to 45 minutes. Occasionally, the Ix BN buffer rinses were repeated twice followed by another layer of FITC avidin- DCS. The slides were rinsed in IX BN and a thin layer of antifade solution (p-phenylenediamine dehydrochloride in

PBS, pH 8.0) containing 0.25 μg/ml of propidium iodide was added to stain the chromosomes. The slides were coverslipped and the corners were sealed with dots of clear nail polish. The fluorescein and propidium iodide were excited at 450-490 mn (filter 0515) . Only chromosomal positions showing label on both chromatids were considered positive. For banding, the slides were destained in an increasing alcohol series and air dried. Chromosome banding was achieved by staining the slide with Wright's

Stain.

As is shown in Figure 2a, hybridization of human

Cot 1 (highly repetitive) DNA to such a preparation results in fluorescence of a small, acrocentric human DNA segment, which is far larger than would be expected from a 10,000 kb piece of human DNA, as was indicated by alu probing of NotI digests. In order to confirm that this DNA is human 17q, inter-alu PCR products (Ledbetter, et al., 1990, Genomics

5: 475; Nelson, et al., 1989, Proc. Natl. Acad. Sci. USA

86: 6686) from the Ll cell line were labelled and hybridized to a human metaphase chromosome spread. As shown in Fig. 2b this PCR product preferentially hybridizes to the entire long arm of a chromosome which can be identified as 17q after Giemsa staining of the same chromosome (Fig. 2c) . The conclusion we reached from this combination of cytogenetic experiments is that at least the majority of 17q is present in the Ll cell line with little or no 17p representation. We estimate the length of the human DNA segment in this hybrid to be no less than 40,000 kb. This indicates that only a subset of the total number of NotI fragments, which must be present in this domain of

17q, are detected by Alu probing. A tenfold higher Alu repeat density in a subset of the ^"Q I fragments generated from the human DNA in this cell, line, a possibility suggested by statistical inference of a bimodal alu repeat distribution in human DNA sequences in the Genbank database (Moyzis et al., 1989, Genomics 4: 273), could account for the observed simplification of human DNA complexity.

In order to determine the physical order of the

Alu rich NotI fragments on the chromosome arm, microcell ^* mediated fusion hybrids produced by fusion of Ll derived microcells to a mouse LMTK- recipient cell line was examined. Hybrid human: mouse cell lines containing fragments of human chromosome 17q were constructed as follows. Ll cells were infected with retroviral vector pZipNeoSV(x)l (Cepko et al., 1984, Cell 37:1053; Miller and Buttimore, 1986, Mol. Cell Biol. 6: 2895) derived from the packaging cell line PA317 (Cepko et al., 1984, Cell 37:1053; Miller and Buttimore, 1986, Mol. Cell Biol. 6:2895) . Cells which received the virally encoded neomycin gene were selected in medium containing 800 μg/ml G418. Resistant colonies were pooled and used as described (Lugo et al., 1987, Mol. Cell Biol. 7: 2814; Fournier, 1981, Proc. Natl. Acad. Sci. USA 78:6349; Leach et al., 1989, Genomics 5:167) . Microcell hybrids were selected sequentially, for neomycin resistance and then for thymidine kinase, first in medium containing 800 μg/ml G418, until resistant colonies were visible, and then in medium containing 100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) and 800 μg/ml G4118. Hybrid clones were then picked, expanded and used as a source of DNA.

DNA was prepared in agarose plugs by adding 1.5% low melting point agarose (F.M.C.) to an equal volume of cells suspended in phosphate buffered saline at a concentration of 2xl0⁷ cells/ml. This suspension was then pippeted into 100 μl plug molds and the molds were placed at -20°C to solidify the agarose. The solidified plugs were removed from the molds and placed in ESP (Schwartz and Cantor, 1984, Cell 37:67; Carle et al., 1986, Science 232:65; Patterson, et al., 1987, Som. Cell Gen. 9:359-372) lysis solution (0.5 M EDTA, pH 9.5, 1% sodium laurylsarcosine, 2 mg/ml proteinase K [Boehringer Mannheim] ) , and incubated with gentle shaking in a 50°C water bath for 24 hours. Plugs were then transferred to fresh ESP and stored at 4°C until use. In preparation for restriction enzyme digestion, agarose plugs were washed twice at room temperature for 20 minutes in sterile TE (10 mM Tris [pH 7.5], 1 mM EDTA) . Plugs were then washed twice for 20 minutes in TE containing 1 mM PMSF (phenylmethylsulfonyl fluoride) . Two final washes in TE were then employed to remove the PMSF. At this point a 15- 20 ul slice was removed from the 100 μl plug and incubated at room temperature for 30 minutes in NotI buffer. Digestions were carried out in 100 μl containing 50 units of restriction enzyme for 8-12 hours at 37°C. Prior to electrophoresis the reactions were terminated by a 30 minute incubation of the plug slice at 50° C. If not immediately loaded onto a gel the sample was stored at 4°C in ESP until needed. Pulsed electrophoresis was carried out using an LKB Pulsaphor apparatus . Pulsed field gels in Figure 3a were 1.0% agarose run at 275 volts with a pulse time of 100 sec. for 48 hours. Gel shown as Figure 3b was 1.5% agarose run at 275 volts employing a 60 sec. pulse time for 48 hours.

This strategy results in transfer of large chromosome pieces in addition to whole chromosomes ( (Lugo et al., 1987, Mol. Cell Biol. 7: 2814; Foumier, 1981, Proc. Natl. Acad. Sci. USA 78:6349; Leach et al., 1989, Genomics 5: 167) and generates a reduced chromosome hybrid panel similar to that restriction mapped by Patterson and colleagues (Gardiner et al., 1990, EMBO J. 9:25; Gardiner et al., 1988, Som. Cell Mol. Gen. 12:185) for human chromosome twenty-one. Note that the sequential selection process employed must produce at least one common human DNA fragment (containing the human tk gene - 17q23-17qter

(Haines et al., 1990, Genomics 8: 1 and Nakamura et al.,

1988, Genomics 2:302) in all survivors and the human chromosome pieces are expected to be "marked" with the retrovirus (Leach et al., 1989, Genomics 5:167). Inclusion of the infection step allowed us to directly confirm the clonal independence of daughter hybrids by examination of restriction sites flanking the inserted provirus.

In Figure 3a, the Alu hybridizing NotI fragments present in nine daughter hybrid cell lines are shown.

Examination of the NotI patterns reveals that most of the daughter clones lack a number of the NotI fragments visible in the parental hybrid cell line. In addition, the NotI fragment patterns differ from clone to clone. Some of the

NotI fragments are poorly resolved under the electrophoresis parameters used in the experiments shown in

Figure 3a. Fragment patterns from both the parental and daughter lines were therefore examined at different pulse times to better resolve ambiguous fragments. An example of such an experiment, in which the low molecular weight NotI restriction fragments in several of the daughter clones are resolved, is shown as Figure 3b.

From the combined electrophoretic experiments described above, a summary table of all consensus fragments identified for each clone was constructed and is shown as

Figure 4a. The maximum likelihood relative positions of the remaining Alu rich NotI fragments, with respect to each other, was deduced by inspection of the ]^~~±.I restriction fragment patterns observed in the parent and daughter hybrids to identify "contigs" (Staden, 1980, Nucleic Acids

Res. 8: 3673) . This type of map construction process has been employed successfully in the nematode and yeast genome mapping projects (Coulson et al., 1986, Proc. Natl. Acad.

Sci. USA 83: 7821; Olson et al., 1986, Proc. Natl. Acad. Sci. USA 83:7826; Lander, and Waterman, 1989, Genomics 2: 231), among others. In this data set, the contig procedure is simplified by the availability of the option to either assign the fragment order by subdividing the ^"Ω£^~ fragments present in the parent using the daughter fragment patterns or to build the order by comparing the NotI fragments in the various daughter hybrids with each other. In this example, the fragment interrelationships were built by subdividing the parent Not I fragment set, although either choice generates the same end result. This process is referred to as "bucket" contig construction. The nomenclature seemed appropriate considering that the startpoint for the process involves grouping the unordered parental fragment list. When daughter clone information is subsequently compared to the parent bucket list, the bucket list is split into new bucket lists, where the relationship between buckets is known but where, once again, the fragments within each bucket are unordered lists. In Figure 4b, the process is graphically illustrated. The order of fragments relative to one another is deduced by comparison of clones containing subsets of fragments with Alu detected NotI fragments in the parent hybrid. NotI restriction fragments which are. co- localized with respect to the entire data set, but whose order with respect to one another cannot be determined because none of the clones in the current data break up the restriction fragment grouping are left in an unordered buckets in the summary map. The entire Alu NotI fragment list from the Ll and 9sp-14 only by the presence of centromeric DNA as determined in an independent experiment) is originally placed in one large bucket. The ordering process then seeks to subdivide the fragments into smaller and smaller buckets without creating inconsistencies (gaps) within the inferred order. From the original fragment bucket, inclusion of the data from clones 9sp-10, 9sp-15 and 9sp-18 splits the Ll, 9sp-14 bucket into two buckets containing the indicated fragments. The fragment list for clone 6sp-l (fragments in the respective clone being used to subdivide the larger buckets are shown in bold) , which contains fragments common to and missing from both of these buckets, splits these two buckets into four new buckets as shown. Inclusion of fragment information for clones 9sp-2,

9sp-6 and 9sp-7 further resolve the data into six buckets - three of which contain single NotI fragments.

Independently derived clones 9sp-19 and 6sp-23 are either missing the 410 fragment or, alternatively, the three independently isolated (as determined by independence of retroviral insertion position - see legend to Figure 4a) clones 9sp-2, 9sp-6 and 9sp-7 are missing the 350 fragment.

These two fragments were regrouped into a bucket mapping to the distal right - in a positioning which is not in doubt relative to all other clones in the final presentation

(shown at the bottom of the figure) . Some rearrangement of transferred DNA in microcell fusion strategies is not uncommon (Leach et al., 1989, 5: 167).

The consensus bucket map shown at the bottom of the figure includes both the position of the centromere and tk genes. The position of the centromere is consistent with both the cytogenetic experiments described previously and with probing of DNA from all clones with centromere probe. No clones appear to contain 17ρ as determined from the cytogenetic experiments presented in the text and, independently, using the p arm probe tρ53 (ATCC) in similar experiments (Leach et al., 1989, Genomics 5: 167). Finally, the fragments at 220 kb in clones 9sp-18 and 9sp-19, 630 kb in clones 9sp-15, 6sp-l and 6sp-023 and 195 kb in clones

9sp-7 and 9sp-19 are of unknown origin. The most likely origin of these fragments is partial methylation at NotI site common to the respective clones displaying these anomalous fragments (note, for example the low intensity of the 630 kb fragment in lane 9 of Figure 3a relative to the bands above and below it) . Partial methylation of CG containing restriction sites has been described in other somatic cell hybrids (Gardiner et al. , 1990, EMBO J. 9:25;

Gardiner et al., 1988, Som. Cell Mol. Gen. 12:185) . The distance between buckets or the distance between the centromere and the proximal (left side) cannot be determined from our current data set however the cytogenetic experiments demonstrated that all of 17q is present in the parent. Note that a single fragment can represent a bucket. In separate experiments it was determined that that only the parent hybrid contained a chromosome 17 centromere and that the tk gene was located on the Alu identified 195 kb NotI fragment.

To directly verify whether the bucket map shown in

Figure 4b reflected the actual sequence organization of 17q and to examine precisely the 17q and to examine precisely the 17q distal end point covered by the hybrid panel, a clone, 9sp-18, mapping to the distal right of the reconstructed Not I fragment order, using the same cytogenetic approaches described above for the parental Ll cell line was next examined. As is shown in Figure 2d, human Cot 1 painting of a metaphase chromosome spread from this line reveals that, as expected from the lower total alu detectable Not I fragment sum, a much smaller piece of chromosome 17q is present (fused to a mouse chromosome) in this line relative to that detected in Ll (Figure 2a) . In order to determine the origin of this human DNA on chromosome 17, inter-alu PCR products derived from 9sp-18 were "painted" onto a human metaphase chromosome preparation (Figure 2e) revealing that this clone contains

DNA homologous to DNA at the distal tip of 17q as identified by Giemsa banding of the same chromosomes

(Figure 2f) . This result supports both the validity of the bucket mapping process, as 9sp-18 was placed distal on the map, and independently confirms that our map extends the length of 17q (eg 17qcen - 17q25) , although the position on the chromosome giving rise to each alu detected NotI fragment has not yet been determined.

The physical mapping methodology that has been described, the use of Alu repeat density variation as a means to reduce the overall complexity of chromosome size domains of human DNA, reduces long range human genome mapping to a tractable number of DNA fragments. These results demonstrate that when human DNA is cleaved with enzymes which cut the human genome infrequently enough to produce fragments approaching the size of cytogenetically visible chromosome bands, long range organization of underlying DNA sequences becomes apparent in the restriction fragments produced. Though it has been known for some time that R bands are rich in Alu repeat DNA relative to G bands (Chen and Manuelidis, 1989, Chromosoma

98:309; Holmquist "DNA Sequences in G Bands and R Bands"

In: Chromosomes and Chromatin. Adolph, ED. 1988 (CRC)

Press. Boca Raton, FL; Bernard!, 1989, Annu. Rev. Genet.

23:637; Korenberg and Rykowski, 1988, Cell 53:391;

Moyzis et al., 1989, Genomics 4:273; Avdolovic and Furst,

1989, Amer. Biotechnol. Lab. 7:26; Minoshima et al., 1990,

Cytometry 11:539; Houck et al., 1979, J. Mol. Biol. 132:

289; Rose et al., 1990, Cell 60:495; Glaser et al., 1990,

Genomics 6:48), it is still a widely held view that Alu repeat DNA is ubiquitously present in all human DNA fragments at a constant average density. This view is probably based on the observation of Alu DNA in the overwhelming majority of clones in human genomic libraries

(plasmid, lambda, etc.) . This observation is, however, not directly relevant to regional variation in Alu density as it is not the presence or absence of Alu in genomic domains but rather the observable variation in Alu hybridization intensity (Moyzis et al., 1989, Genomics 4:273) of library members which must be considered in interpreting the results of the alu hybridization experiments presented here. For relatively long Alu rich segments of the genome

, Alu hybridization is an ideal method for constructing relatively high resolution physical maps (Rose et al.,1990,

Cell 60:495; and Glaser et al. , 1990, Genomics 6:48) .

However, cytogenetic verification of the size of a given human chromosome segment, to confirm actual genome distance covered in such experiments, would appear requisite in light of the experiments presented here.

A combination of cytogenetic and molecular methodologies capable of mapping large human genomic domains and which should be applicable to the genomes of most other eukaryotes has been described in this example.

Since cytogenetic bands rich in alu repeat are, in general, poor in the Line repeat kpn and vice versa in the human genome (Chen and Manuelidis, 1989, Chromosoma 98:309;

Holmquist "DNA Sequences in G Bands and R Bands" In:

Chromosomes and Chromatin. Adolph, ED. 1988 (CRC) Press.

Boca Raton, FL; Bernardi, 1989, Annu. Rev. Genet.

23:637; Korenberg and Rykowski, 1988, Cell 53:391;

Moyzis et al., 1989, Genomics 4:273), it is likely that with use of the available bank of human single-copy DNA . probes as well as other human specific DNA repeats it will be possible to generate reasonably high resolution physical maps of the human genome using existent human: rodent hybrid panels . The process employed is referred to as

"framework" physical mapping, since the physical "maps" produced with only a somatic hybrid panel are at low resolution. However, additional probes can improve upon resolution.

There are several features of the results disclosed in this example that should impact the human genome project as it proceeds toward the goal of producing high resolution genetic and physical maps of the human genome. First, the existence of Alu-rich genome domains suggests that strategies which attempt to build genome or chromosome maps from clone libraries (cosmid, YAC) , etc.) would benefit by first segregating clones based on intensity of hybridization to Alu repeat DNA, as clones with similar Alu density should be found in the same or similar regions of the genome. In this fashion, contig formation should be greatly facilitated, at least early in such projects . A second, and not unrelated point is that Alu-rich regions of the human genome appear to contain two- thirds or more of the known human genes (Gardiner et al., 1990, EMBO J. 9:25; Gardiner et al., 1988, Som. Cell Mol. Gen. 12: 185) making it appear reasonable to begin mapping and eventually sequencing in these regions . Third, Alu insertion position appears to have been maintained during primate evolution (Ryan and Dugaiczyk, 1989, Proc. Natl. Acad. Sci. USA 86:9360) and thus mapping information from these regions can serve as startpoints for mapping of other primates. Finally, regionally mapped alu-rich fragments should be a ready source of new polymorphic probes (inter- alu PCR (Ledbetter et al., 1990, Genomics 5:475; Nelson et al., 1989, Proc. Natl. Acad. Sci. USA 86:6686) to facilitate fine genetic mapping of the human genome.

Claims

WHAT IS CLAIMED IS:

1. A method for obtaining a physical map of a genome or portion thereof from a eukaryotic organism comprising the steps of:

(b) isolating DNA from the hybrid cells of step (a) to isolated DNA;

(d) separating the DNA fragments of step (c) to obtain separated DNA fragments;

(h) generating a physical map of the genome or portion thereof therefrom.

2. The method according to claim 1 in which step (a) further comprises providing a hybrid cell comprising the genome or portion thereof to be mapped in which the hybrid cell is obtained from the fusion of a first progenitor cell from a first species comprising the genome or portion thereof to be mapped and a second progenitor cell from a second species.

3. The method according to claim 1 in which the first progenitor cell is a mammalian cell.

4. The method according to claim 1 in which the mammalian cell is a human cell.

5. The method according to claim 1 in which the second progenitor cell is a non-human mammalian cell.

6. The method according to claim 5 in which the non-human mammalian cell is a rodent cell.

7. The method according to claim 6 in which the rodent cell is a mouse cell.

8. The method according to claim 1 in which the DNA fragments are separated by pulse field gel electrophoresis.

9. The method according to claim 1 in which the presence of at least one type of repetitive DNA sequence at high density on the separated DNA fragments is detected by hybridizing each of the separated DNA fragments to a known repetitive DNA sequence specific to the first species.

10. A method for obtaining a physical map of a chromosome or portion thereof from a eukaryotic organism comprising the steps of: (a) providing a plurality of hybrid cells in which each hybrid cell is obtained from the fusion of a first progenitor cell from a first species and a second progenitor cell from a second species and in which each hybrid cell comprises no more than one piece of one chromosome or portion thereof from the first species, which chromosome or portion thereof is the chromosome to be mapped, and in which the sum of the pieces in the plurality of hybrid cells represent the total chromosome or portion to be mapped;

(b) isolating DNA from the hybrid cells of step (a) to isolated DNA;

(d) separating the DNA fragments of step (c) to obtain separated DNA fragments;

(h) generating a physical map of the chromosome or portion thereof therefrom.

11. The method according to claim 10 in which step (a) further comprises providing a hybrid cell comprising the chromosome or portion thereof to be mapped in which the hybrid cell is obtained from the fusion of a first progenitor cell from a first species comprising the chromosome or portion thereof to be mapped and a second progenitor cell from a second species.

12. The method according to claim 10 in which the first progenitor cell is a mammalian cell. 5

13. The method according to claim 10 in which the mammalian cell is a human cell.

14. The method according to claim 10 in which the

10 second progenitor cell is a non-human mammalian cell.

15. The method according to claim 14 in which the non-human mammalian cell is a rodent cell.

15

16. The method according to claim 15 in which the rodent cell is a mouse cell.

17. The method according to claim 10 in which the chromosome or portion thereof to be mapped is a human 0 chromosome.

18. The method according to claim 17 in which the human chromosome is human chromosome 17. 5

19. The method according to claim 17 in which the human chromosome is human chromosome 21.

20. The method according to claim 10 in which the chromosomal DNA fragments are separated by pulse field gel 0 electrophoresis.

21. The method according to claim 10 in which the presence of at least one type of repetitive DNA sequence at _S high density on the separated DNA fragments is detected by hybridizing each of the separated DNA fragments to a known repetitive DNA sequence specific to the first species .

22. The method according to claim 10 in which the known repetitive DNA sequence is a human repetitive DNA sequence.

23. A method for obtaining a physical map of a human chromosome or portion thereof comprising the steps of:

(a) providing a plurality of hybrid cells in which each hybrid cell is obtained from the fusion of a human cell and a non-human cell and in which each hybrid cell comprises no more than one piece of the human chromosome or portion thereof to be mapped, and in which the sum of the pieces in the plurality of hybrid cells represent the total chromosome or portion to be mapped;

(b) isolating DNA from the hybrid cells of step (a) to isolated DNA;

(d) separating the DNA fragments of step (c) to obtain separated DNA fragments;

(g) ordering the separated DNA fragments in which the relative location of the human repetitive DNA has been determined; and

(h) generating a physical map of the human chromosome or portion thereof therefrom.

2 . The method according to claim 23 in which the non-human mammalian cell is a rodent cell.

25. The method according to claim 24 in which the rodent cell is a mouse cell.

26. The method according to claim 23 in which the human chromosome is human chromosome 17.

27. The method according to claim 23 in which the rare restriction enzyme is selected from the group consisting of NotI. S_aj^*ιII, Mlul_r and BssHII.

28. The method according to claim 23 in which the rare restriction enzyme is NotI.

29. The method according to claim 23 in which the chromosomal DNA fragments are separated by pulse field gel electrophoresis.

30. The method according to claim 23 in which the presence of at least one type of human repetitive DNA sequence on the separated DNA fragments is detected by hybridizing each of the separated DNA fragments to a known human repetitive DNA sequence.

31. The method according to claim 30 in which the human repetitive DNA sequence is selected from the group consisting of a short interspersed DNA sequence and a long interspersed DNA sequence.

32. The method according to claim 30 in which the human repetitive DNA sequence is a short interspersed DNA sequence.

33. The method according to claim 32 in which the short interspersed DNA sequence is an Alu sequence.

34. A method for obtaining a physical map of a human chromosome or portion thereof comprising the steps of:

(a) providing (i) a hybrid cell which comprises no more than one human chromosome or portion thereof, the human chromosome to be mapped and (ii) a plurality of hybrid cells in which each hybrid cell is obtained from the fusion of a human cell from and a non-human cell and in which each hybrid cell comprises no more than one piece of the human chromosome or portion thereof to be mapped, and in which the sum of the pieces in the plurality of hybrid cells represent the total chromosome or portion to be mapped;

(b) isolating DNA from the hybrid cells of step (a) to isolated DNA;

(d) separating the DNA fragments of step (c) to obtain separated DNA fragments;

35. The method according to claim 34 in which the non-human mammalian cell is a rodent cell.

36. The method according to claim 35 in which the rodent cell is a mouse cell.

37. The method according to claim 34 in which the human chromosome is human chromosome 17.

38. The method according to claim 34 in which the rare restriction enzyme is selected from the group consisting of NotI, ≤afill, Mini, and BssHII.

39. The method according to claim 34 in which the rare restriction enzyme is NotI.

40. The method according to claim 34 in which the chromosomal DNA fragments are separated by pulse field gel electrophoresis.

41. The method according to claim 34 in which the presence of at least one type of human repetitive DNA sequence on the separated DNA fragments is detected by hybridizing each of the separated DNA fragments to a known human repetitive DNA sequence.

42. The method according to claim 41 in which the human repetitive DNA sequence is selected from the group consisting of a short interspersed DNA sequence and a long interspersed DNA sequence.

43. The method according to claim 41 in which the human repetitive DNA sequence is a short interspersed DNA sequence.

44. The method according to claim 43 in which the short interspersed DNA sequence is an Alu sequence.

45. A method for obtaining a physical map of human chromosome 17 or portion thereof comprising the steps of:

(a) providing a plurality of hybrid cells in which each hybrid cell is obtained from the fusion of a human cell from and a non-human cell and in which each hybrid cell comprises no more than one piece of human chromosome 17 or portion thereof to be mapped, and in which the sum of the pieces in the plurality of hybrid cells represent the total human chromosome 17 or portion to be mapped;

(b) isolating DNA from the hybrid cells of step (a) to isolated DNA;

(d) separating the DNA fragments of step (c) by pulsed-gel electrophoresis to obtain separated DNA fragments;

(e) detecting the presence of an Alu human repetitive DNA sequence on each of the separated DNA fragments of step (d) to obtain separated DNA fragments comprising an Alu human repetitive DNA sequence;

(f) determining the relative locations of the Alu human repetitive DNA sequence on the separated DNA fragments comprising at least one human repetitive DNA sequence of step (e) ;

(g) ordering the separated DNA fragments comprising an Alu human repetitive DNA sequence of step (f) ; and

(h) generating a map of human chromosome 17 or portion thereof therefrom. 46. The method according to claim 45 in which step (a) further comprises providing a hybrid cell comprising human chromosome 17 or a portion thereof in which the hybrid cell is obtained from the fusion of a human comprising human chromosome 17 or portion thereof to be mapped and a non-mammalian species.

47. The method according to claim 45 in which the non-human mammalian cell is a rodent cell.

48. The method according to claim 46 in which the rodent cell is a mouse cell.

49. The method according to claim 45 in which the rare restriction enzyme is selected from the group consisting of ^"~±^~-, 2^{~ ~}II, Mini, and BssHII.

50. The method according to claim 45 in which the rare restriction enzyme is NotI .

51. A physical map of a genome or portion thereof from a eukaryotic organism prepared according to the method of claim 1.

52. A physical map of a chromosome or portion thereof from a eukaryotic organism prepared according to the method of claim 10.

53. A physical map of a human chromosome or portion thereof prepared according to the method of claim 23. 54. A physical map of a human chromosome or portion thereof prepared according to the method of claim 34.

55. A physical map of human chromosome 17 or portion thereof prepared according to the method of claim-

45.