Gene mapping Gregory M. Lathrop*, Dora Cherift, C cile Julier* and Michael James* *Centre d'l~tude du Polymorphisme Humain, 7501 Paris, France and $INSERM, Unit~ 301, F-75475 Paris 10, France Current Opinion in Biotechnology 1990, 1:172-179

Introduction In recent years, linkage investigations using DNA markers have led to the discovery of the chromosomal assignments of many genes responsible for human diseases. In several instances, a candidate cDNA has been isolated on the basis of the known genetic assignment of the disease. New techniques for the development of highly polymorphic loci, construction of somatic cell hybrids, and highresolution in situ mapping have had a large impact on these studies. Here we will review several recent technical developments, and their application to studies in human genetics.

Genetic maps Genetic linkage is the essential step that leads to the chromosomal assignment of the gene responsible for a genetic disease of unknown biochemical origin. Polymorphic DNA markers are the primary tools for linkage studies, and much attention has been given to producing primary linkage maps of these loci. The characterization of large numbers of classic restriction fragment length polymorphisms (RFLPs) and other markers in reference families has led to the completion of primary genetic linkage maps of all human chromosomes. Over 70 laboratories worldwide are collaborating to map markers in reference families, for which DNA is distributed by the Centre d'l~tude du Polymorphisme Humain (CEPH) [1]. The original CEPH panel of 40 reference families (656 potentially informative meioses) has been extended to 58 (1212 potentially informative meioses) to provide sufficient material for the construction of high-resolution linkage maps. As of September 1990, 1941 systems (probe/enzyme combinations) had been characterized in the original or extended panel, and added to the database. This represents an increase of 70% over 1 year. Consortium chromosome maps hayer'been constructed from the combined marker data for two chromosomes: chromosome

1 [2] and chromosome 10 [3]. The first public database containing 799 systems has been distributed to the general scientific community. Most of the markers that have been mapped so far are RFLPs based on the presence or absence of restriction sites. Although these polymorphisms are widely distributed in the human genome, they are of relatively low informativeness for genetic linkage studies, with an average heterozygosity of 30-40%. Locus-specific minisatellite polymorphisms, or variable number of tandem repeats (VNTRs) based on repeat motifs of 5-100 bases in length have higher average heterozygosity [4,5]. Mapping studies have shown that minisatellite clusters occur in the human genome, often in telomeric regions [6]. Recently, microsatellite polymorphisms, based on tandem repeats of shorter sequences such as GT, have been described [7-',8"*]. These can be characterized as genetic markers in families by the polymerase chain reaction (PCR). These markers are both highly polymorphic and widely distributed in the human genome. Efficient screening of cosmids from chromosome-specific libraries has proved a powerful method for the rapid definition of classic RFLP markers to obtain high-resolution genetic and physical maps of human chromosomes, in combination with somatic cell hybrids, linkage studies, and the new mapping methods described below. For example, Yamakawa et al. (personal communication) and Tokino et al. (personal communication) have isolated and mapped 75 RFLP markers to chromosome 3, and 62 RFLP markers to chromosome 11, respectively, using this approach. In addition to site polymorphic markers, many with heterozygosity of more than 50%, they also identified four VNTR loci on chromosome 11, and five VNTR loci on chromosome 3 (Y Nakamura, personal communication). Interestingly, in situ studies of the five VNTR markers on chromosome 3 showed that three were located in different interstitial regions. An interesting strategy for determining new VNTR markers has been developed by Vergnaud [9]. He has produced a series of synthetic tandem repeats (STRs), based on 14-16bp motifs, which can be used either to de-

Abbreviations CEPl-II~Centre d'l~tude du Polymorphisme Humain; PCR--polymerasechain reaction; PIC--polymorphism information content; RFLP--restriction fragment length polymorphism; RH--radiation-reducedhybrid; STR--synthetic tandem repeat; VNTR--variable number of tandem repeats;YAC--yeast artificial chromosome. 172

(~) Current Biology Ltd ISSN 0958-1669

Gene mapping Lathrop, Cherif, Julier,James 173 tect polymorphic loci directly, or to isolate other minisatellite sequences by screening in genomic libraries. Although the STR motifs described are different from the sequences from which other VNTRs have been isolated, preliminary linkage studies suggest that they detect loci that have a similar distribution pattem (G. Vergnaud, personal communication). As microsatellite loci are widely distributed in the human genome and are also highly polymorphic, they are a source of marker loci that avoid some of the limitations of classic site polymorphisms and VNTRs. Recently, Weber [10 °] studied the informativeness of a series of 55 microsatellite, CA-repeat sequences obtained from searches in sequence databases, and 57 others obtained from screening genomic libraries. From the distribution of the frequency and polymorphism information content (PIC) values as functions of the size class (average number of tandem repeats), he concludes that the human genome will contain over 7000 microsatellites with PIC > 0.75; this approaches the number that would be needed to construct a I cm map throughout most of the human genome, assuming a uniform distribution [ 11]. The possibility of obtaining many highly informative, PCR-based markers has led some laboratories to adopt new strategies for the construction of high-resolution linkage maps of human chromosomes (R. White, personal communication; J. Weissenbach, personal communication). Highly polymorphic markers, spaced at approximately 10 centiMorgans throughout the genome, will be localized by reference to existing primary linkage maps. The markers should be of sufficient informativeness to allow the identification of most recombination events in reference families, such as those in the CEPH panel. Other markers can be mapped to a particular interval in a small subset of families; once several markers have been assigned to the same interval, their order can be determined by characterizing a small number of meioses from the total panel, as only chromosomes that recombine in the interval defined by the highly informative flanking markers provide information about order. This strategy substantially reduces the number of marker characterizations needed to order loci, as it requires that genotypes be determined for recombinant offspring, parents, and only some other family members (either two grandparents, or a single non-recombinant offspring).

Radiation hybrid mapping Somatic cell hybrids containing portions of particular human chromosomes have been important tools in determining regional locations of cloned genes and DNA markers. Classically, the derivative chromosomes are usually obtained from naturally occurring translocations, or random fortuitous breakages in the hybrid cell. Thus, somatic cell mapping has generally been restricted to assignments to large chromosome regions as a prelude to in situ or meiotic mapping.

Recently, a systematic approach to the production of somatic cell hybrids containing fragments of specific human chromosomes, has been shown to be a powerful method for high-resolution mapping of human chromosomes. This method involves treating an HPRT + (hypoxanthine phosphoribosyl transferase-positive) rodent cell hybrid, containing a single target human chromosome, with ionizing radiation to break the chromosomes into fragments. The lethally irradiated cells are fused to h p r t - hamster cells to allow rescue of some of the fragmented human and rodent chromosomes and elimination of non-fused cells by selection in HAT (hypoxanthine, aminopterin and thymidine) medium. Although this strategy was first proposed and implemented by Gross and Harris [12,13], recent studies [14 °°,15 ° ] have demonstrated its application to obtain radiation-reduced hybrids (RHs) that systematically retain a high frequency of random human fragments spaning the target human chromosome. An RH panel containing overlapping fragments that span a human chromosome can be used to produce an ordered map of marker loci. The frequency with which two markers are cotransferred in the hybrid panel is a function of their physical proximity and the frequency of chromosome breakage at the radiation dosage applied to the cells. An important feature of RHs is that human fragments are not rearranged (as seen with chromosome-mediated gene transfer). Although high radiation does produce smaller fragments--and, in principle, higher resolution--a counterproductive effect is that more independent fragments are obtained in any one RH [17 °°]. Doses of 60--80 Gy have been found to be sufficient for the construction of maps of several human chromosomes, including chromosomes 4, 21, 22 and 11 (C Richards and D Cox, personal communication) [14°°,16°,17°°]. Mapping data obtained from RH panels suggest that significant, non-random frequencies of marker cotransfer are observed for physical distances of approximately 5 Mb to ones of less than 0.2 Mb. Thus, RH mapping is a useful adjunct to meiotic mapping, which can resolve gene order at greater than i centiMorgan (approximately 1 Mb). At shorter intervals, consistent relationships have been observed between distances estimated from RH data, and physical distances determined from pulsed-field gel electrophoresis. RH panels also provide an important source of material for refined mapping and isolation of genes responsible for human genetic disease. Although many hybrids will contain multiple human chromosome fragments at doses of 60-80 Gy, usually one or more are found to retain only a single region of interest. DNA probes can be isolated from a chosen hybrid, by screening cosmid or phage libraries constructed from the cell line, or by humanspecific Alu PCR [18°]. The clones can be used in four ways: to construct physical maps of the region; in in situ studies to characterize the p a n d further [19 °]; to develop polymorphisms for linkage studies; and to screen in other libraries, such as yeast artificial chromosome (YAC) libraries. A recent application of an RH panel has led to the discovery of a candidate gene responsible for Wilms' tumour by

174

Mammalian gene studies Glaser, Rose and their colleagues [19"]. Previously, they had used a cell surface antigen closely associated with the Wilms' tumour locus to select for hybrids retaining segments of the chromosome surrounding the locus (Fig. 1) [17".]. One hybrid only retained a small region in the vicinity of the gene, and facilitated cosmid mapping. A region of less than 345 kb was found to be deleted in all Wilms' tumour patients, and a candidate cDNA was discovered [19",20]. A more general approach to target a particular chromosome region for selection has been described by Dorin et al. [21 .]. These authors integrated a selectable drug-resistance gene into a chosen locus by selection of appropriate plasmid constructs into a somatic cell hybrid. Correctly targeted cells were chosen as donors for chromosome-mediated gene transfer, with selection for drug resistance in the recipient cells. The resultant cells had a reduced amount of the chromosomal region surrounding the drug-resistance gene. A similar strategy should be applicable to RH production. Furthermore, if the genetic disease locus that is sought has a phenotype that can be studied in cell culture, correction of the defect in vitro by chromosome transfer from the RH panel and drug selection should allow precise localization of the complementing gene.

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I n situ hybridization by fluorescence microscopy has bec o m e an important technique for a regional localization and ordering of cosmid and YAC clones. As a result of advances in the labelling of non-radioactive probes, and in methods for amplification and detection of hybridization signals, unique DNA segments of more than 1 kb can now be localized on metaphase chromosomes [22--24]. Non-specific signals due to the presence of repeated sequences can be suppressed if total human DNA is present as a competitor. Several recent studies have shown the effectiveness of these approaches in obtaining high-resolution maps of human chromosomes. Lichter et al. [25 " ] have used fluorescent in situ techniques to obtain a physical map of 50 chromosome 11 markers (13 DNA segments corresponding to genes and 37 other clones). They relied on confocal microscopy and image analysis software to obtain high-precision localizations in the absence of chromosome banding. Positions are expressed as distances calculated as fractions of the chromosome length. Fluorescent techniques for high-resolution R-banding that are compatible with in situ hybridization of cloned DNA segments have also been described. In one such

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1. Example of a radiation hybrid panel from the region of Wilms' tumour gene on chromosome 11p13. The ideogram on the right is the cytological representation of the short arm of chromosome 11. On the left is a map of genetic markers; single-copy probes are listed on the left side and repeat elements are listed (in italics) on the right of the crossed bar. On the far left, the arrow indicates the position of the Wilms' tumour and associated complex locus, WAGR. MICI (boxed) is a gene which is very proximal to the WAGR locus, and codes for a cell surface protein; an antibody against this antigen was used to select amongst the radiation-treated cells for those which retained this marker. The DNA retained in the selected radiationreduced hybrids is represented by the solid bars in the centre identified by a number. The shading indicates relative gene dosage (m, strong; [], weak; D, very weak) which may reflect stability in the hybrid. Hybrid 3A is particularly useful in delimiting the Wilms' tumour locus in relation to a detailed restriction map. Adapted from [17 ° "].

Gene mapping Lathrop, Cherif, Julier, James method (Fig. 2), R-bands are prepared after hybridization and detection of specific signals; identification of the bands and specific signals is made possible by the spectral characteristics of the two fluorochromes that are chosen for visualization of the probe (fluorescein) and chromosome staining (propidium iodide) [26]. In addition to Tonserving the classic reference scheme, this system has the advantage of allowing chromosome localizations to be obtained by conventional fuorescence microscopy. Cherif et al. [26] described a high-resolution in situ map of chromosome 11q, in the region of the gene responsible for ataxia telangiectasia (Fig. 3). A similar approach was used for the regional localization of 75 cosmid markers to chromosome 3, and 11 cosmid markers to chromosome 11 (Y Nakamura, personal communication).

hanced by simultaneous hybridization, and labelling with different fluorophores. Higher resolution mapping has been described within interphase nuclei [27 oo]. Mthough the order of DNA sequences separated by 50 kb-1 Mb has been reported from these studies, the usefulness of routine application of this mapping strategy has yet to be evaluated. Fig. 4 shows an application of mapping to interphase nuclei with confocal microscopy for the resolution of gene order. In this example, the order of two cosmids that map to the same band by in situ hybridization on a metaphase chromosome is resolved by comparison of their positions with respect to a third cosmid.

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Fig. 2. Chromosomal localization of a cosmid by competitive in situ hybridization. (a) Simultaneous visualization on both chromosomes 11 of R-bands and hybridization signal observed with filter combination 13 (from Leitz). Chromosomes were counter~ stained with propidium iodide. (b) Pattern of R-bands in the same metaphase observed with filter combination N2.1 (from Leitz).

Resolution of clones within bands may be aided by simultaneous hybridizations of pairs of cosmids on YACs labelled with different fluorophores. In situ hybridization on metaphase chromosomes appears to allow ordering at distances of more than 1 Mb, and resolution may be en-

Fig. 3. Physical map of 11q22-q23 region by in situ hybridization of biotinylated cosmids.

In addition to providing a rapid method for regional localizations, in situ techniques allow direct comparison of physical and genetic maps. One such application has led to the identification of a potential bias in the distribution of markers derived from cosmid libraries, which are often found in the regions of R-positive bands (Y. Nakamura, personal communication). In situ hybridization is also important for the identification of the hu-

175

176

Mammalian gene studies Fig. 4. Example of in situ hybridization of three cosmids from chromosome 11q in interphase nuclei analysed by confocal microscopy: two biotinylated cosmids, (a,b) depicted in red, were cohybridized with a digoxigeninlabeled cosmid (c,d) depicted in green. The images (a) and (c) were computationally sharpened and filtered and in (b) and (d), the effect of contrast enhancement brings out the hybridization signals. (e) is obtained by additior~ of the two images (b) and (d).

man component of somatic cell hybrids, as discussed above. A new method, with many potential applications,

involves amplification of DNA from the hybrid by PCR with primers that are specific for human Alu and L1 repet-

Gene mappingLathrop, Cherif, Julier, James itive sequences, followed by hybridization of the PCR products to metaphase chromosomes [18 ,].

Disease studies The widespread availability of mapped DNA markers has recently led to chromosomal assignments of the genes responsible for many different genetic diseases. In some instances, the mutation responsible for the disease has been characterized, as for example, in neurofibromatosis type 1 [28-30]. In principle, similar approaches should be successful for any Mendelian disease, if a sufficient number of multiplex families can be found. In practice, several types of complication may be encountered. One problem is locus heterogeneity, i.e. when different genes cause the same disease. Recent examples of the discovery of unexpected locus heterogeneity include X-linked retinitis pigmentosa [31] and tuberous sclerosis [32]. In other instances, genetic linkage studies have confirmed that a single locus (or a tightly linked group of loci) is responsible for syndromes with different clinical classifications, such as the hereditary childhood spinal muscular atrophies [33]. Phenotype definition is one of the many problems for the application of the linkage approach to so-called complex, or multifactorial diseases, such as psychiatric traits. The importance of assessing the robusmess of linkage results, especially in the case of complex phenotypes, has been demonstrated by the failure to confirm the assignment of a gene responsible for bipolar depression to chromosome 11p in a large Amish pedigree after slight modifications of disease assignment and the addition of new family members to the study [34]. On the other hand, confirmation of linkage has been obtained for another complex phenotype: a gene related to atopy has been assigned to chromosome 11q [35] (B. Cookson, personal communication). Careful application of linkage methods should lead to similar confirmed assignments for other multifactorial disease traits. Once an initial localization has been obtained, high-resolution mapping is needed to obtain a precise definition of the placement of the gene. Usually, this requires a dense map of highly polymorphic marker loci in the region of the disease gene. In addition to the techniques described above for construction of RH cells, screening in YAC libraries with cosmid or other clones is a particularly attractive scheme, both for physical mapping and for obtaining highly polymorphic markers (from microsatellite repeats contained in the YAC). Several large YAC libraries are now available for screening [36,37°]. Microdissection of human chromosomes regions is another potential source of small clones and of candidate expressed sequences in the region of a disease gene [38,39 "°].

Genetics of model organisms Studies of the genetics of complex diseases and of Mendelian diseases for which multiplex families are dif-

ficult to find can be greatly aided by studies of animal models. If a trait resembling a human disease for which the genetic cause is unknown exists in an inbred mouse or rat model, experimental crosses can be defined for linkage studies in these species. In principle, experimental models can be rapidly investigated by linkage studies because the number of segregating disease-susceptibility genes is limited ff crosses are appropriately selected, environmental factors are easier to control, and a large number of meioses are usually available for study. Linkage results from a model system can be used to define candidate chromosome regions or candidate genes for investigation in the human disease on the basis of homology. There is often uncertainty about the similarity between the genetic causes of a disease in humans and in a model system. In some instances, close genetic relationships have been found. For example, malignant hypothermia, an inherited muscle disorder, and one of the main causes of death due to anaesthesia, has been mapped by linkage to chromosome 19q12-13.2 in a small number of multiplex human families, as a result of a similar disease being mapped to a homologous region in the pig [40]. The ryanodine receptor gene has also been assigned to this region, and is a candidate susceptibility locus for malignant hypothermia [41]. In the mouse, progress in genetic mapping has been greatly aided following the introduction of interspecific crosses involving Mus spretus. These crosses have great potential for genetic mapping because of the ease with which polymorphic differences between M. spretus and inbred laboratory strains of mice can be detected [42]. However, linkage studies in many experimental mouse crosses that are of interest for disease models have been hampered by the paucity of genetic markers that detect polymorphisms between inbred laboratory strains. Recently, both minisatellites (VNTRs) and microsatellite tandem repeats have been described that may overcome this difficulty [43,44]. Interestingly, minisatellites seem to be widely distributed in the mouse genome. Cross-hybridization with human VNTR probes under conditions of low stringency produces complex 'fingerprint' patterns that allow linkage studies to proceed rapidly in experimental design pedigrees [43]. Alternatively, genomic mouse libraries can be screened with human VNTR probes to generate clones that detect single-locus VNTRs or multilocus fingerprint patterns in the mouse under various hybridization conditions (C. Julier, unpublished data). The techniques for the construction of RHs and in situ hybridization that have been described above should also be powerful tools for these genetic investigations, if they are appropriately adapted to meet the requirements of new experimental conditions. For example, speciesspecific PCR has been applied successfully to obtain murine probes from somatic cell hybrids on a hamster background, and RH mapping has been applied to mouse chromosomes ( P Avner, personal communication).

177

178

Mammalian gene studies Conclusion

This is an important paper which provides many details on the distribution of informativeness in microsatellite potymorphisms.

The gene mapping strategies described here provide powerful methods for localizing and characterizing the genes responsible for human genetic diseases. The catalogue of mapped genetic traits is growing rapidly as these techniques become widely applied in many laboratories. In the future, large-scale programmes on the human genome will produce mapped sets of overlapping cosmid and YAC clones that cover many regions of the human genome. These and other technological developments are likely to accelerate the pace of gene mapping, and shorten the time between chromosomal assignment of a disease locus and the discovery of the corresponding gene.

11.

LATHROPGM, LALOUELJM, WHITE R: The n u m b e r of meioses n e e d e d to resolve gene order in a 1% linkage map. H u m a n gene m a p p i n g 9. Cytogenet Cell Genet 1988, 46:643.

12.

GROSS SJ, HARMS H: N e w m e t h o d for mapping g e n e s in h u m a n c h r o m o s o m e s . Nature 1975, 255:680~584.

13.

GROSSSJ, HARRIS H: Gene transfer by m e a n s of cell fusion. The m a p p i n g of 8 loci o n h u m a n c h r o m o s o m e 1 by statistical analysis o f gene assortment in somatic cell hybrids.J Cell Sci 1977, 25:39-57.

References and recommended reading

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15.

BENHAM F, HART K, CROLLAJ, BOBROW M, FRANCAVILL3/I, GOODFELLOWPN: A m e t h o d for generating hybrids contalning nonselected fragments of h u m a n c h r o m o s o m e s . G~ nomics 1989, 4:509-517. Confirmation of the study of Cox et aL [16"], but demonstrates retention of small, multiple fragments in the RHs at high radiation doses. .

• ••

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1.

DAUSSET J, CANN HM, COHEN D, LATHROP GM, LALOUEL JM, WHITE R: Centre d'l~tude du Polymorphisme H u m a n (CEPH): Collaborative genetic m a p p i n g of t h e h u m a n genome. Genomics 1990, 6:575 577.

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NAKAMURAY, LEPPERT M, O'CONNELL P, WOLFF R, HOLM T, CULVER M, MARTINC, FUJIMOTOT E, MOFF M, KUMLtNE, WHITE R:Variable n u m b e r o f t a n d e m repeat (VNTR) markers for h u m a n g e n e mapping. Science 1987, 235:16161622.

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ROYLENJ, CLARKSONR, WONG Z, JEFFREYS AJ: Preferential localization of hypervariable minisatallites near h u m a n telomeres. H u m a n Gene Mapping 9. Cytogenet Cell Genet 1988, 46:685.

WEBERJL, MAY PE: Abundant class of h u m a n DNA polymorp h i s m w h i c h can be typed using t h e polymerase chain reaction. A m J H u m Genet 1989, 44:388-396. This paper, with [ 8 " ] , shows that CA tandem repeats are highly polymorphic, and that they can be characterized as genetic markers in families by PCR. 7.

•,

8. •.

laWW M, LUTY JA: A hypervariable microsatellite revealed by in vitro amplification of a dinucleotlde repeat within t h e cardiac muscle actin gene. A m J H u m Genet 1989, 44:397-401. Published simultaneously with [ 7 " ] , this is a fundamental paper describing microsatellite po~ymorphisms.

9.

PmTCHARDCA, CASHERC, UGLUME, COX DR, MYERS RM: Isolation and field-inversion gel electrophoresis analysis of DNA markers located close to t h e Huntington disease gene. Genomics 1989, 4:408418. The RH panel produced by Cox et al. [ 1 4 " ] is used to produce new DNA markers near the Huntington's disease locus. This study also shows that hybrids containing more than one fragment of h u m a n DNA provide probes of interest. 16. .

17.

GLASERTi ROSE E, MORSE H, HOUSMAN D, JONES C: A panel of irradiation-reduced hybrids selectively retaining h u m a n c h r o m o s o m e l l p 1 3 : their structure and use to purify t h e WAGR g e n e complex. Genomics 1990, 6:48454. An exhaustive study of RHs that had been physicaUy selected to retain the 11p13 locus. The hybrids were analysed for retention of 56 11p probes, and characterized cytogenetically and by pulsed-fieM gel electrophoresis. , •

18. .

LICHTERP, LEDBETTERSA, LEDBETTERDid, WARD DC: Fluoresc c n c e in situ hybridization w i t h Alu and L1 polymerase chain reaction probes for rapid characterization of h u m a n c h r o m o s o m e s in hybrid cell lines. Proc Natl A ca d Sci USA 1990, 87:66344638. Outlines a general method using human-specific PCR products from somatic cell hybrids as probes for in situ hybridization to normal hum a n metaphase chromosomes to identify the h u m a n components of the hybrid. 19. .

ROSE EA, GLASER T, JoNEs C, SMITH C, LEWIS WH, CALL KM, MINDENM, CHAMPAGNE E, BONETTA L, YEGER H, HOUSMAN D: Complete physical m a p of t h e WAGR region of 11p13 localizes a candidate Wilms' t u m o r gene. Cell 1990, 60:495-508. The RHs identified in [ 1 7 " ] were screened for new probes that could detect deletions c o m m o n to Wilms' tumour. Particularly useful was the ability to detect and quantify the h u m a n DNA content of the hybrids by probing pulsed field gel blots with human-specific repetitive DNA probes. 20.

CALLKM, GLASERT, ITO CY, BUCKLERAJ, PELLETIERJ, HABERDA, ROSE F,A, KRALA, YEGER H, LEWISWH, JONES C, HOUSMANDE: Isolation and characterization of a zinc finger polypeptide at t h e h u m a n c h r o m s o m e 11 Wilms' t u m o r locus. Cell 1990, 60:509-520.

21. •

DORINJR, INGLISJD, PORTEOUSDJ: Selection for precise chromosomal targeting of a d o m i n a n t marker by h o m o l o g o u s recombination. Science 1989, 243:1357-1360.

VERGNAUD G: Polymers of r a n d o m short oligonucleotides

detect polymorphic loci in t h e h u m a n genome. Nucleic Acids Res 1989, 17:7623~7630.

10. •

WEBERJL: Informativeness of h u m a n (dC-dA)n (dG-dT)n polymorphisms. Genomics 1990, 7:524--530.

Gene mapping Lathrop, Cherif, Julier, James 179 A selectable marker (neo) was targeted to a pre-determined chromosome region in a somatic cell hybrid. Subsequent chromsomal transfer to a rodent cell, and neomycin resistance selection produced secondary hybrids with reduced amounts of h u m a n DNA around the locus. An example of a general strategy for directed mapping. 22.

23.

24.

GAe6ONJA, VAN DEN BERGHEJA, KEMSnEADJT: Novel non-iosotopic in situ hybridization t e c h n i q u e detects small ( l k b ) unique s e q u e n c e s in routinely G-branded h u m a n chromosomes: fine mapping of N-myc and beta-NGF genes. Nucleic Acids Res 1987, 15:4761-4770. BHATr B, BURNS J, FINAERY D, MCGEE J: Direct visualization of single copy genes on b a n d e d m e t a p h a s e c h r o m o s o m e s by non-isotopic in situ hybridization. Nucleic Acids Res 1988, 16:3951-3961. CHERIFD, BERNARD O, BER6ER R: Detection of single-copy g e n e s by nonisotopic in situ hybridization on h u m a n c h r o m s o m e s . H u m Genet 1989, 81:358-362.

25. ••

LICnTER P, TANG CC, CALL K, HERMANSON G, EvANs GA, HOUSMAN D, WARD DC: High-resolution mapping of hum a n c h r o m o s o m e 11 by in situ hybridization w i t h cosmid clones. Science 1990, 247:64-49. The first paper to demonstrate the efficiency of large-scale gene mapping with fluorescent in situ hybridization. A map of 50 markers, span ning all of chromosome 11, was produced. 26.

CHEmFD, JULIER C, DELATTREO, DERRE I, LATHROPGM, BERGER R: Simultaneous localization of cosmids and c h r o m o s o m e R-banding by fluorescence microscopy: Application to regional m a p p i n g of h u m a n c h r o m o s o m e 11. Proc Natl A c a d Sci USA 1990, 87:6639-6643.

27. ••

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29.

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