GENOMICS

12,183-189

(1992)

A Genetic Linkage Map of Human Chromosome 20 Composed Entirely of Microsatellite Markers JAMIL~ H&%N,*-t CHRISTOPHER DUBAY, *St MARIE-PASCALE NOELLE BECUWE,*~ AND JEAN WEISSENBACH*

PANKOWIAK,

*

*Unit& de Gbnbtique Mokulaire Humaine, CNRS URA 1445 and lnstitut Pasteur, 28 rue du Docteur Roux, 75015, Paris, France; KEPH, 27 rue Juliette Dodu, 75010 Paris, France; and $G&x?thon, 1 rue de I’lnternationale, 97002 Evry, France Received

July 26, 1991;

revised

September

15, 1991

rated by intervals greater than 30 CM. However, loci for several diseases have been linked recently to markers on chromosome 20. These diseases include Alagille syndrome (Schnittger et al., 1989; Zhang et aZ., 1990), a form of MODY (Bell et aZ., 1991), a form of Fanconi’s anemia (Mann et al., 1991), and benign familial neonatal convulsions (Leppert et al., 1989; Malafosse et aZ., 1991). High-resolution mapping of these diseases requires the isolation of additional informative markers on chromosome 20. Highly informative polymorphic DNA markers corresponding to simple sequence repeats (microsatellites or VNDRs) (Litt and Luty, 1989; Weber and May, 1989) were isolated from a flow-sorted library of chromosome 20. Because the analysis of size variations in microsatellite alleles frequently requires separations on polyacrylamide sequencing gels, a procedure derived from the multiplex sequencing technique of Church and Kieffer-Higgins (1988) has been developed to reduce the number of sequencing gels and gel loadings. Twenty-six polymorphic (CA),, microsatellites from chromosome 20 have been mapped using linkage analysis in CEPH families. Our results indicate that microsatellites can be used efficiently to construct a high-resolution linkage map of the human genome, but that they might not be uniformly distributed on chromosome 20.

Twenty-six (CA), polymorphic microsatellites were isolated from a flow-sorted chromosome 20 library. To reduce the number of sequencing gels, these microsatellites were genotyped on 15 CEPH families using a procedure derived from the multiplex sequencing technique of G. M. Church and S. Kieffer-Higgins (1988, Science 240:185-188). A primary map with a strongly supported order was constructed with 15 (CA), markers. Regional localizations for the 11 other microsatellites were obtained with regard to the primary map. The 26 loci span approximately 160 CM. Regional localizations for a set of index markers (D20S4, D20S6, D20S16, and D20S19) and for other markers from the CEPH Public Database (D20S5, D20S15, D20S17, and D20S18) have also been determined. The total map spans a genetic distance of approximately 195 CM extending from the (CA), marker IP20M7 on 2Op to D20S19 on 20q. The density of microsatellite markers is markedly higher in the pericentromeric region, with an average distance of 3 to 4 CM between adjacent markers over a distance of 43 CM. Two-thirds of these randomly isolated microsatellites are clustered in that region between D20S5 and D20S16 representing approximately one-fourth of the linkage map. This might suggest a nonrandom distribution of (CA), simple sequence repeats on this chromosome. o 1992 Academic POW, IW.

INTRODUCTION

Chromosome 20 has remained one of the most poorly mapped human chromosomes, and only a limited number of polymorphic markers have been localized on linkage maps to date (see Grzeschik and Skolnick, 1990). Only five markers from chromosome 20 with a polymorphic information content (PIC) above 0.5, including two with a PIC above 0.7, have been reported (DNA reports HGM 10 and 10.5). The previously published maps comprise no more than five or six markers (Donis-Keller et al., 1987; Nakamura et al., 1989; Summar et al., 1990), some of which are sepa-

MATERIALS

AND

METHODS

Isolation and Sequencing of Human (dC-dA), * (dG-dT), Repeats A human (LLBONSOl, Collection) transferred sham), and

flow-sorted chromosome 20 library available from American Type Culture was plated on Escherichiu coli LAlOl, to nylon membranes (Hybond-N, Amerprobed with a synthetic nick-translated

183 All

o&x3-7543/92 $3.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.

184

HAZAN

poly(dC-dA) * poly(dG-dT) (Pharmacia). Hybridization was carried out in 1 mM EDTA, 0.5 M sodium phosphate buffer, pH 7.0, 7% SDS (Church and Gilbert, 1984) for 3 h at 65°C. Filters were washed at low stringency in 2~ SSC, 0.1% SDS, twice at room temperature and once at 55°C. Among many CA/GT positive X recombinant phages, 130 clones were randomly chosen, isolated, and amplified. The inserts prepared from these clones were purified by low-melting agarose gel electrophoresis and digested with AluI. DNA fragments (ranging in size from about 100 to 500 bp) were ligated into a SmaI-cut M13mp18 vector, and the resulting recombinant phages were used to transform competent E. coli SURE (Stratagene). Nylon plaque lifts were hybridized with nick-translated poly(dC-dA) * poly(dG-dT) as described above. About 100 single-stranded DNA samples were isolated from the positive clones and sequenced by the dideoxynucleotide chain termination method (Sanger et al., 1977) on an Applied Biosystems Model 373A DNA sequencer. Polymerase

Chain Reaction

Oligonucleotide primers flanking the tandem repeat were chosen with the computer program OLIGO (Rychlik and Rhoads, 1989) and matched against human repetitive sequences to avoid primers containing repeat sequences. The primers were synthesized on an Applied Biosystems Model 380B DNA synthesizer and were used for PCR reactions without purification. To estimate the polymorphism of the 88 selected tandem repeats, PCR reactions were first carried out on DNA of five unrelated individuals. Amplifications were performed in a total volume of 50 ~1 containing 40 ng of genomic DNA, 50 pmol of each primer, 6.25 nmol of each dNTP, 50 mJ4 KCl, 10 mM Tris, pH 9, 1.5 m&f MgCl,, 0.01% gelatin, 0.1% Triton X-100, and 1 unit of Z’aq polymerase (NBL). Samples were overlaid with light mineral oil (Sigma) and were processed through one step of denaturation (94’C for 5 min), followed by 30 cycles of denaturation (94°C for 1 min), annealing (55°C for 1 min), and elongation (72°C for 2 min), and a last step of elongation (5 min at 72“C). The polymorphic CA/GT repeats were then amplified for linkage analysis on 15 CEPH families (about 200 meioses): 884, 1332, 1346, 1362, 1377, 1418,1420, 1423, 1424, 1444, 1454,1456,1458,1463, 1582. Primer sequences of the 26 polymorphic microsatellite markers from chromosome 20 have been communicated to GDB. Analysis

of Polymorphic Markers

To check for the presence of polymorphism, aliquots of the amplified DNA were mixed with 2 vol of formamide and loaded on 6% polyacrylamide denatur-

ET

AL. FM

FF FA

MO MF

Cl MM

C3 C2

FA C4

C5 MO

C7 C6

CB

IP20M29

too-

IPM31

FIG. 1. Autoradiograph illustrating two microsatellites (IP20M29 and IPM31) amplified in CEPH family 1444, coprecipitated, comigrated on a sequencing gel, transferred electrophoretically, and revealed by oligonucleotide probes labeled by terminal deoxynucleotidyl transferase. The samples are identified according to the CEPH Public Database: FF, father’s father; FA, father; FM, father’s mother; MF, mother’s father; MO, mother; MM, mother’s mother; C (from 1 to 8), child. The additional bands, less intense and smaller in size than the major bands, are sometimes detected and may be due to a slippage of Taq polymerase during the amplification reaction.

ing gels. To estimate sizes of the alleles, PCR reactions were performed, for each microsatellite, on the recombinant Ml3 template that had been sequenced, and the amplification products were coprecipitated and loaded on the gels as DNA size markers. Up to 15 PCR products, produced with separate primer sets on identical DNA samples, were coprecipitated and comigrated on a single gel lane to reduce the number of sequencing gels. Gels were transferred electrophoretitally on Hybond N’ nylon membranes (Amersham) in 50 mA4 Tris-borate-EDTA according to Church and Gilbert (1984). Nylon membranes were then processed, following the manufacturer’s protocols, and directly hybridized with radiolabeled (CA),, oligonucleotide or, to reveal the coprecipitations, one of the primers used in the PCR reaction (as shown for two markers in Fig. 1). Labeling of oligonucleotides was performed with 12.5 units of terminal deoxynucleotidyl transferase (Boehringer) for 30 min at 37°C in a total volume of 100 ml containing 200 mM potassium cacodylate, pH 7.6,lOO PM DTT, 1 miJ4 CoCl,, 30 &i of [a-32P]dCTP, and 100 niV oligonucleotide primer. Hybridizations were carried out at 42°C for 3 h in 0.13 M sodium phosphate buffer, pH 7.2,0.25 M NaCl, 7% SDS, and 10% PEG (M, 6000) (Amasino, 1986; Church and Kieffer-Higgins, 1988). Blots were

A LINKAGE

MAP

washed once in 2X SSC, 0.1% SDS for 10 min at room temperature and autoradiographed. Chromosome assignment of the markers was performed prior to linkage mapping using amplifications, as described above, on a panel of human/rodent somatic cell hybrids (NIGMS Human Genetic Mutant Cell Repository panel 1). Aliquots of the PCR products were analyzed on 3% NuSieve agarose (FMC Corp.), 1% agarose (Sigma) gels. Allele sizes and frequencies in 50 to 120 chromosomes from the grandparents of the 15 CEPH families were determined and used to calculate PIC values (Botstein et al., 1980). The LINKAGE programs (Lathrop et al., 1985) were used to construct and test the linkage map. Two-point linkage analysis was first performed between all pairs of markers. Markers were then divided into linkage groups based on two-point results, and 15 markers that showed strong linkage and were well distributed were used to create a primary linkage map. We then calculated odds for permutations and orientations of three marker subgroups in the sex-averaged primary map. Odds against any of the alternative orders were greater than lOOO:l, with the exception of the distal long arm, where the most likely order gave odds of only 7O:l against inversion. Regional localizations were determined for the 11 remaining markers and for 8 previously mapped markers from the CEPH Public Database; support intervals for odds of placement of lOO:l, with respect to the maximum likelihood, were calculated (Lathrop et al., 1986).

RESULTS Development

AND

of Microsatellite

DISCUSSION Markers

About 20% of h phages from a flow-sorted chromosome 20 library gave a positive signal when screened with a poly(dC-dA) .poly(dG-dT) probe. We digested inserts from 115 CA/GT positive X clones with a frequent-cutter enzyme and subcloned the digests into M13mp18. Only 85 CA/GT positive subfragments could be successively cloned in M13, and these were subsequently sequenced. The failure to subclone 26% of the (CA), microsatellites from the X phage inserts is not understood, but might be related to the presence of (CA), sequences that have been shown to adopt a Z-DNA conformation (Arnott et al., 1980; Hamada and Kakunaga, 1982; Haniford and Pulleyblank, 1983). Similarly, several of the Ml3 subclones tended to rearrange and to lose their (CA), repeats in our first sequencing experiments when templates were prepared using the TGl host strain. These rearrangements were overcome using a sbcC, recB, recJ host strain (E. coli SURE

OF

CHROMOSOME

20

185

strain, Stratagene). Among the 85 sequenced clones, 8 originated from X phages present in duplicate in the fraction of the flow-sorted library that was screened. Primer sets, designed to amplify the dinucleotide repeat, could be defined in 57 sequences, but not in the 20 remaining instances because of either misplacement of the repeat with respect to the cloning sites or presence of repetitive elements (Sines or Lines) in the sequences. Our attempts at PCR amplification were unsuccessful for 11 primer sets, and products from 16 primer sets amplified in 5 unrelated individuals were not polymorphic. An additional (CA), polymorphic microsatellite, IP20M61, was isolated from the probe IP20K061 (locus D20S25; Rouyer et d., 1990). To increase the number of polymorphic markers, we directly subcloned an AZuI digest of a pool of 12000 X phages from the library of chromosome 20 into M13mp18. Screening of these subclones with poly(dC-dA) . poly(dG-dT) resulted in 38 positive clones, which were then sequenced. Eleven clones were duplicates, 7 had misplaced repeats, and the remaining 20 were used to define primer sets. Amplification was successful with 10 sequences, and 6 turned out to be polymorphic. One-third of the 37 polymorphic markers could be unambiguously assigned to chromosome 20 by amplification on DNAs from a panel of rodent/human somatic hybrids; one marker originated from the X chromosome and the results for the remainder were inconclusive. Three other markers giving inconsistent PCR results were discarded from the linkage study, which thus included a total of 33 microsatel-, lites. Linkage analysis has shown that 7 of those 33 markers are not linked to markers for chromosome 20 in the CEPH V4 Database, indicating that the flowsorted library of this chromosome is approximately 80% pure. Among these 7 markers, 2 proved to be linked to chromosome 15,2 others to chromosome 19, and 1 to chromosome 13. The number of observed alleles is directly correlated with the number of repeated elements present in the original clone (see Fig. 2 and Tables 1 and 2). Similarly, as observed by Weber (1990), informativeness increases with the number of dimeric elements in a (CA), repeat, but interruptions tend to reduce informativeness to the value expected for the longest uninterrupted part of such motifs (Fig. 3). The PIC values for the 26 markers from chromosome 20 range from 0.19 to 0.83. Twenty-one markers (81%) show a PIC above 0.5 and 13 (50%) above 0.7 (Table l), confirming the high informativeness of (CA), microsatellites in general. Likewise, the PIC values for the 7 remaining markers are included between 0.18 and 0.78, but only 3 show a PIC above 0.5 (Table 1).

186

HAZAN

ET

AL.

and female linkage maps with 15 markers selected for their strong linkage and their distribution on chromosome 20 are shown in Fig. 4A. These 15 loci span 94 CM in males and 170 CM in females. The male map shows a cluster of microsatellites in the pericentromerit region, which is less pronounced in the female map. Both maps have some regions that are only poorly covered by the microsatellites. The sex-averaged genetic map of the selected 15 markers spans a genetic distance of 128 CM and is FIG. 2. Variability ble 1) correlated with sponds to the longest sequence.

Multiplex

of 33 (CA),, microsatellites (described repeat length. The number of repeats stretch of uninterrupted CA repeats

Genotyping

in Tacorrein the

TABLE Characteristics Isolated from brary

To reduce the number of sequencing gels required to detect genotypes, PCR products for multiple loci, amplified separately in one individual DNA, were coprecipitated and run in a single lane on a sequencing gel. After separation by electrophoresis, the PCR products were transferred onto a nylon membrane by electroblotting (Church and Gilbert, 1984). The amplified markers were successively probed either by PCR primers (as shown for two markers in Fig. 1) or by oligonucleotides internal to the PCR products. Up to 15 cycles of strippings and probings have been performed without loss of hybridization signal. Electrotransfers were not always uniform and some areas of the gels were only poorly transferred, resulting in patchy hybridizations. These irregularities in transfer might be overcome by simple capillary blotting of the sequencing gels. Detection of amplification products using 32P-labeled primers sometimes results in detection of spurious bands, which does not usually interfere with the genotyping procedure. Occasionally, oligonucleotides internal to the PCR product were used as probes to reduce the number of spurious bands revealed. PCR products, ranging in size from 66 to 253 bp, were coprecipitated in two groups (above and below 130 bp) and run on separate gels. The use of this multiplex procedure appears reliable and efficient. Up to 15 markers can be readily coprecipitated and typed on a single gel. This procedure reduces the tediousness of typing microsatellites to the levels found in techniques using classical RFLPs. Linkage

of 33 CA/GT Polymorphic Loci a Flow-Sorted Chromosome 20 Li-

Procedure Marker”

Maps

Our maps were established by multipoint linkage analysis using the LINKAGE programs (Lathrop et al., 1985). Two-point recombination estimates and lod scores calculated between all 33 loci after scoring of 15 CEPH families were first determined. The male

IP19Mll IPM38 IP20M78 IP20M37 IP20M73 IP20M25 IP15M68 IP20M43 IPlSMlO IP20M66 IP20M14 IP20M41 IP20M71 IPM19 IP20M6 IP2OMl IP20M7 IP20M23 IP20M85 IP20M29 IP20M83 IP20M17 IPM31 IP20M3 IP20M28 IP15M9 IP20M48 IP20M12 IP20M77 IP20M61 IP20M62 IP20M57 IP20M5

1

Locus

D20S53 D20S51 D20S47 D20S62 D20S40 D20S43 D20S58 D20S39 020844 D20S56 D20S60 D20S57 D20S61 D20S54 D20S50 D20S45 D20S59 D20S55 D20S63 D20S52 D20S48 D20S46 D20S25 D20S42 D20S41 D20S64

Number of dinucleotides” 6+6+6+4 7+5 5+4+3 10+8+7+5 10 13 16 12flO 12 17 13 13 16 21+14 17 20 19+5 19 17 21 21 17 19 17 10+4 25 21 19 20+9+6 27 33 22 24+10

Number alleles 2 2 2 3 2 4 5 5 6 8 4 6 6 9 5 8

6 12 11

8 8 10 8 9 10 10 11 12 19

of PIG 0.18 0.19 0.19 0.22 0.34 0.37 0.41 0.47 0.49 0.56 0.57 0.57 0.58 0.61 0.63 0.64 0.64 0.69 0.70 0.71 0.72 0.73 0.74 0.76 0.77 0.78 0.79 0.81 0.81 0.82 0.82 0.83 0.87

’ D-numbers were assigned for the 26 markers located on chromosome 20. Primer sequences of these 26 polymorphic markers have been deposited at GDB. ’ The number of dinucleotides corresponds to the number of CA/ GT repeats in the sequence. When two or more stretches of dinucleotide repeats are separated by one or several consecutive nonrepeated bases, interruptions are marked by 2+2. IP20M5, IPM19, and IP20M77 have compound repeat sequences (a stretch of CA/ GT repeats being adjacent to a stretch of GA/CT or AT/TA repeats).

A LINKAGE

TABLE

MAP

OF

CHROMOSOME

2

1 pter I

Number of CA/GT Repeats in Nonpolymorphic Markers Isolated from a Flow-Sorted Chromosome 20 Library Marker

Number of dinucleotides

IPM33 JPMGO IPM59 IPM64 IPM55 IPM15 IPM16 IPM30 IPM45 IPM39 IPM74 IPM53 IPM88 IPM42 IPM81 IPMZO IPM26 IPM49 IPM70 IPM56

5 5 7 10 11 12 13 13 14 22 4+5 6+4 6+5+. . 7+4+. . 7+9 ?3+3+. . . 13+6 6+5+5f.. 8+3+4 16+12

187

20

1pter

1 pter

+lP20M7

17

--

IP20M17

--

iP2OM67

--

IPmw

--

IPalM

--

IP20t.463

--

IP20t.477

--

IP20f.466

IP20M29

5 --IP20M41 14 14

--IP2OM146 3--lP20M61

Note. The number of dinucleotides corresponds to the number of CA/GT repeats in the sequence. When two or more stretches of dinucleotide repeats are separated by one or several consecutive nonrepeated bases, interruptions are marked by +.

13

I qter MALE

’ qter I

presented in Fig. 4B. The order of the markers is reliable since their odds against alternative orders are greater than 1OOO:l (Fig. 4B) except in one case of tightly linked markers, IP20M48-IP20M61 (odds = 7O:l).

IPZOt.341

SEX AVERAGE

+ 13 IP20M48 7i IP20M61

t , W

PIG

FEMALE A

e

FIG. 4. (A) Male and female multipoint genetic maps of chromosome 20 with 15 markers selected for the construction of a primary map. (B) Sex-averaged genetic map of 15 chromosome 20 markers with well-supported order. Odds against inversion of adjacent loci for markers selected to build this primary map are indicated on the right side. The distances in centimorgans (CM), scaled using the Haldane mapping function, are indicated in italics on the left side of the maps.

10

20

Number

30

1 40

of Repeats

FIG. 3. Informativeness of 33 (CA), polymorphic microsatellites (described in Table 1) as a function of repeat length. The number of repeats corresponds to the longest stretch of uninterrupted CA repeats in the sequence.

Support intervals (100~1 odds) of the maximum likelihood location were calculated for the 11 remaining microsatellite markers and are shown in Fig. 5. Regional localizations for index markers (D20S4, D20S6, D20S16, and DZOS19) and for 4 other markers from the CEPH Public Database (D20S5, D20S15, D20S17, and D20S18) are also provided (Fig. 5). Phys-

188

HAZAN

mo.s16

13.1 13.2

ET

AL.

spans a genetic distance of approximately 195 CM, extending from microsatellite IP20M7 on 2Op to D20S19 on 20q. The density of (CA), markers is very high in the pericentromeric region with an average distance of 3 to 4 CM between adjacent markers over a distance of 43 CM. Two-thirds of the microsatellites that we have isolated are clustered in a region, between D20S5 and D20S16, representing approximately one-fourth of the linkage map. The markers are separated by much larger genetic distances in the distal regions. This is particularly noticeable for the long arm, where a number of previously isolated markers map distal to ours. Similarly, there are only few microsatellites in the distal part of 20~. Since our markers were isolated at random from a flow-sorted library, this underrepresentation of microsatellites in subterminal parts of the chromosome was not expected. Several nonexclusive explanations could account for this observation: (i) The nonrandom distribution might reflect a bias in the library, as observed for chromosome 1 (Dracopoli et al., 1988). (ii) Since linkage distances tend to increase in the subtelomeric parts of chromosomes, especially in male meiosis (Rouyer et aZ., 1990), a regular distribution of (CA), repeats would result in larger intervals for the more distal regions of a chromosome linkage map. (iii) The distribution of (CA),, or of polymorphic (CA), might not be uniform, at least on chromosome 20. A slight decrease in density in the more distal regions combined with an increase of linkage distances could result in the distribution we observe here. The systematic use of randomly picked microsatellites in mapping entire chromosomes is still poorly documented and some additional studies will be necessary to draw more general conclusions. CONCLUSION

FIG. 5. Genetic linkage map of chromosome 20 with 26 (CA), microsatellites. The 15 markers that were selected for the primary map are placed on the right. Support intervals (10&l odds) of the maximum likelihood location for the 11 remaining microsatellite markers are shown on the left. Support intervals (10&l odds) for the index markers DZOS4, D20S6, D20S16, and D20SlS and for 4 other markers that are also included in the CEPH Public Database D20S5, D20S15, D20S17, and D20S18 are shown on the right. Locus D20S25 (Ref. (lQ)), from which microsatellite IP20M61 was isolated, is also indicated on the right. The physical locations of the previously mapped markers D20S4, D20S5, and D20S6 are indicated on the karyogram. The map is scaled in centimorgans (CM) using the Haldane mapping function.

ical locations for the markers DZOS4, D20S5, and D20S6 based on previously published data (HGM 10.5, 1990) are indicated in Fig. 5. The final map

Because (CA),, polymorphic markers can be readily developed, it was tempting to consider the possibility of their exclusive use to construct linkage maps of entire chromosomes. We show here that microsatellite typing can be greatly facilitated by multiplex procedures. However, it has not been proven that microsatellites isolated at random, which may be less densely distributed in distal regions of chromosome arms, can be used exclusively for construction of high-resolution linkage maps. ACKNOWLEDGMENTS We are grateful to Dr. Mark Lathrop for helpful advice in using the LINKAGE programs and critical review of the figures. We thank Patricia Rodriguez-Tome and Claude Discala for assistance in choosing PCR primers, VBronique Wunderle and Nathalie

A LINKAGE

MAP

Cheron for synthesis of oligonucleotides, Francois Iris and Philippe Millasseau for precious advice in sequencing, and Christophe Person and Bob Cottingham for their help in using LINKAGE programs. This work was supported by Association Francaise contre les Myopathies (AFM). J.H. is a recipient of a fellowship from AFM.

OF CHROMOSOME

52. 14.

337:

16.

MALAFOSSE, A., LEBOYER, M., LAKLOU, H., DULAC, O., SCHNITTGER, S., HANSMANN, I., PLOUIN, P., MOUCHNINO, G., GRANDSCENE, P., NAVELET, Y., VALL~E, L., GUILLOU-BATAILLE, M., SAMOLYK, D., FEINGOLD, J., AND MALLET, J. (1991). Localisation of Benign Familial neonatal convulsion gene to 20q13.3. Hum. Genet., in press. MANN, W. R., VENKATRAJ, V. S., ALLEN, R. G., LIU, Q., OLSEN, D. A., ADLER-BRECHER, B., MAO, J., WEIF’FENBACH, B., SHERMAN, S. L., AND AUERBACH, A. D. (1991). Fanconi Anemia: Evidence for linkage heterogeneity on chromosome 20q.

LIT-T,

Genet.

17.

9: 329-337.

NAKAMURA, Y., LEPPERT, M., O’CONNELL, P., LATHROP, M., LALOUEL, J. M., AND WHITE, R. (1989). A genetic linkage map of markers for human chromosome 20. Genomics 5: 945947.

19. ROUYER, F., DE LA CHAPELLE, A., ANDERSSON, M., AND WEISSENBACH, J. (1990). An interspersed repeated sequence specific for human subtelomeric regions. EMBO J. 9: 505514. 20. RYCHLIK, W., AND RHOADS, R. E. (1989). A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids 21.

22.

23.

9. GRZESCHIK, K. H., AND SKOLNICK, M. H. (1990). Report of the committee on the genetic constitution of chromosome 20. Cell Genet.

44: 397-401.

Genomics 18.

470.

55: 229-234.

10. HAMADA, H., AND KAKUNAGA, T. (1982). Potential Z-DNA forming sequences are highly dispersed in the human genome. Nature 298: 396-398. 11. HANIFORD, D. B., AND PULLEYBLANK, D. E. (1983). Facile transition of poly(d(TG) 0d(CA)) into a left-handed helix in physiological conditions. Nature 302: 632-634. 12. LATHROP, G. M., LALOUEL, J. M., JULIER, C., AND O?T, J. (1985). Multilocus linkage analysis in humans: Detection of linkage and estimation of recombination. Am. J. Hum. Genet. 37: 482-498.

647-648.

M., AND LUTY, J. A. (1989). A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am. J. Hum.

307.

Cytogenet.

LEPPERT, M., ANDERSON, V. E., QUATTLEBAUM, T., STAUFFER, D., O’CONNELL, P., NAKAMURA, Y., LALOUEL, .J. M., AND WHITE, R. (1989). Benign familial neonatal convulsions linked to genetic markers on chromosome 20. Nature

15.

1. AMASINO, R. M. (1986). Acceleration of nucleic acid hybridization rate by polyethylene glycol. Anal. Biochem. 152: 304ARNO?T, S., CHANDRASEKAFUN, R., BIRDSALL, D. L., LESLIE, A. G. W., AND RATLIFF, R. L. (1980). Left-handed DNA helices. Nature 283: 743-745. 3. BELL, G. I., XIANG, K., NEWMAN, M. V., Wu, S., WRIGHT, L. G., FAJANS, S. S., SPIELMAN, R. S., AND Cox, N. J. (1991). Gene for non-insuline-dependent diabetes mellitus (maturity-onset diabetes of the young subtype) is linked to DNA polymorphism on human chromosome 20q. Proc. Natl. Acad. Sci. USA 88: 1484-1488. 4. BOTSTEIN, D., WHITE, R. L., SKOLNICK, M., AND DAVIS, R. W. (1980). Construction of a genetic linkage map in man using restriction fragment length polymorphism. Am. J. Hum. Genet. 32: 314-331. 5. CHURCH, G. M., AND GILBERT, W. (1984). Genomic sequencing. Proc. Natl. Acad. Sci USA 81: 1991-1995. 6. CHURCH, G. M., AND KIEFFER-HIGGINS, S. (1988). Multiplex DNA sequencing. Science 240: 185-188. 7. DONIS-KELLER, H., GREEN, P., HELMS, C., CARTINHOUR, S., WEIFFENBACH, B., STEPHENS, K., KEITH, T. P., BOWDEN, D. W., SMITH, D. R., LANDER, E. S., BOTSTEIN, D., AKOTS, G., REDIKER, K. S., GRAVIUS, T., BROWN, V. A., RISING, M. B., PARKER, C., POWERS, J. A., WA?T, D. E., KAUFFMAN, E. R., BRICKER, A., PHIPPS, P., MULLER-KAHLE, H., FULTON, T. R., NG, S., SCHUMM, J. W., BRAMAN, J. C., KNOWLTON, R. G., BARKER, D. F., CROOKS, S. M., LINCOLN, S. E., DALY, M. J., AND AEIRAHAMSON, J. (1987). A genetic linkage map of the human genome. Cell 51: 319-337. 8. DRACOPOLI, N. C., STANGER, B. Z., ITO, C. Y., CALL, K. M., LINCOLN, S. E., LANDER, E. S., AND HOUSMAN, D. E. (1988). A genetic linkage map of 27 loci from PND to FY on the short arm of human chromosome 1. Am. J. Hum. Genet. 43: 462-

189

13. LATHROP, G. M., LALOUEL, J. M., AND WHITE, R. (1986). Construction of human linkage maps: Likelihood calculations for multilocus linkage analysis. Genet. Epidemiol. 3: 39-

REFERENCES

2.

20

24.

25. 26.

Res. 17: 8543-8551.

S., AND COULSON, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc. Natl. SANGER,

F., NICKLEN,

Acad. Sci. USA 77: 5463-5467. SCHNITTGER, S., HOEFERS, C.,

BEERMANN, F., HEIDEMANN, P., AND HANSMANN, I. (1989). Alagille-Watson Syndrome is assigned to 20 (pl.l-~1.2) and provisionally to the region p11.23-~12.1. Cytogenet. Cell Genet. 51: 1074. SUMMAR, M. L., PHILLIPS III, J. A., BATTEY, J., CASTIGLIONE, C. M., KIDD, K. K., MANESS, K. J., WEIFFENBACH, B., AND GRAVIUS, T. C. (1990). Linkage relationships of human arginine vasopressin-Neurophysin-II and OxytocinNeurophysin-I to prodynorphin and other loci on chromosome 20. Mol. Endocrinol. 4: 947-950. WEBER, J. L., AND MAY, P. E. (1989). Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Hum. Genet. 44: 388-396. WEBER, J. L. (1990). Informativeness of human (dCdA), . (dG-dT), polymorphisms. Genomics 7: 524-530. ZHANG, F., DELEUZE, J.-F., AURIAS, A., DUTRIUAUX, A.-M., HUGON, R.-N., ALAGILLE, D., THOMAS, G., AND HADCHOUEL, M. (1990). Interstitial deletion of the short arm of chromosome 20 in arteriobepatic dysplasia (Alagille syndrome). J. Pediatr. 116: 73-77.