Characterization and chromosomal assignment of yeast artificial chromosomes containing human 3p13-p21-specific sequence tagged sites

ELSEVIER

and Chromosomal Assignment of Yeast Artificial Chromosomes Containing Human 3pl3-p21-Specific Sequence Tagged Sites Characterization

Susanne C. Michaelis,l Walter Bardenheueql Andreas Lux, Alexander Schramm, Anja Gockel, Reiner Siebert, Christoph Willers, Katja Schmidtke, Birgit Todt, Annemarie H. van der Hout, Charles H. C. M. Buys, Amanda C. Heppell-Parton, Pamela H. Rabbit& Sylvia Ungar, David Smith, Denis LePaslier, Daniel Cohen, Bertram Opalka, and Jochen Schtitte

ABSTRACT: Human chromosomal region 3pl2-p23 is proposed to harbor at least three tumor suppressorgenes involved in the development oflung cancer, renal cell carcinoma, and other neoplasias. In order to identify one ofthese genes we defined sequence tagged sites (STSs) specific for 3p13-~24.2 by analyzing a chromosome 3~14 microdissection library. ST% were used for isolating yeast artificial chromosome (YAC) clones from the Centre d’Etude du Polymorphisme Humain (CEPH) YAC libraries. Thirty-eight YACs were assembled into a contig approximately 2.5 Mb in size spanning the t(3;8) and t(3;6) translocation breakpoints associated with hereditary renal cell carcinoma and hematologic malignancies, respectively. Chromosomal localization and chimeric status of 126 YACs was analyzed by fluorescence in situ hybridization (FISH). The order of 17 YACs determined by double-color FJSH was in agreement with the STS-based arrangement of the YAC-contig.

INTRODUCITON Chromosomal aberrations of the short arm of human chromosome 3 are frequently observed in different types of tumors, including renal cell carcinoma (RCC) [l, 21, smallcell (SCLC) and non-small cell lung cancer (NSCLC) [3-51, head and neck carcinoma [6], breast cancer [fl , ovarian cancer [a], cervix carcinoma [9], and bladder cancer [lo]. In a hereditary RCC the chromosome 3 breakpoint of a diseaseFrom the Deportment of Medical Oncology (Cancer Research) (S. C. M., W. B., A. L., A. S., A. G., R. S., C. W., K. S., B. T., B. 0.) J. S. J, University ofEssen Medical School, West German Concer Center Essen, Essen, FRG, Deportment of Medical Genetics (A. H. v. d. H., C. H. C. M. B.), University of Groningen, Groningen, Netherlands; Clinical Oncology and Rodiothempeutics Unit [A. C. H.-P, I? H. R.), Medical Research Council Centre, Cambridge, UK, Institute ofMolecular Biology CD.S.), Wayne State University, Detroit, Michigan, and Centre d’Etude du Polymorphisme Humoin @. L. P, D. C.), Paris, France, German Cancer Research Center,

Deportment

of Developmental

Genetics, Heidelberg,

FRG (S. U.).

Address reprints requests to: Susanne Michaelis and Walter Bordenheuer, Deportment ofMedico Oncology (Cancer Research), University of Essen Medical School, West German Cancer Center Essen, Hutelondstr. 55, 45122, Essen, FRG. 1 S. C. M. and W. B. contributed equally to this work. Received July 25, 1994, accepted October 11, 1994.

Cancer

GenetCytogenet81:1-12

0 Elsevier 655

Science

Inc.,

related constitutional translocation, t(3;8), was mapped to 3~14.2 [ll]. In association with familial hematologic malignancies a translocation breakpoint, t(3;6)(3p14.3;pll), was reported [12]. Analysis of cell line U2020 established from a SCLC revealed an interstitial homozygous deletion in 3p12-p13 [13]. Overall, the compilation of published data stresses three distinctive regions to harbor putative tumor suppressor (TS) genes: 3p12-~13, 3~14, and 3p21-~23. A common strategy for investigating a large genomic region consists of the isolation of YAC clones and their analysis for the presence of genes. Although the number of 3pspecific markers ordered into genetic as well as physical maps has increased [14], a higher density of region-specific markers suitable for PCR-based YAC -isolation would be valuable for this strategy. In this work we focused on chromosome region 3~14. As an initial approach we defined region-specific STSs by analyzing a chromosome 3~14 microdissection library [15]. These STSs were used for a PCR-based isolation of YACs from the CEPH-YAC libraries [16, 171. Here we report the construction of a YAC-contig consisting of 38 YACs spanning tbe t(3;8) and t(3;6) translocation breakpoints. Moreover, we present data on size, localization, and chimeric status of isolated YACs determined by pulsed

(1995)

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S. C. Michaelis et al.

2

field gel electrophoresis bridization (FISH).

MATERIALS

(PFGE) and fluorescence in situ hy-

AND MEI’HODS

Definition of 3p13-p24.2-speci&

STSs and Isolation of YACs

The strategy and techniques for defining 3p13-p24.2-specific STSs using a chromosome 3~14 microdissection library and SDS-based screening of the CEPH-YAC libraries were described previously [15]. Deletion Hybrid Panel for Mapping For ordering STSs into subchromosomal regions we used DNA of cell hybrids containing parts of chromosome 3 as their only human chromosomal material in a rodent background. Localizations of breakpoints are included in Table 2. ARl, AR38, ADl, and Aph50 are aphidicolin-induced deletion hybrids whose breakpoints were characterized by localization of markers including reference markers [15, 181. The breakpoints of AD1 (formerly mapped to 31314.2) and Aph50 (formerly mapped to 3~13) have been found to be localized more proximal. The hybrids 3;WUC-1, 3;7/UC-1, 3;6, and TL12-8 are somatic cell hybrids with breakpoints characterized previously [ll, 19, 20, 211. Pulsed Field Gel Electrophoresis of YACs

and FISH Analyses

Yeast DNA containing YACs was isolated in plugs as described previously [15]. Yeast chromosomes were separated by PFGE using the ‘Xotaphor” PFGE apparatus (Biometra, Gottingen, FRG) following the manufacturer’s instructions. For FISH analysis, plugs containing total yeast DNA were digested with agarase (Boehringer-Mannheim, Mannheim, FRG) according to the supplier’s recommendations, followed by phenol/chloroform extraction and ethanol precipitation. Biotin-14-dATP (Life Technologies, Eggenstein, FRG) or digoxigenin-(DIG)-ll-dUTP (Boehringer-Mannheim) was incorporated by nick-translation. Labeled YAC-DNA (330 ng) was denatured and pre-annealed for 3 hours at 37°C in the presence of 3.3 ng Cot-l DNA (Life Technologies) in a volume of 10 pl hybridization buffer (50% formamide, 10% dextrane sulfate, 2 x SSC, 0.5 mM Tris pH 7.6, 100 pg/mL sonicated salmon-sperm DNA) for painting the chromosome 3 centromeres, a DIG-labeled chromosome 3-specific alpha-satellite probe (Amersham, Braunschweig, FRG) was used. Metaphases fmm normal lymphocytes were prepared in the presence of bromo-desoxyuridine following the thymidine method for obtaining extended chromosomes [22]. Prior to hydridization, slides were incubated sequentially in acetone for 10 minutes and in 70% formamidel0.6 x SSC at 70°C for 150 seconds followed by dehydration in ethanol. Hybridization was carried out at 37°C overnight. Post-hybridization washes and detection of hybridized probes were performed according to published methods [23]. Biotin-labeled probes were detected by fluorescein isothiocyanate-conjugated avidin (Vector Laboratories, Burlingame, CA) and DIG-labeled probes were detected with tetramethyl-rhodamine-isothiocyanate (TRITE)-conjugated antibodies (Sigma, Deisenhofen, FRG). Chromosomes were counterstained with diamidino2-phenylindole (DAPI) (10 ng/mL) added to mounting

medium (Vectashield, Vector). Chromosomes were additionally stained with propidium iodide (PI) (10 ng/ml) when biotin-labeled probes were detected with FITS exclusively. Chromosomal localizations of YACs were determined using a FISH workstation comprising a fluorescence microscope with attached cooled CCD camera and filter wheel (Photometrics). Image acquisition was achieved using a Macintosh Quadra 950, BIOSoftware (Digital Scientific, Cambridge, UK), and a Mitsubishi color printer S3600. For a limited number of experiments a confocal microscope with attached MRCGOOworkstation (Biorad) and a Sony video color printer UF5000P was used. FISH data of YACs are based on a minimum of 15 independent determinations. Additionally, hybridization signals occurring in interphase nuclei were taken into account. RESULTS Definition of 3pl3-p21-Specific STSs and Isolation of YACs For isolating YACs we used 3pl3-p21-specific STSs derived fmm a recently described chromosome 3~14 microdissection library [WI. Sequences of PCR primer pairs and PCR conditions are given in Table 1. SI’Ss were assigned to seven subregions on chromosome 3p whose borders were defined by breakpoints in a hybrid deletion mapping panel. ST& for the loci D3S2, D3S3, D3S6, and D3S32 [20] were integrated as reference markers. So far, we isolated 180 single CEPHYACs corresponding to 67 STSs (63 micmdissection clones plus four reference markers). Construction of a YAC-Contig Spanning the t(3;8) and t(3;6) Translocation Breakpoints Single-YAC clone analysis with 3pl4-specific STSs enabled us to identify overlapping YAC clones and to assemble them into a YAC-contig consisting of 38 YACs spanning 26 STSs without the necessity of isolating YAC end-probes. This contig covers approximately 2.5 Mb of DNA (Fig. 1). The chromosome 3 translocation breakpoints t(3;6) and t(3;8), as well as the breakpoints of the aphidicolin-treated hybrids ARI and AR38, are localized within this contig. Analysis of YACs Containing STSs by PFGE and FISH

3pl3-p21-Specific

YAC sizes for the YAC library I (coordinates lAl-735H12) ranged from 150 kb to 1800 kb, with an average length of 490 kb, and for the Mega-YAC library (coordinates 736Al-984H12) from 310 kb to 2500 kb, with an average length of 1400 kb, as determined by PFGE (Table 2). In order to confirm the cbromosomal localization of YACs and determine their chimeric status, FISH analyses were performed on 126 YACs. The frequency of chimerism was 46%) with 13 of 57 (23%) chimeric YACs showing signals only on chmmosomal regions other than their corresponding STS localization. A diffuse painting pattern occurring with 11 of 57 (19%) chimeric YACs was displayed by short arms of the chmmosomal groups D and G only. The results of FISH analyses are listed in Table 2 and examples are shown in Figure 2. For double-color FISH 36 different combinations of YAC-

Analysis

of 3pl3-p21-specific

Table 1 MDC

Sequences

Locus

YACs

of PCR primer

pairs

Annealing temperature

for 3p13-p24.Zspecific PCR product

(“C)

Primer sequences

(bp)

XIDl

D3S1438

52

114

VA812

D3S1396

52

71

IIIE5

D3S1385

65

176

W2.8

52

111

3A7

54

106

IIIBZ

D3S1374

65

107

HA12

D3S1412

62

95

VIB7

D3S1376

65

141

VD8

D3S1415

56

72

IGlo

D3S1411

58

72

55

47

2Bll IVG6

D3S1414

50

60

IB4

D3S1407

62

97

60

127

lA3 VDl

D3S1375

62

74

ND6

D3S1413

62

81

IIG3

D3S1378

52

98

IVA9

D3S1381

54

207

IIH9

D3S1379

64

128

WA2

D3S1380

58

126

LB3.6

62

68

LB4.1

56

102

61

79

52

122

60

59

50

73

54

126

56

259

ID7

D3S1377

H9L IVH9

D3S1382

fib2 XIEl

D3S1387

SY.

STSs

IIIE2

D3S1384

62

99

VA4

D3S1386

62

81

(5’+3’)

ACAGAAAAATAAGAAAGGGG ACTCTATCTTGTAATCACTC TTAAGTCTGCCATTTTGG GAAAGGGGAAATAGAGGC CCTGACTGCCTGGCTCAGCA CCCCACCCTCAGTGATGTCC CCTTAGGTGCCTTGCTTG ATGGCAGTCATGATTTGG AGTGTTAGAGGGACAGGG TCTTGCCGTGTTTTGAGG ACCTGCTACCACCCAGCCCA CAGCATCACCAGGCCAGGCC TCCCACTAATGGTGCAGCCA TTCCAAGGCTTCTCTGGGCA AGGACCACTACCCTGCCC GCTACCCCAGCCCAGAAC ACTCTCTGCTGCTCCAGGTC TGGTGGTATAGGTAGATTGT TGCACTATAGCCTGGGTGAC CCAAACTCAGTCCTCTAGGG ATGTAGGGATTCTGGAGC ACTCAAATCATAACCACC TGAAATAATGCCTAGCAC CTTGTATAACAACAGTGG ACTCCAGAAGGACCGTTCCA CACACCTCATCCACCCTCCG ACACACAGGCACCCAGGC CCAGAGGAGCTAGCAGCA TAATGGTGCAGCCATGGCCT GGGCAGCGAGGAGGGAAGAC CACCATGGCCAGCCCTATCC CCCTGCCTGGAAGTGGTCTA TCCACATCAAACTCTTGC GTTGGTTTTATCTCCTGC AAATTCAAGAGCAAATCAAA AGAGGGAAGACCATGTGAAG TTGAGTCTGGGCTCCCCACG CCTCACCAGGCCCTGGATAT CCCTGGATATACAAAGCTGA ATGGACCTTTGAGTCTGGGC AGGAGGCCACAGAACCTG CCAGGCACAAAAGGAGGG CACATGGCCAGAATGACC TTCCAAAACATGGCTGTG AGGCTAAGCACCCTTTGACC CACGACACCAATTGCTGTGA GGAGAGAGGAATTTAGTG AGGACTGGGAAGTTTAAC GGGGATTTGTGACTTGGC AGTAGGCAGGAGGCTGGG TCGTGYTTTCGGATATGC TGTGGCTTTGAGAAAAGG TCACCACTACTCATCTCC AAGTAGAATGGTGTCTGC AGTAGAGCGCGTTTTGCC TGGAGTGGTCCCCAATCC CTGGGCTGGTGGGGAAGCCT GTGCTGGGTGTCAAATGGCA GGAAACCAGAGGCGCAACAT TTGTGGCATTGTCTCTGGGC continued

S. C. Michaelis

Table

1

Continued Annealing

MDC

Locus

LB5.6

temperature

55

PCR product (“C)

Primer sequences

(bp)

63

(Y-3’)

TTGTCTCTGGGCCTTTGG GGCGCAACATTTGGGTAG

XIF3

D3S1436

52

101

ID5

D3S1383

64

72

50

69

CATCTGCTCAGTTCCTCC TAACTGTCTATCTGTGGC tctaagcccactgcccctgc tgggcccctcttgcagaaga

LB5.2

GCAGAAGAGCTTACTATC AGTAAAAGTTCTAAGCCC

BIB5

D3S1388

56

112

58

68

CAAGCACAGGCTCAGAGTCC TTCAAATGCATCAAGGTGGC

1A2

ATCAAGGTGGCCTCTGAC GACCTGCTTTACCGTGAC

IVHlo

D3S1401

55

85

CCTTGGATCCTGGGAGGG

IF8

D3S139l

54

66

IVA6

D3S1397

52

98

50

96

TCAGATGATATGGCTACC

55

118

TGCCCAGGGTCACAAAGC

62

97

AAGGAGACTCCACAAAGG GGAGCATACCAAAATGAGAA AATGACTAAGACAGGACCCC TTCCAAAGTAACCAAAATCA TGGGAGGGAAGGCTCAGCTT 3B6

GGTGCTTTGAGAGCTAGG IVHl

D3S1400

CTCTGGGTCGATCAACCG 2A5

TGGAAAGGTTCTGAGCTGCC TGGGGAAGGGATGGAAAGTT

VBll

D3S1403

56

71

TCATTGGCACTTTGATGGGT

vc4

D3S1404

56

88

ATTCTTCCATGACGTTAGGT

W3.2

54

103

2B6

56

120

GGCAGACACACCTGAATG CCTGTCCAGCCCACCACC

AGTCCTTAAGAGCCAAATCT TGATGGGTCTAGGCTGAGGT CGCCTTTGCATCCTTCAG ATACAGGTGGCCTAACAG CAGCCAGTATTCCATGGG IVD2

D3S1398

56

118

IA3

D3S1389

62

81

IVA4

D3S1395

54

114

IIIEl

D351393

60

80

56

>200

GGGATGGCATTAGGTTCC TCCAGGTCCTCTGAGAAGCA ACCCCACTTACCCCTGCACT CAAATTTACCATCCAACCTC ACTACTTTTGGAGAGACTTT GGTTGAAACCTGGCCCCACT CCCCATCTGCAGCAACAAAA 2c5

CAGAGTCTCTCTACCAGC ATATCTTTAAAATATTTCAAATATC

VIGlo

D3S1405

52

118

IA1 1

D3S1390

57

62

52

133

GCATAGTAAGCCTCTAGC ATTTCACCTCAGGCTCTG GAATGTATGTTGGGGATGGG ACTGATGAAAGATTCTTGAG

2Hl

AAAGCAGAAGCATAGTGC CCAGTGCTATATTCCAGC

IIIE2

D3S1392

58

117

TCCATTCCCTAAATACCACA

IIIE12

D3S1394

56

128

GGTATATGCCATCTCTTCAT

XIDl1

D3S1437

52

59

IVEl

D3S1399

65

81

BIB4

D3S1406

50

124

VB1o

D3S1408

62

48

ACCTGCCTCTTCTTCCTAAA ATCAGTTTTAGTCCCAGTGC GGTAGGTATTATTTTTTGGG ATGTCCTCTGTCTAATGC ACCCCACTTACCCCTGCACT TCCAGGTCCTCTGAGAAGCA TGTGCCACCCTTAAAATC CCTTCCTTCTTTACCTGG TCGCTCACCCCCAGGGTTGC GTGGCGCGGGTTGATCAGGT continued

et al.

Analysis

of 3pl3-p21-specific

Table 1

YACs

Continued

MDC

Locus

Annealing temperature (“C)

VIC2

D3S1409

63

70

VIH4

D3S1410

63

92

52

88

3Bl

pairs were analyzed, revealing the following order of YACs: centromere-324F’l-821ClO-(757Hl0, 808BlO)-169B5-(85OA6, 17181, 65E7)-(237E3, 268Fl, l26E9)-21lF4-18lD3-(128B8,87OE5, 708A10)-103F2-te1omere. The limited resolution of FISH did not allow the determination of the exact order of those YACs given in brackets. The double-color FISH results were in accordance with the STS-based order of the YAC-contig.

DISCUSSION The goal of our research is to convert the putative TS-gene region 3~14 into a contig of non-chimeric YACs which can then be analyzed for the presence of candidate TS genes. This region is of interest because loss of heterozygosity within 3~14 has been observed in a variety of neoplasias. Moreover, it harbors the tumor-associated translocation breakpoints t(3;8) and t(3;6) and the constitutive fragile site FRA3B [24-261. Although both translocation breakpoints were recently cloned [27, 281, no candidate TS genes adjacent to them have yet been published. Thus, a 3pl4-specific YAC-contig including these translocation breakpoints may be valuable for further investigation of this putative TS gene region. Here, we demonstrate that the strategy of defining regionspecific STSs by analyzing microdissection clones is an efficient way to obtain markers suitable for the isolation of corresponding YAC clones. The high density of our 3pl4-specific STSs enabled us to assemble a YAC-contig without timeconsuming chromosomal walking procedures. For future functional analyses and the isolation of 3p14specific cDNAs non-chimeric YACs mapping to this region will be preferred. Therefore, YACs were analyzed for their localization and chimeric status by FISH. We used total yeast DNA containing the entire YAC rather than Alu-PCR products which may not represent the entire YAC and compromise reliability of results. Combining the results of FISH and STSPCR analyses the frequency of chimeric YACs in this study was 46%) including 11 YACs showing a diffuse painting pattern on the short arm of chromosome groups D and G which may result from rRNA homologous repeats contained within these regions [28]. Overall, the frequency of chimerism in our analysis is comparable to published data for the CEPH and other YAC libraries [30-321. Ten percent of the YACs showed no signal at the chromosomal region the corresponding STS was assigned to. This suggests that the STScontaining part of these YACs was too small to be detected by FISH. Similarly, non-chimeric YACs as determined by

PCR product (bp)

Primer sequences (5’+3’) TCCACTGTTGATGGGCAC GTCTGTTCATCACAGCAC ACAGATGGGATTTTGGTG AGCAAACTCCAACAGACC CTGGGACACAGCTAAAGC GAGTGTCAATTTCAGACC

FISH might in fact be chimeric, possibly increasing the frequency of chimerism to 56%. In summary, we isolated and characterized a large number of YACs by FISH and constructed a YAC contig spanning the t(3;8) and t(3;6) translocation breakpoints from the putative TS-gene region 3~14. Double-color FISH with nonchimeric YACs confirmed the order of YACs as determined by STS-PCR. The 3pl3-p21-specific YACs described here will provide a valuable tool for the identification of candidate TS genes.

Susanne Michaelis was supported by a fellowship of the Graduiertenkolleg “Normale und maligne Zellen:’ University of Essen, FRG, Walter Bardenheuer by a fellowship from the Boehringer Ingelheim Fonds, Stuttgart, FRG. FISH analyses were supported by an EMBO fellowship, Heidelberg, FRG (ASI’F 7667). This work was supported by Deutsche Forschungsgemeinschaft, Bonn (SFB 354).

REFERENCES 1. Vander Hout AH, Van der Vlies P, Wijmenga C, Li FP, Oosterhuis JW, Buys CHCM (1991): The region of common allelic losses in sporadic renal cell carcinoma is bordered by the loci D3S2 and THRB. Genomics 11:537-542. 2. Yamakawa K, Morita R, Takahashi E, Hori T, Ishakawa J, Nakamura Y (1991): A detailed deletion mapping of the short arm of chromosome 3 in sporadic renal cell carcinoma. Cancer Res 51:47Oi-4711. 3. Brauch H, Tory K, Kotler F, Gazdar Z, Pettengill OS, Johnson B, Graziano S, Winton T, Buys CHMC, Sorenson GD, Poiesz BJ, Minna JD, Zbar B (1990): Molecular mapping of deletion sites in the short arm of chromosome 3 in human lung cancer. Genes, Chromosomes and Cancer 1:240-246. 4. Rabbitts P, Douglas J, Daly M, Sundaresan V, Fox B, Haselton P, Wells F, Albertson D, WatersJ, Bergh J (1989): Frequency and extent of allelic loss in the short arm of chromosome 3 in nonsmall-cell lung cancer. Genes Chmm Cancer 1:95-105. 5. Whang-Peng J, Bunn PA, Kao-Shan CS, Lee EC, Carney DN, Gazdar A, Minna JD (1982): A non-random chmmosomal abnormality, de13 (p14-~23) in human small cell lung cancer (SCLC). Cancer Genet Cytogenet 6:119-134. 6. Latif F, Fivash M, Glenn G, Tory K, Orcutt ML, Hampsch K, Delisio J, Lerman M, Cowan J, Beckett M, Weichselbaum R (1992): Chromosome 3p deletions in head and neck carcinomas: statistical ascertainment of allelic loss. Cancer Res 52:1451-1456. z Sato T, Akiyama F, Sakamoto G, Kasumi F, Nakamura Y (1991): Accumulation of genetic alterations and progression of primary breast cancer. Cancer Res 51:5794-5799.

D3Sl375

Ml

c D3S1413 IvlM -----

3 Fi

--

-----

c P

- D3S1378 IIG3

-

m ------_

pp81

- D3S1379 nH9 - D3S1380 lvA2

3P

- LB36 LB4.1

26

ES1377

25 24.3 24.2

L

24.1 23

l--

- $-p82 .---_ - 2B2

22

21.3

H9L

I

I

- D3S1387 XlEl . SY. &Sl3a4 D3S1386 VA4

14.2

LB56

14.1

D3S1436 xm

D3S1383 IDS

E w&

LB52 e-m-_ D3S1388 JIus5 lA2 -----

E -__------_

3

D3S1401 NE10

_

D3Sl391 IFa D3S1397 NA6

Figure 1 YAC-contig spanning the t(3;8) and t(3;6) translocation breakpoints. Distances between loci shown in this figure are not drawn to physical scale. YAC clones are drawn as lines, and CEPH coordinates of YACs are shown. YAC lengths only reflect the coverage of ST&, not the YAC sizes. The YACs 85OA6 and 195F3 span the chromosome 3 translocation breakpoints t(3;8) and t(3;6), respectively [ZZ 281. The breakpoints of the aphidicolin-treated hybrids AR1, AR36 and translocation breakpoints are indicated by dashed lines. No order could be determined for STSs indicated by brackets.

Analysis

of 3pl3-p21-specific

Table 2 Breakpoint of hybrid

Chromosomal

STS

Telomere 3;21/uc-1 31324.2

localizations

Locus

of YACs YAC coordinate

isolated

with

3pl4-p21-specific Size

Chimeric

(kb)

STSs

FISH signals at chromosomal

region

D3S32

16ClO 334El 238B7 292F2 126ElO

nd nd nd nd nd

nd nd nd nd nd

nd nd nd nd nd

D3S1374

407All

Yes

320

3pZlplusDq

D3S2

160A3 159A4 357E1 loOGl0

nd nd nd nd

nd nd nd nd

nd nd nd nd

VIB7

D3S1376

372B8 252F12

Yes No

310 nd

3~24 3p21-p23

VD8

D3S1415

245ElO 331c4 400D4 378F2

nd nd Yes No

1800 200 300 nd

nd nd 3p21pIus3qpIus5q2pIu~5q3pI~~4q 3p14-~21

IB4

D3S1407

104E2 241BlO

No No

250 280

3~21 3~21

IIA12

D3S1412

103F2

No

180

3p14-p21

1A3

nd

66H6 104E2 134D5 241BlO 722ClO 705E8 677H12 279G2

No No No No nd No nd nd

480 250 520 280 260 nd 900 560

3p14-p21 3p14-p21 3p14-p21 3p14-p21 nd 3~21 nd nd

VDl

D351375

42H7 43C2 103F2 19OCl

nd nd No nd

420 420 180 680

nd nd 3p14-p21 nd

ND6

D3S1413

49A12 9lH12 120B7 323C6 367A12 226B7

Yes nd Yes nd No nd

580 nd 320 260 340 400

3~24 nd 3p2lplusG/D-p* nd 3~21 nd

IIG3

D3S1378

418F5 705F4 327G12

Yes No Yes

300 1500 320

E-P 3p14 3p14plus1p

IVA9

D3S1381

103G8 269ClO 327G12 37168 705F4

nd No Yes Yes No

nd nd 320 270 1500

nd 3p14-p21 3p14-p21pIuslp 3p143-p21pIuslp 3p14-p21

IIH9

D3S1379

212B2

No

400

3p14-p21

WA2

D3S1380

212B2

No

400

3p14-p21

LB3.6

nd

454G4 679C12 700A10

No nd No

250 700 980

3p14-~21 nd 3~14~~21

BIB2 3;7/uc-1 3p21.1

AR1 3~21.1

YACs

continued

S. C. Michaelis

Table 2 Breakpoint of hybrid

3;6 3p14.3

AR36 3p14.2

Continued

STS

Locus

YAC coordinate

Size Chimeric

Ml

FISH signals at chromosomal

731D5 776AlO 87015 960Dll

nd nd No nd

nd nd nd nd

nd nd 3p14 nd

430 440 300 420

3p14-p21 3p14 3p14-p21 3p14-p21p1us12q12-q13

D7

D3S1377

128B8 200G1 324B5 370612

No No No Yes

IVH9

D3S1382

186C3 181D3 312H4 348H9 195F3

Yes No Yes Yes nd

320-420 390 980 310 nd

409B3 13oF9 597F4 613E3 60G3

Yes No Yes Yes No

550 380 nd 660 480

C-q 3p14 C-q nd 3p14

2B2

nd

region

3p14-p21plusD-p 3p14-p21 3p14-p21pIuslp C-q nd 3pl4plusl3qplusG/D-p* 3p14.3 3p14.3plusC-q

XIEl

D3S1387

61333 624Gl 870E5

Yes nd No

660 nd nd

SY

nd

15C6 211F4 219D5 252ClO 540B7 581BlO 607F5 253Bll 671A6 87035

No No Yes No Yes No Yes Yes nd No

550 300 350 320 + 580 180 nd 580 250 ud nd

3p14 3p14 3p14pIus2p 3p14 3p14plusD-p 3p13-p14 3pl4plusD-p 3p14plus7p nd 3p14

IIIEZ

D3S1384

126E9 307B9 145F7 222A9

Yes Yes No Yes

430 320 + 420 530 440

3p14plus5q12 3pl4pIusD-p 3p14 loqllpluslq

VA4

D3S1386

126E9 307B9 64E7

Yes Yes Yes

430 320 660

3p14plus5q12 3pl4plusD-p 3pl4plusBqplus8porS

LB5.6

nd

64E7 665A8

Yes nd

660 1000 + 790 + 460

3p14plusB-qplus8or9p nd

XIF3

D3S1436

690B2

Yes

700

3pl4plusC-q

ID5

D3S1383

268Fl 405C8 292Bl1

No No Yes

380 350 nd

3p14 3p14 JpplusB-p

LB5.2

nd

237E3 405C8 521c6

Yes No nd

nd 350 680

3p14plusC-q 3p14 nd

R7K145 IIB5

nd D3S1388

850A8 8537

No No

1300 620

3p14 3p14

1A2

nd

74B2 621H4

nd Yes

440 460

nd 17-q or 18-q continued

et al.

Analysis

of 3pl3-p21-specific

Table z

YACs

Continued

Breakpoint of hybrid

STS

Locus

YAC coordinate

TL 12-8 3p14.2

IF8

D3S1391

vc4

Size

FISH signals at chromosomal

region

Chimeric

(kb)

171Bl

No

640

3p14

D3S1404

194Hll

No

340

3p14

IA3

D3S1389

9OC8 154D3

No No

410 320

3p14 3p14

IVHl

D3S1400

138G6 194c7 144G3 18683 194B5 43OC2 444D6 369B2 371H4 419c5

No nd No Yes nd Yes Yes No nd nd

400 150 700 650 170 440 750 nd 280 + 430 190

3p14 nd 3p14 3pl4plusD-p nd 3pl4plusG/D-p* 3pl4plusD-q 3p14 nd nd

D3S6

308Fll

Yes

880

3pl4plusothers

3B6

nd

130Hll 143c5

No Yes

220 nd

3p14 3pl4plusG/D-p*

VB11

D3S1403

75H2 94Blo 237B7 37638

No No No nd

nd 500 540 nd

3p14 3p14 3p14 nd

W3.2

nd

237B7 2338HlO 446E7 59OG3 825E12 64OC7 698H8 6FlO 131HlO SF1

No No No nd nd nd Yes No nd No

540 400 440 150 nd nd 580 350 nd 420

3p14 3p14 3p14 nd nd nd 3p13-p14plus3p24plusc-q 3p13-p14 nd 3p13-14

IVDZ

D3S1398

169B5 56C5 163A9 408B8 158B6 248ClO 261C12 28OG2 309Cll 160D8

No nd Yes Yes Yes Yes Yes nd Yes No

2B6

nd

248ClO 309Cll 318G6 408B8 515H4 654C5 666F7 714ElO

Yes Yes Yes Yes Yes Yes nd nd

nd

757810 760A5 808BlO 884D6 21D3

No No No No Yes

1000 2500 1500 860 510

2A5

nd

280 + 400 820 440 150 400 400 730 nd 300 200 400 300 320 150 550 510 660

3p13-p14 nd pl3-pl4plusG/D-p* 3p13-p14plusD-q 3p13-p14plusG/D-p* 3pl3-pl4plusG/D-p* 3p13-p14plus1p nd 3p13-p14plusG/D-p* 3p14 3p13-p14plusG/D-p* 3p13-p14plusG/D-p* 3p23-p24 3p14plus14por15p C-P C-P nd nd 3p13-p14 3p13 3p14 3p13-p14 3p13-p14plus14por15p continued

Table 2

Continued

Breakpoint of hybrid

STS

YAC coordinate

Locus

Size Chimeric

79Cll

No

96Gl

No nd Yes No No

201E6 698B6 325F3 67685 714ElO IVA4

IIIElZ

IIIEl

IA1 1

2Hl IIEZ

VIGlo

AD1 3p13 Aph5o 3~12 Centromere

IIIB4

D3S1395

D3S1394

D3S1393

D3S1390

nd D351392

nd

616AlO 707H9 532E4 675F12

nd Yes Yes No

17D2 161Gll 166G8 248A5 28881

Yes nd No Yes nd

181H6 293Dl

No Yes

(kb) 430 630 390 930 370 nd nd 870 nd 850 1300 350 nd nd nd nd nd 250 + 850

415F7

No

420

145F3 146F3 147F3 148F3

Yes

600 350 520 410

393B8 62838

No

89Cll 206B3 210H12 277B12 372F6 393B8 432D9

No

Yes Yes Yes

nd

No Yes No No No No

FISH signals at chromosomal 3p13-p14 3p13-p14 nd

3p13-p14plusD-q 3p14 3p14 nd nd D-P 3p12-p13pIus3p14 3p13-p14 3pl4plusC-q nd 3p13-p14 3pl4plusD-pplusC-q nd 3p13-p14 3p14plu~lp21phrslp31ph1sC-p plusC-qplusG/D-pa 3p14 2q22 3p13-p14plus2q23-q24 4q13 C-q

320 nd

3p14

nd 520 400 340 nd 320 400

3p13-p14 3p14 3p14plus5q 3p14 3p14 3p14 3p14

802Dl 821ClO 981Cll 322F8 729c2

Yes Yes

2000 480 2000 410 nd

D3S1406

324Fl

Yes

280

3p12-p13plusD-p

D3S3

376G4

nd

nd

nd

D3S1405

No No No

region

3p12-p13 3p12-p13 3p12-p13plus18 3p12-p13 3p12-p13plus3q27-q28plusB-q plus9q

Subregions are defined by breakpoints of a cell hybrid deletion mapping panel (described in Materials and Methods). The order of STSs within a subregion is unknown unless marked by arrows which point towards the centromere. Only those YACs are listed which were originally isolated with their corresponding STSs. Brackets connect identical YACs identified by different STSs. Freviously assigned loci [reference markers) are written in bold letters. R7K145 is a hybridization probe [27]. YACs used for double-color FISH are written in bold. Key to FISH signals: Localizations of distinct signals on 3p are described according to Mitelman (331. Localization of distinct signals on chromosomes other than chromosome 3 are indicated by the group of chromosomes (A-G) followed by the chromosome arm or more detailed information. Asterisks indicate diffuse painting of p-arms. Abbreviations:

nd, not determined;

STS, sequence-tagged

site.

Figure 2 FISH analysis revealing chimeric and non-chimeric YACs. A-C) Chimeric YAC 632E4: Biotinylated YACDNA was detected with FITC-conjugated avidin (green), chromosomes were stained with PI (red), and images were achieved using a confocal microscope. A) Complete metaphase spread showing two distinct signals on each chromosome 3, at 3p12-p13 and 3~14. The centromeres of chromosome 3 are marked by arrows. B) Same metaphase as in A, enlarged x 3.4. The hybridization sites at 3p12-p13 and 3~14 can be clearly seen (arrows). C) Interphase nuclei showing two signal pairs per nucleus. Each pair displays identical separation distances, giving evidence for physical linkage on the same chromosome. D-G) Non-chimeric YACs: Images were achieved using a FISH workstation and were recolored in order to reveal more precisely the positions of the signals, D) YAC signals were detected with FITC (blue-green) and the centromeres of chromosome 3 were painted with TRITC (red). YAC 705E8: Complete metaphase

spread and an interphase nucleus showing one distinct YAC signal at 3~21 on each homologous chromosome (see arrows). The right upper corner of the image shows both chromosomes 3, enlarged x 2. E-G) Double-color FISH was performed with YACs either detected with FITC or TRITC. E) 103F2 (FIX) [at 3~21) showing signals distal to 85OA6 (TRI’IC) (at 3~14). F) 169B5 (FIlC) mapping proximal to 171Bl (TRITC) (both at 3~14). G) 757HlO (FIX) overlapping 821ClO (TRITE). No order could be determined for this YAC couple.

S. C. Michaelis

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18.

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