Cytogenetics and Cell Genetics

56/1/91

/

S. Karger ' f Medical and Scientific I Publishers i Basel · M ü n c h e n I Paris · London

New York · New Delhi Bangkok · Singapore Tokyo · Sydney

KARG Ε R

Vol. 56, 1991

Cytogenetics and Cell Genetics

Founded 1962 as 'Cytogenetics' by H.P. Klinger

Editor-in-Chief H.P. Klinger Department of Genetics Albert Einstein College of Medicine 1300 Morris Park Avenue Bronx, N Y 10461, USA Tel: (212) 430-2451 FAX: (212) 430-2454 Bitnet: klinger @aecom.yu.edu

Managing Editor M . Kaelbling

(address as for H.P. Klinger, above)

Associate Editors and Sections Somatic cell genetics

D . Bootsma Department of Cell Biology and Genetics Erasmus Universitv P.O. Box 1738

Rotterdam, The Netherlands

U . Francke Department of Human Genetics Howard Hughes Medical Institute Beckman Center for Molecular and Genetic Medicine Stanford University Medical Center Stanford, CA 94305-5425, USA

Molecular cytogenetics H.J. Evans MRC Human Genetics Unit Western General Hospital, Crewe Road Edinburgh EH4 2XU, Scotland

Editors R. Berger, Paris G.A.P. Bruns, Boston, Mass. W.K. Cavenee, Montrea l , Quebec J. Cervenka, Minneapolis , M i n n . Z.-Y. Cheng, Beijing T. Cremer, Heidelberg K.E . Davies, Oxford C M . Disteche, Seattle, Wash. B.S. Emanuel, Philadelphia, Pa. R.E.K. Fournier, Seattle, Wash. S.M. Gartler, Seattle, Wash. T.W. Glover, Ann Arbor , M i c h . T J . Hassold, Atlanta, Ga. S. He im, Lund

Assistant Editors E.S. Geffner, Hunt ing ton , N .Y . R H . Geffner, Hunt ington , N .Y. B . M . O'Hara, Pearl River , N .Y .

Executive Editors Gene mapping, cloning, molecular genetics

T.B. Shows Department of Human Genetics Roswell Park Memorial Institute 666 Elm Street Buffalo, NY 14263, USA Tel: (716) 845-3108 FAX: (716) 845-8449 Bitnet: V129LQEW @UBVMSA

Human (including clinical) cytogenetics J.L. Hamerton Division of Human Genetics University of Manitoba School of Medicine 250-770 Bannatyne Avenue Winnipeg, Manitoba R3E 0W3, Canada

Genetic regulation of cell malignancy G. Kle in Department of Tumor Biology Karolinska Institute S-104 01 Stockholm 60, Sweden

Cytogenetics and molecular genetics of cancer, gene mapping

M . M . Le Beau Department of Medicine University of Chicago Joint Section Hematology/Oncology 5841 S. Maryland Avenue, Box 420 Chicago, I L 60637, USA

V. van Heyningen, Edinburgh G.P. Holmquis t , Duarte, Calif. R.S. Kucherlapati , Bronx, N . Y . L . M . Kunkel , Boston, Mass. P.A. Lalley, Det ro i t , M i c h . D . H . Ledbetter, Houston, Tex. M.F. Lyon, Oxon E. R. Magenis, Portland, Ore. J.-L. Mandel , Strasbourg P.J. McAlp ine , Winnipeg, Manitoba F. Mi te lman , L u n d R .K. Moyzis , Los Alamos, N . Mex. R .L . Nussbaum, Philadelphia, Pa. S. Pathak, Houston, Tex.

Editorial Secretary B. Callaci, Bronx, N.Y.

Molecular cytogenetics, gene mapping, somatic cell genetics, informatics P L . Pearson Department of Medicine and the Welch Medical Library The Johns Hopkins University 1830 East Monument Street Baltimore, M D 21205, USA Tel: (301) 955 9705 FAX: (301) 955 0054 Bitnet: PEARSON @ WELCH.JHU.EDU

Comparative cytogenetics O.J. Mi l l e r Program in Molecular Biology and Genetics 3216 Scott Hall of Basic Medical Sciences Wayne State University School of Medicine 540 East Canfield Avenue Detroit, M I 48201, USA

Gene mapping and cloning S.L. Naylor Department of Cellular and Structural Biology The University of Texas Health Science Center 7703 Floyd Curl Drive San Antonio, TX 78284, USA

D.J. Porteous, Edinburgh J.D. Rowley, Chicago, 111. M.S. Sasaki, Kyo to W. Schempp, Freiburg A . A . G . L . Schinzel, Zür ich Μ . Schmid, W ü r z b u r g D . Schweizer, Vienna G.R. Sutherland, Nor th Adelaide L.-C. Tsui, Toronto, Ontario D . Warburton, New York, N . Y J.J. Wasmuth, I rvine, Calif. A . Westerveld, Amsterdam

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© Copyright 1991 by S. Karger AG, P.O. Box, CH-4009 Basel (Switzerland) Printed in Switzerland on acid-free paper by Buchdruckerei Schüler AG, Biel

Contents Vol. 56,1991

No.l

Editor's Announcement Calendar o f future events I

Original Articles Aneuploidy induction in mice: construction and use of a tester stock

with 100% nondisjunction Beechey CV, Searle AG 2

No induction of interchromosomal effect in male meiosis of Chinese hamsters with reciprocal translocations Sonta S, Kitayama Κ 9

Analysis of whole-arm translocations in malignant blood cells by non-isotopic i n situ hybridization (With 1 color plate) Speleman F, Mangelschots K, Vercruyssen M , Dal Cin P, Aventin A, Offner F, Laureys G, Van den Berghe H , Leroy J 14

Composition and chromosomal localization of the small multigene family encoding mouse U3B nucleolar RNA Mazan S, Mattet M-G, Passage E, Bachellerie J-P 18

The Chinese hamster cell mutant V-H4 is homologous to Fanconi ane­mia (complementation group A) Arwert F, Rooimans MA, Westerveld A, Simons J W I M , Zdzie-nicka M Z 23

Painting of defined chromosomal regions by in situ suppression hy­bridization of libraries from laser-microdissected chromosomes Lengauer C, Eckelt A, Weith A, Endlich Ν , Ponelies Ν , Lichter Ρ, Greulich Κ Ο , Cremer Τ 27

Gene Mapping, Cloning, and Sequencing Localization of the human T-cell receptor gamma locus (TCRG) to

7p 14->p 15 by in situ hybridization Bensmana M , Mattet M G , Lefranc M-P 31

Localization of the dihydrolipoamide branched-chain transacylase gene (DBT) of the human branched-chain keto acid dehydrogenase complex to chromosome 1 Lau KS, Eddy RL, Shows TB, Fisher CW, Chuang DT, Cox RP . . 33

Animal Cytogenetics and Comparative Mapping Meiosis in a sterile male mouse with an iso Yq marker chromosome

de Boer P, de Jong JH, van der Hoeven FA 36 Α D N A sequence that is present in both sexes of Artiodactyla is

repeated on the Y chromosome of cattle, sheep, and goats Matthews ME, Reed KC 40

The high-resolution GTG-banding pattern of pig chromosomes Yerle M , Galman O, Echard G 45

The effect of heterochromatin on synapsis of the sex chromosomes of Peromyscus (Rodentia, Cricetidae) Hale DW, Hedin MC, Smith SA, Sudman PD, Greenbaum IF . . . 48

Localization of the gene encoding insulin-like growth factor I on mouse Chromosome 10 Taylor Β A, Grieco D 57

AM and HaelW restriction enzyme banding patterns of Macaca fuscata and Cercopithecus aethiops sabaeus chromosomes Stuppia L, Romagno D, Palka G, Guanciali Franchi P, Parruti G, Calabrese G, Bianchi U 59

Brief Report Synaptonemal complexes of the common shrew, Sorex araneus L. , in

spermatocyte spreads Borodin PM 61

Commentary A so-called rare heteromorphism of the human genome

Verma RS, Luke S, Conte RA, Macera MJ 63

Calendar of Events 1991-1992 64

No. 2

Original Articles Ring Y chromosome: molecular characterization by D N A probes

Pohlschmidt Μ, Rappold G, Krause Μ, Ahlert D , Hosenfeld D , Weissenbach J, Gal A 65

The synaptic sequence in hydroxyurea-treated spermatocytes of Pity-mys duodecimcostalus (Rodentia, Microtidae) Carnero A, Jimenez R, Burgos M , Sanchez A, Diaz de la Guardia R 69

A repeating unit of the DYZ1 family on the human Y chromosome consists of segments with partial male-specificity Nakagome Y, Nagafuchi S, Seki S, Nakahori Y, Tamura T, Ya-mada M , Iwaya Μ 74

Achiasmatic sex chromosomes in Pitymys duodecimcostatus: mecha­nisms of association and segregation Carnero A, Jimenez R, Burgos M , Sanchez A, Diaz de la Guardia R 78

Selective digestion of mouse chromosomes with restriction endonu-cleases. I I . X-ray microanalysis of / /fldll-treated chromosomes Gosälvez J, Mezzanotte R, Lopez-Fernändez C, Del Castillo P, Stocken JC, Goyanes V, Sumner AT 82

Random/nonrandom X-chromosome inactivation in Nesokia indica: possible influence of heterochromatin Thelma BK, Tewari R, Juyal RC, Rao SRV 87

Characterization of a B-lymphocyte t(2;14) (p l l ; q32) translocation from an ataxia telangiectasia patient conferring a proliferative advantage on cells in vitro Metcalfe JA, Heppell-Parton A, McConville C M , Taylor A M R . . 91

Gene Mapping, Cloning, and Sequencing The gene for mannose-binding protein maps to chromosome 10 and is

a marker for multiple endocrine neoplasia type 2 Schuffenecker I , Narod SA, Ezekowitz RAB, Sobol H , Feunteun J, Lenoir G M 99

Regional mapping of the parathyroid hormone gene (PTH) by cytoge­netic and molecular studies Tonoki H , Narahara K, Matsumoto T, Niikawa Ν 103

Epidermal type I transglutaminase (TGM1) is assigned to human chro­mosome 14 Polakowska RR, Eddy RL, Shows TB, Goldsmith LA 105

Chromosome 1 l q localization of one of the three expected genes for the human α-3-fucosyltransferases, by somatic hybridization Couill in P, Mollicone R, Grisard MC, Gibaud A, Ravisö Ν , Fein-gold J, Oriol R 108

Relationship of the human protooncogene CBL2 on l l q 2 3 to the t(4; 11), t( 11 ;22), and t( 11; 14) breakpoints Savage PD, Shapiro M , Langdon W Y , Geurts van Kessel A D , Seuanez H N , Akao Y, Croce C, Morse HC I I I , Kersey JH 112

Animal Cytogenetics and Comparative Mapping Heterochromatin and karyotype reorganization in Fish of the family

Anostomidae (Characiformes) Galetti P M Jr, Mestriner CA, Venere PC, Foresti F 116

Genetic mapping of the gene for androgen-binding protein/sex hor­mone-binding globulin to mouse Chromosome 11 Joseph DR, Adamson MC, Kozak CA 122

Mapping of the silver fox genes: assignments of the genes for M E 1 , A D K , PP, PEP A, GSR, M P I , and GOT1 Nesterova TB, Rubtsov NB, Zakian SM, Matveeva V G , Grapho-datskyAS 125

Commentary Sequence of the C H I and hinge-CH2 exons of the human immunoglob­

ul in IGHA2 A2m(2) allele: comparison with the nonallelic and allelic IGHA genes Bensmana M , Chuchana P, Lefranc G, Lefranc M-P 128

Calendar of Events 1991-1992 Loose flyer insert

IV Contents

No. 3-4

Original Articles Characterization of a de novo duplication of 1 lpl4—>pl3, using fluo­

rescent in situ hybridization and Southern hybridization (With 1 color plate) Speleman F, Mannens M , Redeker B, Vercruyssen M , Van Oost-veldt P, Leroy J, Slater R 129

Detection of trisomy 8 in hematological disorders by in situ hybridiza­tion (With 2 color plates) Kibbelaar RE, van Kamp Η, Dreef EJ, Wessels JW, Beverstock GC, Raap A K , Fibbe WE, den Ottolander GJ, Kluin PM 132

Genetic and molecular evidence of an X-chromosome deletion span­ning the tabby (Ta) and testicular feminization (Tfm) loci in the mouse Cattanach BM, Rasberry C. Evans EP, Dandolo L, Simmler MC, AvnerP 137

Molecular cytogenetics of α satellite DNA from chromosome 12: fluo­rescence in situ hybridization and description of DNA and array length polymorphisms Gre igGM, Parikh S, George J, Powers VE, Willard HF 144

Telomeric associations involving Y q l 2 in a lymphoblastoid cell line AbruzzoMA, Miller JA, Singer V L 149

Gene Mapping, Cloning, and Sequencing Deletion mapping of plasminogen activator inhibitor, type I

(PLANH1) and ß-glucuronidase (GUSB) in 7q2 l -»q22 Schwartz CE, Stanislovitis P, Phelan MC, Klinger K, Taylor HA, Stevenson RE 152

Somatic cell mapping of bovine clathrin light chain genes: identifica­tion of a new bovine synthetic group Gallagher DS, Threadgill DW, Jackson AP, Parham P, Womack JE 154

Localization of human tryptophan hydroxylase (TPH) to chromosome 11 ρ 15.3—>p 14 by in situ hybridization Craig SP, Boularand S, Darmon M C Mallet J, Craig IW 157

Chromosomal localization of the gene for AA-type platelet-derived growth factor receptor (PDGFRA) in humans and mice Hsieh C-L, Navankasattusas S, Escobedo JA, Williams LT, FranckeU 160

Assignment of ß-hexosaminidase A α-subunit to human chromosomal region 15q23-*q24 Nakai H , Byers M G , Nowak NJ, Shows TB 164

Regional assignment of the human gene coding for a multifunctional polypeptide (P4HB) acting as the ß-subunit of prolyl 4-hydroxylase and the enzyme protein disulfide isomerase to 17q25 Pajunen L, Jones TA, Goddard A, Sheer D, Solomon E, Pihlaja-niemi T, Kivir ikko K I 165

Chromosomal localization of the human proliferating cell nuclear anti­gen (PCNA) gene to or close to 20pl 2 by in situ hybridization Rao V V N G , Schnittger S, Hansmann I 169

Direct assignment of the human ßB2 and ßB3 crystallin genes to 22q 11.2—»q 12: markers for neurofibromatosis 2 Hulsebos TJM, Bijlsma EK, Geurts van Kessel A H M , Brakenhoff RH, Westerveld A 171

Localization of the human dihydrolipoamide dehydrogenase gene (DLD) to 7q31-»q32 Scherer SW, Otulakowski G, Robinson BH, Tsui L-C 176

Localization of histidase to human chromosome region 12q22—>q24.1 and mouse chromosome region 10C2-»D 1 Taylor RG, Garcia-Heras J, Sadler SJ, Lafreniere RG, Willard HF, Ledbetter D H , Mcinnes RR 1 78

Animal Cytogenetics and Comparative Mapping Chromosomal localization of the gene for late lactation protein (LLP)

in the tarn mar wallaby (Macropus engenii) Westerman M , Spencer J, Collet C 182

Mapping of silver fox genes: chromosomal localization of the genes for GOT2, A K 1 , ALDOC. A C P I , ITPA, PGP, and Β LVR Nesterova TB, Nikitina IV, Zakian SM, Rubtsov NB, Matveeva VG, Radjabli SI 185

The chromosomes of tufted deer (Elaphodus cephalophus) Shi L, Yang F, Kumamoto A 189

Assignment of the rat parathyroid hormone-like peptide gene (PTHLH) to chromosome 4: evidence for conserved synteny be­tween human chromosome 12, mouse chromosome 6, and rat chro­mosome 4 Szpirer C, Riviere M , Szpirer J, Hanson C, Levan G, Hendy GN . 193

Chromatin diminution and chromosome elimination in four Japanese hagfish species Nakai Y, Kubota S, Kohno S 196

Localization of the gene encoding myelin basic protein to mouse chro­mosome 18E3->4 and rat chromosome l p l 1—»pl2 Koizumi T, Katsuki M , Kimura M, Hayakawa J 199

The gene for retinal rod 33-kDa protein is on mouse chromosome 1, near Lamb2 Danciger M , Kozak CA, Abe T, Shinohara T. Farber DB 202

Homologs of eleven loci on human chromosome 11 assigned to two owl monkey chromosomes Ma NS-F, Gerhard DS. Bock SC 206

Brief Report High-resolution banding in chromosomes of Β lymphoblastoid cells

and cultured skin fibroblasts Kuwano A, Kajii Τ 212

Commentary Interphase cytogenetics reveals somatic pairing of chromosome 17 cen­

tromeres in normal human brain tissue, but no trisomy 7 or sex-chromosome loss Arnoldus EPJ, Noordermeer IA, Peters ACB. Raap A K , Van der PloegM 214

Abstracts of the Twenty-Ninth Annual American Cytogenetics Conference 217

Calendar of Future Events 1991-1992 231

Author Index 233 Subject Index 235

Cytogenetics and Cell Genetics

Founded 1962 as 'Cytogenetics' by H.P. Klinger

Editor-in-Chief H.P. Klinger Department of Genetics Albert Einstein College of Medicine 1300 Morris Park Avenue Bronx, NY 10461, USA Tel: (212) 430-2451 FAX: (212) 430-2454 Bitnet: klinger @ aecom.yu.edu

Managing Editor M . Kaelbling

(address as for H.P. Klinger, above)

Executive Editors Gene mapping, cloning, molecular genetics

T.B. Shows Department of Human Genetics Roswell Park Memorial Institute 666 Elm Street Buffalo, N Y 14263, USA Tel: (716) 845-3108 FAX: (716) 845-8449 Bitnet: V129LQEW @UBVMSA

Molecular cytogenetics, gene mapping, somatic cell genetics, informatics P.L. Pearson Department of Medicine and the Welch Medical Library The Johns Hopkins University 1830 East Monument Street Baltimore, M D 21205, USA Tel: (301) 955 9705 FAX: (301) 955 0054 Bitnet: PEARSON @ WELCH.JHU.EDU

Associate Editors and Sections Somatic cell genetics

D. Bootsma Department of Cell Biology and Genetics Erasmus University P.O. Box 1738

Rotterdam, The Netherlands

U . Francke Department of Human Genetics Howard Hughes Medical Institute Beckman Center for Molecular and Genetic Medicine Stanford University Medical Center Stanford, CA 94305-5425, USA

Molecular cytogenetics H . J . Evans MRC Human Genetics Unit Western General Hospital, Crewe Road Edinburgh EH4 2XU, Scotland

Human (including clinical) cytogenetics J .L . Hamerton Division of Human Genetics University of Manitoba School of Medicine 250-770 Bannatyne Avenue Winnipeg, Manitoba R3E 0W3, Canada

Genetic regulation of cell malignancy G . Klein Department of Tumor Biology Karolinska Institute S-104 01 Stockholm 60, Sweden

Cytogenetics and molecular genetics of cancer, gene mapping

M . M . Le Beau Department of Medicine University of Chicago Joint Section Hematology/Oncology 5841 S. Maryland Avenue, Box 420 Chicago, IL 60637, USA

Comparative cytogenetics O.J . Miller Program in Molecular Biology and Genetics 3216 Scott Hall of Basic Medical Sciences Wayne State University School of Medicine 540 East Canfield Avenue Detroit, M I 48201, USA

Gene mapping and cloning S.L. Naylor Department of Cellular and Structural Biology The University of Texas Health Science Center 7703 Floyd Curl Drive San Antonio, TX 78284, USA

Editors R. Berger, Paris G.A.P. Bruns, Boston, Mass. W.K. Cavenee, Montreal, Quebec J . Cervenka, Minneapolis, Minn. Z. -Y. Cheng, Beijing T. Cremer, Heidelberg K . E . Davies, Oxford C M . Disteche, Seattle, Wash. B.S. Emanuel, Philadelphia, Pa. R . E . K . Fournier, Seattle, Wash. S.M. Gartler, Seattle, Wash. T.W. Glover, Ann Arbor, Mich. T.J . Hassold, Atlanta, G a . S. Heim, Lund

Assistant Editors E .S . Geffner, Huntington, N.Y. P.H. Geffner, Huntington, N.Y. B .M. O'Hara, Pearl River, N.Y.

V. van Heyningen, Edinburgh G.P. Holmquist, Duarte, Calif. R .S . Kucherlapati, Bronx, N . Y . L . M . Kunkel, Boston, Mass. P.A. Lalley, Detroit, Mich. D . H . Ledbetter, Houston, Tex. M.F. Lyon, Oxon E . R. Magenis, Portland, Ore. J . - L . Mandel, Strasbourg P.J. McAlpine, Winnipeg, Manitoba F . Mitelman, Lund R . K . Moyzis, Los Alamos, N . Mex. R . L . Nussbaum, Philadelphia, Pa. S. Pathak, Houston, Tex.

Editorial Secretary B. Callaci, Bronx, N.Y.

D . J . Porteous, Edinburgh J .D . Rowley, Chicago, 111. M.S. Sasaki, Kyoto W. Schempp, Freiburg A . A . G . L . Schinzel, Zürich Μ. Schmid, Würzburg D. Schweizer, Vienna G . R . Sutherland, North Adelaide L . - C . Tsui, Toronto, Ontario D . Warburton, New York, N.Y. J .J . Wasmuth, Irvine, Calif. A. Westerveld, Amsterdam

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Cytogenetics and Cell Genetics

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Cytogenet Cell Genet 56:27-30 (1991) © 1991 S. Karger AG, Basel

0301 0171191 /0561 -0027 S 2.75/0

Painting of defined chromosomal regions by in situ suppression hybridization of libraries from laser-microdissected chromosomes C. Lengauer,1 A. Eckelt,2 A. Weith, 3 N . Endlich, 2 Ν. Ponelies,2 P. Lichter, 4 Κ . Ο . Greulich, 2 and Τ. Cremer 1

1 Institut für Humangenetik und Anthropologie, Universität Heidelberg, Heidelberg (FRG) ; 2 Physikalisch Chemisches Institut, Universität Heidelberg, Heidelberg (FRG); • Deutsches Krebsforschungszentrum, Institut für Experimentelle Pathologie, Heidelberg (FRG); and 4 Department of Human Genetics, Yale University School of Medicine. New Haven, CT (USA)

Abstract. "Painting" of defined chromosomal regions provides a powerful tool for cytogenetic analyses. Here, we demonstrate that chromosomal in situ suppression (OSS)-hybridization of DNA libraries derived by microcloning laser-microdissected chro­mosomal regions can be applied to achieve this goal. As an exam­ple, we used unhanded metaphase spreads from a female patient carrying a balanced translocation, t(l;7)(lqter->lp36::7ql 1 7qter). Fragments from the long arms of 130 translocation chromosomes were microdissected. After microcloning, human inserts with an average size of about 3 kb were pooled from 400

recombinant bacteriophage DNA clones and used as a complex probe set in CISS-hybridization experiments. This resulted in painting of the translocation chromosome along the region 7q35 to lp31. Painted chromosomal subregions in normal chromo­somes 1 and 7 were consistent with this finding. This approach may be used to perform painting of any chromosome regions for which microlibraries can be established. Possible applications include the definition of marker chromosomes in clinical and tumor cy­togenetics and studies of chromosomal evolution, as well as studies of nuclear chromosome topography in animal and plant species.

Chromosomal in situ suppression (ClSS)-hybridization of bio-tinylated libraries from sorted human chromosomes, in conjunc­tion with fluorescent or colorimetric detection procedures, has provided a new tool for the specific "painting" of complete in­dividual chromosomes in both mitotic and interphase cells (Lich­teret al., 1988a, b; Pinkel et al., 1988). CISS-hybridization has also been useful for the mapping of genomic DNA sequences (e.g., cloned in cosmid vectors) which contain interspersed repetitive sequences, such as A/u sequences, in addition to chromosome site-specific sequences (Landegent et al., 1987; Lichter et al., 1990a, b). Still, the generation of complex probe sets for the selective staining of desired chromosomal subregions, such as chromosome arms or chromosome bands, has remained a time-consuming task. In ad­dition, the lack of sorted chromosome libraries from nonhuman species has, until now, constituted another limiting factor in the general use of the CISS-hybridization technique.

This study was carried out to investigate whether probe sets useful for the painting of chromosomal subregions could be estab­lished by microcloning of laser-microdissected chromosomes. As a model case, a marker chromosome (lqter—• 1 p36::7q 11 —>7qter) (Fig. 1) from a healthy female carrying a balanced translocation, t(l;7)(p36;ql 1), was microdissected using an excimer laser micro-beam. Chromosomes were not stained to facilitate microcloning procedures, but the marker chromosome could be easily recog-

Supported by a grant (Cr 59 10-1) from the Deutsche Forschungsgemeinschaft. C . L . is a recipient of a scholarship from the Konrad-Adcnauer-Stiftung; T .C . is a recipient of a Heisenberg Stipendium from the Deutsche Forschungsgemeinschaft.

Received 6 April 1990: accepted 24 July 1990.

Request reprints from Dr. T. Cremer. Institut für Humangenetik and Anthropologie, Uni­versität Heidelberg. Im Neuenheimer Feld 328, D-6900 Heidelberg (FRG) .

nized as the largest chromosome of the complement. Laser mi­crodissection was aimed to include the region 7q22->32 (Eckelt et al., 1989). Accordingly, two laser cuts were performed, one to re­move the chromosome 1 material and the other to remove the distal region of 7q. In the absence of banding, it was difficult to reproduce the localization of the laser cuts precisely in marker chromosomes that had undergone various amounts of condensa­tion. While a preliminary investigation of the resulting microli-brary indicated the presence of clones from the desired region (Eckelt et al., 1989), CISS-hybridization should answer the ques­tion of the actual extent of the microdissected region.

Materials and methods Cell material. Phytohemagglutinin (PHA)-stimulated human lymphocyte

cultures were established from healthy (46,XY; 46,XX) donors. Metaphase spreads were prepared for subsequent use in CISS-hybridization experiments (see below) using standart procedures and stored at 4 C. For laser microdissec­tion, Epstein-Barr virus-transformed cells of a female patient carrying a bal­anced translocation. t(l;7)(p36;ql 1), were kindly provided by K. Miller (Medizinische Hochschule Hannover) (Eckelt et al., 1989). Metaphase spreads were prepared and fixed as described by Martinsson et al. (1989).

Laser microdissection. The details of the procedure have been described pre­viously (Monajembashi et al., 1986; Eckelt et al., 1989; Ponelies et al., 1989). Briefly, pulses of an excimer pumped dye laser (Lambda Physik, Göttingen, FRG), at a repetition rate of about 10 Hz, were directed into an inverted I M 35 microscope via the fluorescence illumination path. In the focus, power densities of up to 10" W/cnr were obtained. Laser cutting of the translocation chro­mosome, t( 1 ;7)( 1 qter-> 1 p36::7q 11 -*7qter), was achieved by moving the marker chromosome at preselected sites through the laser focus. A total of 130 frag­ments were collected for microcloning.

Microcloning. Microcloning was performed essentially as described by Scalenghe et al. (1981) and Martinsson et al. (1989), with several modifications described below. Micromanipulation work was done in a paraffin oil-filled

28 Lengauer/Eckelt/Weith/Endlich/Ponelies/Lichter/Greulich/Cremer

charmer under a phase-contrast microscope. After laser dissection, fragments were aken up by a glass needle, using a de Fonbrune micromanipulator, and transferred into a 0.6-nl aqueous droplet contaning 1 mg/ml proteinase Κ and 0.2% SDS. After digestion for 2 h at 55 °C, proteins in the microdrop were extracted three times with four volumes of phenol saturated with TE buffer, folloved by a wash with 0.5 μΐ of chloroform. Restriction digestion was carried out ina 1-nl reaction volume containing 10 m M Tris HCl (pH 7.5), 5 m M NaCl, 0.5 mvl MgCl 2 , and 280 units/μΐ EcoKl. After incubation for 2 h at 37 C, the enzyne was removed by extraction with phenol. The digested D N A was ligated for 2t h at 12°C to 0.56 ng £coRI-cut dephosphorylated λ phage N M 1149 DNA (Murray et al., 1983), using 0.1 units T4 ligase in a total volume of 2.4 nl of ligition buffer (50 mM Tris HCl [pH 7.5], 10 m M MgCl 2 , and 1 m M ATP). The lgation product was packaged in vitro as described by Scherer et al. (1981). Phages were plated on plates with the h f l " mutant NM514 as bacterial host (Murray et al., 1983).

A total of 450 recombinant phage clones from the microlibrary (containing 478 cones) were picked onto LB-agar plates with an overlayer of 3 ml of soft agar ;ontaining the NM514 bacterial host and incubated at 37 C overnight. Plaqie hybridization was carried out with 32P-labeled, sheared human genomic DNA (Church and Gilbert, 1988). After autoradiography, 230 (51 % ) of the 450 phagts showed signals of varying intensity, indicating the presence of highly or modtrately repetitive human sequences. Clones from the remaining fraction were lot further tested individually but would likely contain single or low-copy hurrun sequences. The average insert size of 22 randomly picked clones (11 k,sin^e copy" clones and 11 clones containing highly repetitive sequences) was 3kb range, 0.55-8.2 kb).

ENA probes and preparation. As a probe set for CISS-hybridization experi­ments, 400 recombinant phages from the microcloned phage-DNA library were pooled at random and amplified in liquid culture using E. coli LE 392 as bacterial host. Purification of the phages and extraction of phage DNA were carried out as deicribed by Maniatis et al. (1982). After digestion with EcoKU human inserts were recovered from a 0.8% agarose gel by electroelution into dialysis bags and conctntrated with /i-butanol (Maniatis et al., 1982).

Flasmid DNA clone pu7tl containing the alphoid sequence specific for hu­man jhromosome 7 was established by Waye et al. (1987) and kindly provided by Ptter Devilee (Leiden). Plasmid transformation and DNA preparation were carried out following standard procedures (Birnboim and Doly. 1979).

frobe labeling, CISS-hybridization, and probe detection. Plasmid D N A pct7ti and human inserts from the pooled phages of the microlibrary established from the microdissected chromosome region were labeled with biotin-11 dUTP by nek translation (Langer et al., 1981). CISS-hybridization and detection of hybrdized probes with fluorescein isothiocyanate (FITC)-labeled avidin were performed as described in detail by Lichter et al. (1988a). One cycle of signal ampification was performed as described by Pinkel et al. (1986). Preparations were counterstained with 0.2 μg/ml 4,6-diamidino-2-phenylindole-dihydro-chloide (DAPI) and 1 μg/ml propidium iodide and mounted in fluorescence antitiding buffer (1 mg /7-phenylenediamine in 1 ml of glycerin buffer, pH 8.0). Celh were evaluated with a Zeiss Photomicroscope I I I equipped for epifluores-cenci. Photographs were taken on Agfachrome 1000 RS color slide film. Alter­natively, digitized images were obtained using a laser-scanning confocal micro­scope as described by Lichteret al. (1990b). In the latter case, photographs were taken from a video monitor.

Results

Figure 2a (left side) shows the marker chromosome, t(l;7)(lqter->lp36::7qll->7qter), after painting with the probe set derived from the microlibrary. Fractional length measure­ments (Lichter et al., 1990b) of 10 marker chromosomes, with the telomere of the long arm and the centromere serving as bound­aries, made it possible to determine the extension of the painted region from 7q35 to lp31 (Fig. 1). This result indicates that the miaocloned chromosome fragments frequently included chromo­some lp3 material in addition to 7q material. Consistent with this conclusion was the observation in normal chromosomes 1 that lp3 was painted except for the telomeric region (Fig. 3a). This finding

Fig. 1. Schematic drawing of the translocation chromosome, t(l;7)(lqter->lp36::7ql 1 ->7qter). The bracket on the right indicates the exten­sion of the painted region, as obtained by fractional length measurements of 10 marker chromosomes (see text).

Fig. 2. CISS-hybridization of a biotinylated D N A library derived by micro-cloning of a laser-microdissected chromosomal region (see text) to a metaphase spread (a, b) and interphase nuclei (c) from PHA-stimulated lymphocytes of a female patient with a t(l;7)(p36:ql 1). (a) Translocation chromosome t( 1 ;7)( 1 qter—• 1 p36::7q 11 ->7qter), after detection of biotinylated sequences with avidin-FITC, demonstrates the specific staining of the microcloned chro­mosome region. In an adjacent normal chromosome 7 (right), the long arm is specifically painted. Chromosomes are counterstained with propidium iodide, (b) The same chromosomes counterstained with D A P I . The arrow indicates the border between the painted and nonpainted chromosome regions, (c) Three distinctly stained domains in interphase nuclei from the same experiment. The largest domain likely represents the painted region of the translocation chromo­some, the medium-sized domain the painted long arm of the normal chromo­some 7. and the small domain the painted part of the short arm of the normal chromosome 1 contained in these nuclei.

CISS-hybridization o f libraries from microdissected chromosomes 29

Fig. 3. Typical, enlarged normal chromosomes 1 (a) and 7 (b, c) after CISS-hybridization of the microcloned library, detection with avidin-FITC, and counterstaining with propidium iodide. In a and b the chromosomes were photographed directly from the video monitor after laser-scanning confocal microscopy and digital image enhancement; the photograph in c was taken with a Zeiss Photomicroscope I I I equipped for epifluorescence. In a, specific labeling of chromosome 1 extends from the subtelomeric region to the middle of the short arm. Note that the telomeric region is not labeled (arrow). The chromosome 7 in b demonstrates a banded staining pattern on 7q. Three fluorescent bands are also apparent on the prometaphase chromosome 7 in c (small arrows). The centromeric region of this chromosome is strongly stained by simultaneous in situ hybridization of the alphoid probe pa7t l .

is in accordance with the absence of sequences from this region in the marker chromosome. In normal chromosomes 7 (see Fig. 2a, right side, and Fig. 3b for examples) a region of the long arm (from 7ql 1 to 7q35, according to fractional length measurements) was specifically stained. This chromosome was further identified by simultaneous hybridization of the probe set with an alphoid probe specific for the chromosome 7 centromeric region (Fig. 3c). Paint­ing was most prominent over the middle and proximal parts of 7q (Fig. 2a). This result is in agreement with the fact that, in the mi­crodissected marker chromosomes, material close to the end of the long arm was cut off. Notably, three fluorescent bands were ob­served in more extended painted chromosomes 7 (Fig. 3b, c). Dig­ital imaging microscopy using a laser-scanning confocal micro­scope facilitated the analysis of the hybridized chromosomes. In this way, the signal-to-noise ratio could be enhanced by optical filtering and image processing, while nonspecific background could be removed.

The conditions used for CISS-hybridization in the present ex­periments were optimized for chromosome painting in metaphase spreads but not in interphase nuclei. Still, some nuclei from cells of the translocation carrier clearly showed three distinctly stained domains (Fig. 2c), representing the painted regions of both the marker chromosome and the two normal chromosomes 1 and 7.

Discussion

In this study we demonstrate that a microlibrary established from laser-microdissected fragments of a translocation chromo­

some, t(l;7)(lqter->lp36::7qll->7qter), is sufficient for specific painting of the microdissected chromosome region. In addition to most of the long arm of the normal chromosome 7, a region of the short arm of the normal chromosome 1 (lp3, except for the telomeric region) was also painted, proving that the microdissected fragments included material from both chro­mosomes.

The painted region is estimated to include 100-150 Mb of DNA from the marker chromosome—equivalent to the length of a medium-sized chromosome of the human complement. Pro­vided that all 400 microlibrary clones used for probe preparation carry human inserts from different sites of the microdissected frag­ments, the maximum complexity of sequences contained in the probe pool could equal up to 1.2 Mb of DNA, which might possi­bly include some 600 kb of unique sequences. The actual complex­ity of the probe may be lower, however, depending on the number of clones that carry identical inserts.

The banded pattern seen on the long arms of more extended chromosomes 7 after CISS-hybridization of the microlibrary was observed, although Alu banding of unpainted chromosomes due to the presence of biotinylated Alu sequences in the probe set was efficiently suppressed. The presence of some clustered, 7q-specific, repetitive sequences may provide a possible explanation. Other explanations, such as a chance spatial clustering of the cloned se­quences in the microlibrary or a nonuniform distribution of EcoRl

restriction sites (used for digestion of the microdissected DNA) along 7q, may be taken into consideration. Notably, such a band­ed pattern was not obvious in CISS-hybridization experiments using a library from sorted chromosomes 7 where the DNA se­quence complexity of this chromosome was represented much more completely (Cremer et al., 1988; Lichter et a l , 1988a).

We expect that the approach exemplified here will open intrigu­ing new possibilities in basic and applied cytogenetics. These pos­sibilities include the evaluation of unidentified marker chromo­somes or translocated chromosome material contained, for exam­ple, in tumor cells. Furthermore, microlibraries derived from in­dividually collected complete or microdissected chromosomes may provide a universal tool for chromosome painting in any de­sired species. Recently, the elegant work of Lüdecke et al. (1989) showed that banded chromosomes can be used for microcloning. Since the collection of banded chromosomes by micromanipula­tion can be precisely controlled (Senger et al., 1990), impurities in a microlibrary caused by undesired chromosomes or fragments can be avoided. In contrast, libraries from sorted chromosomes may not only be difficult to achieve for many species, but even the best sorts inevitably contain a certain fraction of unwanted chromosomes or fragmented chromosome pieces.

At present, microdissection and microcloning of chromosomes are highly specialized procedures which may not be easy to estab­lish in all clinical cytogenetic laboratories. We believe, however, that probe sets useful for the painting of chromosome arms and bands, once they are established in a few specialized laboratories, could provide an important service to the cytogenetic community in general. Furthermore, polymerase chain reaction-based meth­ods, when used to amplify DNA from microdissected chromo­some material, have the potential to facilitate or even replace elaborate microcloning procedures (Nelson et al., 1989; Ledbetter et al., 1990; Lengaueretal., 1990).

30 Lengauer/^ckelt/Weith/Endlich/Ponelies/Lichter/Greulich/Cre^

Acknowledgements

We thank Dr. David C. Ward for his generous support, Dr. Lore Zech for helpful comments, Anna Jauch and Susanne Popp for helpful discussions of the

methodological aspects of this study. Dr. Konstantin Miller for providing cell cultures from the female proband carrying the translocation, and Ulrike Bauder and Angelika Wiegenstein for excellent technical assistance and photographic work.

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