Cosmid-derived transcripts and sequence tags mapped to three subregions of human chromosome 22

GENE AN INTERNATIONAL. OOURNAL GENES AND GENOMES

ELSEVIER

ON

Gene 183 (1996) 29-33

Cosmid-derived transcripts and sequence tags mapped to three subregions of human chromosome 22 Carsten Pusch

a ,*

Roman Mtillenbach a ,b, Peter GOtt a, Holger Schmitt a, Zhili Wang c, Bruce Roe c, Nikolaus Blin"

a Division of Molecular Genetics, University of Tiibingen, Wilhelmstr. 27, 72074 Tiibingen, Germany b Institute of Child Health, Oncology, ICRF, London WC1N1EH, UK Department of Biochemistry, University of Oklahoma, Norman, OK 73019, USA Received 21 November 1995; revised 10 April 1996; accepted 10 May 1996

Abstract

Fifty cosmids from the ICRF, London, and Lawrence Livermore Laboratory, California, human chromosome 22 cosmid libraries were isolated, regionally assigned and tested for their ability to detect repeats or single copy sequences. The search resulted in nine cosmids containing repetitive motifs from the pericentric region of chromosome 22. An additional 19 cosmids, that detected single copy sequences in the long arm of chromosome 22q: 7 in the region 22qll.2-q13.1 and 12 in 22q13.1-qter, were mapped more precisely by fluorescence in situ hybridization. Three out of these 19 recombinants displayed restriction fragments containing (CA), repeats, were subcloned and sequenced. One cosmid, representing a region coding for an ubiquitous 300-bp transcript, is localized 600 kb from PDGFB, and four cosmids contained sequences surrounding the ARSA gene at 22q13.3. Presently, long range physical maps, that may be useful for analysing structural alterations of chromosome 22q13, are being constructed from these additional, regionally assigned markers from chromosome 22q13 employing both existing cosmid and new bacterial artificial chromosome (BAC) libraries. Keywords: Cosmids; Genomic sequencing; In situ hybridization; Transcript mapping; STS

1. Introduction

During the past years structural alterations of chrom o s o m e 22 were linked to a series of tumorigenic disorders, the first and, by now, a historical example being the Philadelphia chromosome. Meningioma, NF2, Ewing's sarcoma and soft tissue clear cell sarcoma m a y serve as additional examples. More recently, candidate genes have been identified for these neoplasms by means of positional cloning (Trofatter et al., 1993; Delattre et al., 1992). In addition, several congenital syndromes, e.g. C A T C H 2 2 and cat eye were linked to chromosome 22 abnormalities (for review, see Scambler, 1994). In two N F 2 patients, ring formation of chromosome 22 accompanied by deletions of the A R S A locus in 22ql 3.3 * Corresponding author. Tel. +49 7071 2976401; Fax +49 7071 296409. Abbreviations: NF2, neurofibromatosis type 2; PCR, polymerasechain reaction; PDGFB, platelet-derived growth factor beta chain; STS, sequence tagged site. 0378-1119/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0378-1119(96)00411-8

was reported ( T o m m e r u p et al., 1992). An additional r(22) in a N F 2 case, however, does not seem to involve the region 22q13.3 (Schmitt et al., 1994). A set of 7 patients showing developmental delay and facial dysmorphism was noted to display 22q 13 deletions of varying extension (Nesslinger et al., 1994). Despite numerous efforts to elucidate molecular events in all these deficiencies related to chromosome 22, most of the present knowledge remains focused on the region 2 2 q l l - q 1 2 , leaving sizable gaps in the genetic m a p of 22q13 ( D u m a n s k i et al., 1991; Riley et al., 1994; Collins et al., 1995). Some of the most recent maps, although applying numerous STS markers, do not even cover the distal region in 22q13.3 (Riley et al., 1994; G a y p a y et al., 1994). A new collection of cosmid markers from chromosome 22 was published recently (Kurahashi et al., 1994). Out of 108 cosmids localized, 33 m a p p e d within the 22ql3.2-qter region but neither contig data nor in situ assignment was presented. In a more recent experiment, an additional set of STS markers was regionally assigned ( H u d s o n et al., 1995). In this present

30

c Pusch et al./Gene 183 (1996) 29 33

study, we have m a p p e d b y a p a n e l o f s o m a t i c cell h y b r i d s 28 new c o s m i d s to three s u b r e g i o n s o f c h r o m o s o m e 22. T h e y were f u r t h e r assigned b y fluorescence in situ h y b r i d i z a t i o n . T h e i r restriction f r a g m e n t s were tested for presence o f ( C A ) , r e p e a t s a n d expressed sequence tags.

2. Experimental and discussion 2.1. Selection o f recombinants R e c o m b i n a n t clones were selected f r o m the L a w r e n c e L i v e r m o r e , C a l i f o r n i a , c h r o m o s o m e 22 c o s m i d collection for c e n t r o m e r i c repetitive sequences. A d d i t i o n a l l y , a b o u t 50 c o s m i d s f r o m a s e c o n d c h r o m o s o m e 22 c o s m i d l i b r a r y (established at the I C R F , L o n d o n ) were r a n d o m l y picked. O u t o f these, 28 were r e g i o n a l l y assigned to c h r o m o s o m e 22 b y using g e n o m i c D N A f r o m a r o d e n t / h u m a n s o m a t i c cell h y b r i d panel. These cell lines contain either the complete chromosome 22 ( P g M e - 2 5 N u ) o r its following fragments: 22pter-q13.1 ( 1 / 2 2 A M - 2 7 ) ; 22q12.1-qter ( A 3 E W - 6 A ) . H y b r i d i z a t i o n o f D N A filters with new r e c o m b i n a n t s allows a r e g i o n a l differentiation o f the l o n g a r m o f c h r o m o s o m e 22: 2 2 q l l . l - q 1 2 . 1 ; 22q12.1-q13.1; 22q13.1-qter. A similar a p p r o a c h was used for a newly c o n s t r u c t e d c o s m i d l i b r a r y f r o m c h r o m o s o m e 22 ( J a p a n e s e C a n c e r R e s e a r c h R e s o u r c e s Bank; K u r a h a s h i et al., 1994). All cosmids, due to their specific restriction p a t t e r n s achieved by EcoRI, B a m H I a n d HindIII ( d a t a n o t shown), a p p e a r e d to r e p r e s e n t u n i q u e r e c o m b i n a n t clones.

2.2. Assignment o f pericentric repetitive sequences O u t o f the n u m e r o u s c o s m i d s c a r r y i n g repetitive sequences p r e s e n t in the pericentric region o f c h r o m o s o m e 22, nine were c h a r a c t e r i z e d in m o r e detail b y filter a n d fluorescence in situ h y b r i d i z a t i o n ( T a b l e 1). F i v e o f t h e m c o n t a i n e d the a l p h a satellite r e p e a t specified by the locus D 2 2 Z 2 ( M c D e r m i d et al., 1986). In three a d d i t i o n a l cosmids, a 48-bp r e p e a t m o t i f ( D 2 2 Z 3 ) was present. T h e sequence c o m p o s i t i o n o f the r e m a i n i n g c o s m i d s requires a closer analysis, since it carries repetitive sequences which are n o t limited to c h r o m o s o m e 22 b u t are p r e s e n t in all a c r o c e n t r i c c h r o m o s o m e s , as d e m o n s t r a t e d b y in situ h y b r i d i z a t i o n ( F i g . 1).

2.3. Detection o f expressed sequences A n a d d i t i o n a l g r o u p o f 19 c o s m i d s also was studied. Here, 7 were assigned to 22q12.1-q13.1 a n d 12 to 22q13.1-qter b y h y b r i d i z a t i o n to the s o m a t i c cell h y b r i d p a n e l d e s c r i b e d a b o v e ( T a b l e 1), a n d in several instances, also b y fluorescence in situ h y b r i d i z a t i o n (see Fig. 1). T h e g e n o m i c D N A c o n t a i n e d within c o s m i d

Table 1 Mapping and characterization of 25 chromosome 22-specific cosmids Cosmid

Chromosomal region

FISH localization

Containing

AN14G6 + BN6C 10 + CN4F7 + C0221 H0983 A1048 B0198 C0731 D27E7 A0267 C0116 D0932 Cl155 E0551 H0861 G0943 A061 A1071 A1171 C033 C0290 C0297 D0748 D9H45 Dl150 E027 E1298 G0515

22pter-ql 1.2

22cen 22cen

22pter-q 11.2 22pter-ql 1.2

22cen 22cen

s-satellite ~-satellite ~-satellite ~-satellite ~-satellite D22Z3 D22Z3 D22Z3

(CA),

22pter-ql 1.2 all acrocentric 22q12.1-q13.1 22q12.1-12.3 22q12.1-qlYl 22q12.1-q13.1 22q12.1-q13.1 22q12.l-q13.1 22q12.1-q13.1 22q13.1-qter 22q13.l-qter 22ql 3.1-qter 22q 13.1-qter 22q13.1-qter

22q13.1 22q13.1

(,) D22S16

+ -

22q13.3 22q13.3 22q13.3

ARSA ARSA

-

22q13.2 22q 13.3

ARSA

22ql 3.1-qter 22q13.1-qter

22ql 3.3 22q13.3 22q 13.1-q 13.2

ARSA

+ + -

22ql 3.1-qter 22q13.1-qter

Next to their assignment to a chromosomal subregion by a somatic cell hybrid panel, in situ hybridization was performed. Demonstration of the presence of known sequences (s-satellite, ARSA, D22S16, D22Z3) was achieved by filter hybridization. Cl155 was previously mapped close to PDGFB (*) by pulsed field gel electrophoresis. Methods: The ICRF cosmid library established in London was provided by H. Lehrach, the Lawrence Livermore library was obtained from P. Scambler, London. Both collections are available on gridded filters representing the entire chromosome 22 and were processed as published previously (Schmitt et al., 1994). Clones from the Lawrence Livermore library are marked with (+). For assigning recombinants to chromosome 22 subregions, genomic DNA from somatic cell hybrids was applied for filter hybridizations using PgMe-25Nu, 1/22AM-27 and A3EW-6A cell lines obtained from A. Geurts van Kessel, Nijmegen, The Netherlands. DNA isolation, filter hybridization conditions and mapping were reported previously (Herzog et al., 1991). The following probes were used: D22Z2 (McDermid et al., 1986), D22Z3 (Metzdorf et al., 1988), D22S16 (G6ttert et al., 1989), CP8 (for arylsulfatase; Stein et al., 1989). The DNA probes were labeled using a random primed DNA labeling kit (Boehringer Mannheim). C 1155 c o d e d for an u b i q u i t o u s l y expressed t r a n s c r i p t o f 300 bp, t h a t also was present in R N A derived from v a r i o u s h u m a n a n d r o d e n t cells ( F i g . 2). The sequence o f this t r a n s c r i p t was derived f r o m a h u m a n p a r t i a l c D N A ( s u b m i t t e d by G e n e t h o n to E M B L d a t a b a s e , accession No. Z41097) f o u n d in C l 1 5 5 ( T a b l e 2 ) a n d c o n f i r m e d by P C R using flanking p r i m e r s i n d i c a t e d in Table 2. Previous pulsed field gel e l e c t r o p h o r e s i s p l a c e d

31

C Pusch et al./Gene 183 (1996) 29-33

described cosmids as well as new BAC recombinants (Kim et al., 1994; Schmitt et al., 1996) and additional 22q13 markers, some of which were linked in a small cosmid contig (Xie et al., 1994), should fill several gaps which remain in this region of the present maps of chromosome 22 (Bell et al., 1995; Collins et al., 1995). Together, these mapped cosmid and BAC collections still constitute valuable resources for the isolation of new genes, e.g. by exon trapping.

2.4. Assignment of sequence tags Fig. 1. Non-isotopic in situ hybridization of human metaphase chromosomes with cosmid D27E7 (10 centromeric signals). Biotinylated cosmid probes were labeled by nicktranslation (BRL bionick kit) and hybridized in presence of competitor DNA (cotl; Gibco-BRL). The fluorescence signals (biotin/FITC) were detected as reported previously (Schmitt et al., 1994) using a Leitz DMRB46 microscope equipped with corresponding fluorescence filters. An assignment was considered unambiguous after evaluating 30 mitoses and observing > 90% of all chromosomes 22 with clearly distinguishable signals.

E. coli; rat liver; i

rat kidney; Caco-2; Capan- 1;

D

MCF7;

Finally, EcoRI, BamHI and HindlII restriction fragments from all of the above cosmids were tested for presence of microsatellite clusters formed by the repetitive motif (CA),. Three out of 19 cosmids hybridized with the (CA)12 probe (Fig. 3B). Ten of the hybridizing subfragments obtained by the Tsp509(I) restriction endonuclease were subcloned for further sequence analysis and three sequences are examplified in Table 3. Our present cosmid collection covers approximately 1 Mb of chromosome 22. These as well as the new STSs will aid in establishing regional contigs, as outlined for the ARSA region and already have been used in mapping a new translocation breakpoint, t(17;22) in a dermatofibrosarcoma (Pedeutour, Nice, personal communication). Finally, these additional chromosome-22-specific markers will be valuable for mapping other, newly discovered chromosome 22 specific rearrangements, such as those recently reported in the ARSA region that might be involved in idiopathic mental retardation (Flint et al., 1995).

Y- 1.

I

1

2

3

4

5

6

t h e C 1 1 5 5 s e q u e n c e at a d i s t a n c e o f a b o u t 600 k b f r o m the locus PDGFB ( B l i n et al., 1993). T h e c o s m i d s A1071, A1171, D1150 and C0297 encompass the arylsulf a t a s e g e n e ( A R S A ) , p r e s e n t l y t h e m o s t distal l o c u s in 22q and constitute a regional contig covering sequences s u r r o u n d i n g this g e n e ( d a t a n o t s h o w n ) . T h u s , t h e a b o v e

8

9

10 2000 bp

0.3 kb Fig. 2. Expression of a 300-bp transcript encoded by C1155. RNA was separated by denaturing gel electrophoresis, blotted to support filters and hybridized with labeled C 1155 DNA. For Northern blots, standard membranes and techniques were applied as published previously (Blin et al., 1993). The channels represent total RNA from, from top to bottom: E. coli; rat liver; rat kidney; human colon carcinoma cell line Caco-2; human pancreas carcinoma cell line Capan-1; human mammary carcinoma cell line MCF7; mouse adenoma cell line Y-l, all cell lines available through ATCC (Rockville, MD, USA). The quality of RNA and efficiency of electrophoretic separation and blotting were controled by ethidium bromide staining of the blotting membrane.

7

900 bp 470 bp

w

220 bp

80 bo Fig. 3. DNA filter hybridization and autoradiographic detection of hybridizing bands. Original EcoRI fragments were restricted by Tsp509(I) and hybridized with the (CA)12 repeat. Lanes: 1-3, fragments from cosmid C1155; 4-6, fragments from cosmid D0748; 7-10, fragments from cosmid D9H45. The sizes are given in base pairs (bp).

32

C. Pusch et al./Gene 183 (1996) 29-33

Table 2 Genomic DNA sequence from C1155 coding for an anonymous cDNA and resulting in an ubiquitous transcript shown in Fig. 2 1

agncgtttaa

tgctgtacac

aggtgtcCCT

CTTCATTTCG

ACCTTTATTA

tcngcaaatt

acgacatgtg

tccacagGGA

GAAGTAAAGC

TGGAAATAAT

GTGACTTGAA

TGAGGCTATT

CAAGTTTGCA

GATACATAAA

GCTCTAAAGG

CACTGAACTT

ACTCCGATAA

GTTCAAACGT

CTATGTATTT

CGAGATTTCC

AGAGCTAAGT

TCTAAAAAGT

TTAATACAGG

TGGGGAAATG

GGTCAAACCC

TCTCGATTCA

AGATTTTTCA

AATTATGTCC

ACCCCTTTAC

CCAGTTTGGG

AACAAGGTGA

GATCTGTTAA

AGATCAGTGT

AAAGTCCTGC

TTTTATCTAA

TTGTTCCACT

CTAGACAATT

TCTAGTCACA

TTTCAGGACG

AAAATAGATT

AACAAAACAA

AAACAGCATA

CATACAATTT

GGATATGACT

TTGCAATGGA

TTGTTTTGTT

TTTGTCGTAT

GTATGTTAAA

CCTATACTGA

AACGTTACCT

TATTGTCTAA

AAGATCGGGG

TTTCGGTTAC

CATCCAGCTT

CACGGGAACC

ATAACAGATT

TTCTAGCCCC

AAAGCCAATG

GTGCCCTTGG

AGCAGTTTTG

GGGAGGTTGC

TTGAAGAGCT

GTAGGTCGAA 307 TCCAtgatgc

TCGTCAAAAC

CCCTCCAACG

AACTTCTCGA

AGGTactaqg

tccaggatct

23501

23551

23601

23651

23701

23751

aggtcctaga

23801

The genomic sequence in one cosmid (C1155) was determined by a shotgun-based approach (Bodenteich et al., 1993) (GenBank Accession No. L48815). Underlined: primer sequences for PCR-synthesis. Bold: cDNA sequence (clone c-2oa07 from EMBL database, accession No. Z41097).

Table 3 DNA sequences from 3 cosmids (Table 1) containing (CA), repeats accession No. L48815 GGGGG CAATA accession No. X95657 ACATT ACACA GCGGN accession No. X95658 GGGTC TGTAT CCCGA

TGGGG TGCTC

GGAGC ACACA

AGACA CACAC

CTGGA AACCA

ACACA GCCAN

CACAC ACAAT

ACACA CGGGC

CACAC CAGTG

ACACA TGTTT

ATGGC CACAC ACCA

TGGCT ATACA

CGGGA CACAC

CAGAC ACACA

CACAC CACAC

ACACA AGCAC

CACAC CCCTG

ACACA GAGGA

CACAC GAGAG

CTGGT GTGTG GCCAG

GCCGC TGTGT NCATA

CTCTC GTGTG ATGTA

TCCTC TGTGT

CAGGG GTGTG

GTGCT TGTGT

GTGTG GTNNN

TGTGT NGGNC

GTGTG TTNTC

Acknowledgement

References

The authors are grateful to Drs. H. Lehrach and P. Scambler, London, for the cosmid libraries, to H. Shizuya and M. Simon, Pasadena, for access to the BAC library and unpublished data and to P. Fegert for expert help with cell cultures. The project was aided by a university instrumentation grant, EC Concerted Action program travel support (Biomedl B14), and grants from the D A A D exchange program (H.S.), D F G (B L 166/14-1; Mu 1153/1 - 1 ) and the National Institutes of Health H u m a n Genome Project (HG00313) (B.R.).

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