Development of a somatic cell hybrid mapping panel and molecular probes for human chromosome 3

l

GENOMICS

8,435-446

(1990)

Development of a Somatic Cell Hybrid Mapping Panel and Molecular Probes for Human Chromosome 3 H. DRABKIN,*+’ M. WRIGHT,* M. JONSEN,* T. VARKONY,* C. JONES,t S. GOLD,* H. MORSE,*.t M. MENDEZ,t AND P. ERICKSON* *University

M. SAGE,*

of Colorado Medical Center, 4200 East 9th Avenue, Denver, Colorado 80262; and Roosevelt Institute for Cancer Research, Denver, Colorado 80206

tEleanor

Received

February6,

1990;

RevisedJune

13. 1990

arm material have been demonstrated in the majority of cases (Whang-Peng et al., 1982; Kovacs et al., 1988). At the molecular level, loss of 3p heterozygosity has corroborated these cytogenetic changes (Kovats et al., 1988; Brauch et al., 1987; Naylor et al., 1987). Similarly, loss of 3p heterozygosity has been demonstrated in a subset of breast, ovarian, cervical, testicular, and non-small-cell lung carcinomas (Devilee et al., 1989; Panani and Ferti-Passantonopoulo, 1985; Yokota et al., 1989; Lothe et al., 1989; Popescu et al., 1988; Kok et al., 1987; Weston et al., 1989). These and other data would appear to suggest that there may be two suppressor loci on the short arm of chromosome 3. Other recurrent chromosome 3 rearrangements occur in acute myelogenous leukemia, and there are several developmental disorders due to translocations involving chromosome 3 in a secondary fashion (Bitter et al., 1985; Tommerup and Nielsen, 1983; Drabkin et al., 1989; Reilly et al., 1988). However, the relative paucity of regionally mapped or genetically linked molecular probes has hindered the analysis of these disorders. The size of this chromosome poses a considerable challenge to the construction of a map that would greatly facilitate identification of these various disease loci. In addition, it might be anticipated that the genes responsible for perhaps 7% of human genetic disorders will be located here. As a first step in constructing such a map, we have developed a somatic cell hybrid mapping panel and have isolated and localized to varying degrees 432 molecular probes. These probes have been isolated from flow-sorted libraries, as well as Not1 boundary and random digest (partial MboI) lambda libraries. The Not1 probes are of added interest, since they not only facilitate construction of pulsed-field physical maps, but identify many HTF islands and therefore gene sequences as well. These studies also demonstrate the utility of isolating molecular probes from different types of libraries, or with

A somatic cell hybrid mapping panel and molecular probes have been developed for human chromosome 3. This panel defines 11 regions for the short and long arms of the chromosome. Four hundred thirty-two probes have been mapped using these hybrids. One hundred thirty-one of these probes were derived from EcoRI and HindID flowsorted libraries. The remaining 301 probes were isolated from Not1 boundary and random (partial MboI) libraries constructed from a hybrid that provided a relative enrichment in 3p DNA sequences. For some regions of the chromosome, significant differences in the distribution of probes were noted. This was observed for both the unique sequence flow-sorted and Not1 probes. These differences are in agreement with previous suggestions that Giemsa light bands are G&rich, and therefore gene-rich (especially housekeeping genes), and that the Giemsa dark bands may contain DNA that is more highly condensed. The isolation of probes from different types of libraries, or by different screening strategies, appears to reduce deficiencies that might arise from the use of probes derived with a more limited approach. These hybrids and probes should facilitate the construction of physical and genetic linkage maps to identify various disease loci involving chromosome 3. 0 isso Academic PWS, IUC.

INTRODUCTION

Chromosome 3 is composed of approximately 210 million bp of DNA representing 7% of the human genome. Specific rearrangements involving chromosome 3 have been described for several malignant disorders, most notably for small-cell lung cancer and renal carcinoma. In these tumors, deletions or nonreciprocal translocations resulting in the loss of short-

1 To whom correspondence should be addressed at the Division of Medical Oncology, University of Colorado Cancer Center, 4200 E. 9th Ave., Denver, CO 80262. 435

OSSS-7543/90$3.00 Copyright 0 1990 by Academic Prees, Inc. All rights of reproduction in any form reserved.

436

DRABKIN

varied screening procedures, in order to obtain a more random distribution of markers. MATERIALS

AND

METHODS

Somatic Cell Hybrids The CHO mutant Urd-C is deficient in uridine monophosphate synthetase (UMPS) and has been previously described (Patterson et al., 1983). This gene has been localized to the long arm of human chromosome 3, thereby providing a specific selection for the retention of intact or derivative 3 chromosomes. The CHO mutant Q131-91A is deficient in both uridine (UMPS) and de nouo purine biosynthesis. This mutant was derived from Urd- C by mutagenesis with ethylmethanesulfonate (EMS) and selection for purine auxotrophy using the bromodeoxyuridine/visible light procedure (Puck and Kao, 1967). By complementation analysis, the purine mutation is in the Ade- G group (David Patterson, unpublished data), which is complemented by a gene on human chromosome 21 (Patterson et aZ., 1981). The hybrids UCTP 2A3, 3;7/UC2E-1, and UCHl2 have been previously described (Gerber et al., 1988; Drabkin et al., 1989; Miller et al., 1987; Patterson et al., 1983). UCTP 2A3 and UCHl2 contain, as the only identifiable human material by cytogenetic analysis, the intact 3 and long arm of chromosome 3, respectively. The hybrid 3; 7/UC2E-1 contains the der(3) chromosome, 7pter+7p13: :3p21.1+3qter, from the Greig polysyndactyly 3; 7 translocation. Human lymphoblastoid cells (GM9528) containing a 3;21 translocation, t(3;21)(p24.2;q21), were grown in RPM1 medium containing 20% fetal calf serum (FCS) and then fused to the CHO mutant Q131-91A using uv-inactivated Sendai virus as previously described (Moore et al., 1977). The hybrid 3;21/Q13191A-1 was selected simultaneously in uridine- and purine-deficient medium (F12D) containing 5% dialyzed fetal calf serum plus 1 X lo-’ M oubain. The hybrid R158-6A was isolated by fusing Q131-91A cells with the human lymphoma cell line RC-KS Cl50 (Kubonishi et al., 1986), kindly provided by Dr. Jorge Yunis, and selected in uridine-deficient medium (F12) with 5% dialyzed FCS. Lymphoblastoid cells containing an X;3 translocation with breakpoints at 3~14.1 (or 3~13) and Xp13 were kindly provided by Dr. Tom Glover. These were fused with the mouse cell line RAG [obtained from the American Type Culture Collection (ATCC), Rockville, MD], deficient in HGPRT (hypoxanthine-guanine phosphoribosyltransferase), and the hybrid Rl-1 was HAT selected (hypoxanthine, 3 X lo-’ iVl; aminopterin, 2 x lop7 iVl; thymidine, 3 X 10e5M) in McCoy’s 5A medium. Acute myelogenous leukemia cells containing a 3; 3 translocation, t(3;3)(q21;q26.2), were kindly pro-

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vided by Dr. Manuel Diaz and Dr. Janet Rowley. These were fused with Urd- C cells, and hybrids were selected in uridine-deficient F12 medium with 5% dialyzed FCS. Hybrid H3-4 was found to contain the 3q- chromosome (3pter+3q21: :3q26.2-t3qter) as the only identifiable human material by cytogenetic analysis. This hybrid provides a relative enrichment for isolating 3p DNA sequences due to the absence of 3q21+q26 material and was used for additional genomic library constructions (see below). The hybrids R227-3A, R148-3, and 273-2-3B4 contain terminal 3q deletions and were selected for loss of the human transferrin receptor (3q26+qter) by growth in medium containing anti-human transferrin receptor antibodies plus complement. Hybrids R227-3A and R148-3 were derived from the CHO-human hybrid 314-1, which contains an intact chromosome 3 as the only identifiable human material. Hybrid 273-2-3B4 was derived from the CHO-human hybrid UCHB, which contains the long arms of human chromosomes 3 and 21, separately translocated onto CHO chromosomes, as the only identifiable human material. Prior to selection with anti-transferrin receptor antibodies, UCH2 was first exposed to lowdose (300 rads) gamma irradiation to induce doublestrand chromosome breaks. R227-3A and R148-3 were selected without prior irradiation.

Cytogenetic and in Situ Hybridization

Analysis

Human chromosomes in the somatic cell hybrids were identified and analyzed by sequential G-11 staining and GTG banding (Morse et al., 1982). In some cases, distamycin A was used to obtain elongated chromosomes. For this, distamycin A hydrochloride (Sigma) was dissolved in distilled water at 0.150 mg/ml and 0.5 ml of this solution added to a 60-mm plate of dividing cells containing 4 ml of culture medium. After 24 h, colcemid was added to a final concentration of 0.05 pg/ml for 1 h prior to harvest. In situ hybridization (Harper and Saunders, 1981) was performed under conditions outlined by Cannizzaro and Emanuel (1984).

Isolation

of Probes from Flow-Sorted

Libraries

The EcoRI and Hind111 chromosome 3 libraries (Nos. 57748, 57751) were obtained from the ATCC Repository. To enrich for clones with detectable inserts, the libraries were amplified and fractionated on a cesium chloride gradient. Since the density of DNA is greater than the density of protein, those phage with more DNA move farther through the gradient than do phage without inserts (Barker et al., 1987). Repetitive sequence negative phage were tested for the presence of detectable inserts from individual fractions. Those fractions producing a relatively high percentage of unique sequence inserts were selected

HUMAN

CHROMOSOME

for further use. The phage miniprep procedure of Grossberger (1987) was used to prepare DNA, as described, except that bacteria were removed by centrifugation at 7000 rpm and the phage pellet after ultracentrifugation was resuspended in 0.470 ml 1X TE (10 mM Tris, pH 8.0, 1 m&f EDTA), added to a 1.5ml microcentrifuge tube containing 0.025 ml 10% SDS and 0.005 ml Proteinase K (10 mg/ml), and placed at 50°C for 3 h to overnight. Following phenol-chloroform extraction and ethanol precipitation, the DNA pellet was dissolved in 0.030-0.050 ml TE. A second more sensitive screen to eliminate those inserts containing repetitive sequences was carried out by Southern blot analysis on DNA digested with EcoRI or Hind111 and hybridized with 32P-labeled total human DNA. Repetitive sequence negative inserts were excised from a low-melting-point agarose gel. The gel fragment was melted at 65°C and 0.012 ml was used in an oligolabeling reaction scaled down to 0.020 ml. The entire reaction mixture was used as probe without separation of unincorporated [32P]dCTP. This did not result in background problems and was quite economical for screening large numbers of probes. All probes isolated from the flow-sorted libraries were tested with an initial mapping panel containing DNAs from the UCTP 2A3, UCH12, and CHO (Urd- C) cell lines. A fragment that hybridized uniquely to UCTP 2A3 DNA confirmed the human chromosome 3 nature of the probe, and in combination with UCHl2 DNA allowed for the determination that the DNA sequences were derived from either 3p or 3q. Construction and Use of Random (Partial NotI Libraries from Hybrid H3-4

MboI)

3 PHYSICAL

437

MAPPING

Recombinant phage containing human DNA were identified by hybridization to 32P-labeled total human DNA (Gusella et al., 1980) and further plaque-purified. DNA minipreps were as described above. Hybridizations were performed after 32P-labeling the entire phage DNA, and repetitive sequences were blocked with excess unlabeled total human DNA (Sealy et al,, 1985). Initially, 123 probes were tested with the primary panel used to screen the flow-sorted probes (UCTP 2A3, UCH12, CHO). Of these, 4 (3%) were of CHO origin. Since the percentage of non-human recombinants was quite low, this primary screening panel was replaced by the following hybrids: R158-6A, 3;21/ Q131-91A-1, 3; 7/UC2E-1, Rl-1, UCH12, and R227-3A. This approach allowed discrimination among the six 3p regions, and between the two 3q regions contained in hybrid H3-4 (cen*q21 and 3q26.2+qter). Except for the cen+q21 region, at least one CHO hybrid was also negative for the human fragment(s), excluding the possibility that the probe was not of human chromosome 3 origin. For probes in the cen+q21 region, it is possible that 3% could be of CHO origin. For the 36 Not1 probes in this region, it is unlikely that more than 1 would be of CHO origin. Hybridization

Conditions

Hybridization was carried out at 65°C in 5~ SSC, 2~ Denhardt’s, 0.5% SDS, 5% dextran sulfate, and 0.1 mg/ml salmon sperm DNA. The final wash was in 0.1X SSC, 0.1% SDS at 55-65°C.

and

For the random library, DNA from hybrid H3-4 was partially digested with Mb01 and size fractionated on a lo-40% sucrose gradient (Frischauf, 1987). Individual fractions were dialyzed against 1X TE, followed by 0.1X TE, concentrated to approximately 0.5 ml with iso-butanol, then phenol-chloroform extracted, ethanol precipitated, and redissolved in 0.020 ml 1X TE. Individual fractions were ligated to BamHI-digested EMBL3 (Frischauf et al., 1983) phage arms and packaged in vitro using extracts prepared from the lysogens BHB 2680 and BHB 2690 as outlined (Scalenghe et al., 1981). Packaging reactions were plated on the P2 lysogen/mcrA-Bhost NM646 (Whittaker et al., 1988) kindly provided by Dr. Noreen Murray. Not1 boundary libraries were similarly constructed by first digesting the DNA with NotI, followed by a partial Mb01 digest and size fractionation. Inserts were ligated to BamHI-NotI-digested EMBLG (Frischauf et al., 1987) phage arms, packaged, and plated as above. The EMBLG vector was kindly provided by Dr. Hans Lehrach and Alister Craig.

RESULTS Development

of Somatic Cell Hybrids

The somatic cell hybrid mapping panel used for these studies is shown schematically in Fig. 1. The chromosome 3 material present in each hybrid is represented by the heavy vertical lines. Six regions have been delineated for the short arm and a minimum of five for the long arm. Metaphase chromosomes of selected hybrids containing either an interstitial deletion (R158-6A) or terminal deletions (R227-3A, R148-3, and 273-2-3B4) are shown in Fig. 2. The karyotype of those hybrids containing defined translocations has not been included for the purpose of brevity. Hybrid R158-6A contains an apparent interstitial deletion in the short arm of chromosome 3. The cytogenetic analysis indicates that the 3p terminal Giemsa dark band, 3~26, is present, but that the next more proximal dark band, 3~24, is abnormally light and the length of the short arm is reduced by approximately 20%. We interpret these results to be an interstitial deletion of bands 3~24.1-3~24.3 and perhaps

438

DRABKIN

ET

AL.

7.6 25 24.3 24.2 24.1 23 22

21.2 21.1 14.3 14.2 14.1

1 1.2 11.1 1 1.2 12 13.1 13.2 13.3

22 23 24 25.1 :::f 26.1 26.2 26.3 27 28

2A3*

FIG. 1. Schematic of the chromosome heavy vertical bars. Hybrids that contain

R 6

3;2 YlC

3 somatic cell hybrid only a single human

1131 *

RI-1

!E-1

UC

l

w-41

R227 3A*

273-2 384

mapping panel. The chromosome 3 content chromosome are indicated by an asterisk.

all, or a portion of 3~25. Chromosome 3 is also unusual in that it terminates in a Giemsa dark band. Thus, it is unlikely that this terminal material is derived from other than chromosome 3. The molecular data also confirm this interpretation (below). Hybrids R227-3A, R148-3, and 273-2-3B4 were derived from preexisting hybrids containing either an intact chromosome 3 (R227-3A, R148-3) or the long arm of chromosome 3 (273-2-3B4) by selection for loss of the chromosomal region containing the transferrin receptor. In each hybrid there has been a translocation of CHO material onto a region of 3q accompanied by a terminal deletion. It seems likely that the translocated CHO material provides stability through telomere sequences and that this event is more likely than an interstitial 3q deletion. By Giemsa-11 staining, there is no evidence for retention of terminal 3q material. Giemsa-11 staining and GTG banding were

Rl483+

of each hybrid

is indicated

by the

used to determine the most likely breakpoints of the 3q deletion hybrids. While our best estimates have been made, these could vary at least by a chromosome band. Hybrid R148-3 appears to contain the 3q26.1 and 3q26.3 dark bands. This would place the breakpoint in band 3q27 or below. In contrast, hybrids R227-3A and 273-2-3B4 do not contain the 3q26 dark bands and appear to be broken in band 3q25. Cytogenetically, we cannot distinguish between these latter two breakpoints. At the molecular level, the R148-3 breakpoint is clearly distal to the breaks in R227-3A and 273-2-3B4, and it appears that the 273-2-3B4 break is distal to that in R227-3A (below). Three of the new hybrids were derived from naturally occurring translocations. Hybrid 3;21/Q13191A-1, the result of the double UMPS/Ade G selection, contains the der(3) chromosome, 2lqter+ 21qll: : 3p24.2+3qter, as the only identifiable human

HUMAN

CHROMOSOME

material. Similarly, hybrid H3-4 contains, as the only identifiable human material, the 3pter-*3qZl: : 3q26.2-*3qter chromosome derived from the AML 3;3 translocation, t(3;3)(q21;q26.2). Hybrid Rl-1 contains the der(3) chromosome, Xpter+Xpl3:: 3p14.1(13)+3qter along with other human chromosomes, but does not contain the normal 3, normal X, or der(X) chromosomes. At the molecular level, no inconsistencies have been detected with any of the hybrids containing breaks in the short arm, and hybrid 3;21/Q131-91A-1 has been similarly tested with many chromosome 21 probes, again with no inconsistencies.

Flow-Sorted Probes and Characterization Chromosome 3 Mapping Panel

of the

Initially, in screening for unique sequences, we were able to identify inserts in only approximately 50% of the phage. This was presumably due either to the presence of nonrecombinants or to difficulties in identifying small inserts less than approximately 500 bp. The isolation of inserts was necessary, because use of the intact phage as a hybridization probe produced weak signals on Southern blots of genomic DNA. By amplifying and fractionating the library, it was possible to identify phage without repetitive sequences that had a high probability of containing detectable inserts. A miniprep procedure that produced adequate quantities of DNA cleavable with either EcoRI or Hind111 greatly facilitated the use of these probes. We found the method of Grossberger to be convenient and highly reliable. One hundred thirty-one unique sequence probes (MJ, MS, or SG designation) from the flow-sorted libraries have been localized on chromosome 3 (Fig. 3 and Table 1). Sixty are located on 3p, and 71 are on 3q. These probes have been characterized using the somatic cell hybrids that contain a reduced amount of human material. It is possible that when used against total human DNA some of the probes will require blocking. Fifteen probes, localized to 3q on our primary screening panel, have been omitted from this report because they contained enough repetitive elements that blocking was required. We have consistently obtained more probes from the long arm of chromosome 3, even when using the libraries constructed from H3-4 (below). No inconsistencies have been detected with any of the short arm probes. Six probes present in hybrid R158-6A (3pter+3p25: : 3p23+3qter) are absent in hybrid 3; 21/Q131-91A-1 (3p24.2-+3qter), confirming the presence of the distal 3pter material suggested by the cytogenetic analysis. One probe, MS21, is absent in hybrid R158-6A and present in 3;21/Q131-91A-1, suggesting that the interstitial deletion in R158-6A extends proximal to the 3~24.2 breakpoint in the 3; 21

3 PHYSICAL

MAPPING

439

translocation. However, this overlap appears to be small since MS21 is the only probe identified to date with this pattern. For the long arm, two inconsistencies have been noted and are described in more detail. There are 32 flow-sorted probes in the cen+q21 region. These are defined by their presence in H3-4 (3pter+q21: : q26.2+qter), as well as in 273-2-3B4 (3q11.2+q25) and R227-3A (3pter+q25). All probes are similarly present in R148-3 (3pter-*q27 or 28). One exception is probe MJ1484, which was missing in hybrid 273-2-3B4. Other data (below) indicate that the 273-2-3B4 break occurs distal to the breakpoint in R227-3A. This suggested that 273-2-3B4 might have a small deletion in the cen-*q21 region. This hybrid was derived from UCH2, which contains the long arm of chromosome 3 translocated onto a CHO chromosome. Since the original chromosome 3 centromere was likely lost in the generation of UCH2 (dicentric chromosomes are unstable), a small peri-centromeric deletion might have occurred and not been detected cytogenetically. Alternatively, UCH2 was exposed to 300 rads prior to selection with anti-transferrin receptor antibodies, which may have produced one or more deletions (below). It is also possible that probe MJ1484 might be derived from q26.2+q27(28), i.e., present in H3-4 and R148-3, and the R227-3A hybrid may have retained an unrecognized noncontiguous fragment from this region. Localization to the more distal 3q27(28)-tqter region would require that both R148-3 and R227-3A contain additional unrecognized noncontiguous distal fragments. We have not seen any discrepancies to suggest that hybrid UCH12 (the primary discriminator between 3p and 3q) contains any material from the short arm. An in situ hybridization experiment on normal human metaphase chromosomes was therefore carried out. Of 121 grains (18%) present on chromosome 3, 30 grains (25%) were present at 3q13.2. These results suggest that probe MJ1484 is located in the cen+q21 region, although secondary grain accumulations in 3~26 (10%) and 3q29 (13%) prevent any more definitive conclusions. The region 3q21+q25 is represented by 15 probes that are all absent in H3-4, i.e., 3q21+q26.2 but present in both 273-2-3B4 and R227-3A as well as in R148-3. There are no inconsistencies in this group of probes. The next more distal region is 3q25-*q26.2. There are three probes, MJ1523, MS1222, and MJ1567, that are present in R148-3 (3pter+q27 or 28), absent in H3-4, and absent in both R227-3A and 273-2-3B4. These probes are therefore below the 3q25 breakpoints in R227-3A and 273-2-3B4, but above the 3q27(28) break in R148-3. Their absence in H3-4 further localizes these probes to 3q25-*q26.2. Two probes, MS60 and MS416, are also absent in H3-4 and R227-3A but present in 273-2-3B4 and R148-3.

DRABKIN

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AL.

a

FIG. 2. Metaphase chromosomes from selected the 3q deletion hybrids indicates the approximate 3pter+p25::p23+qter; (b) R227-3A, 3pter+q25;

hybrids as described in the text. The arrow indicates the chromosome of interest and for site of recombination between the human and hamster chromosomes: (a) R158-6A, (c) 273-2-3B4, 3qll+q25; (d) R14&3,3pter+q27(28).

This would indicate that the 273-2-3B4 break is distal to that which occurs in R227-3A. However, one probe, MS89, clearly has the opposite pattern, that is, absent in 273-2-3B4 (and H3-4) but present in R227-3A (and

R148-3). Thus, probe MS89 would suggest that the R227-3A break occurs distal to 273-2-3B4. Since we found evidence that 273-2-3B4 sustained a deletion in the cen+q21 region, it is possible that more deletions

HUMAN

CHROMOSOME

FIG.

3 PHYSICAL

2-Continued

MAPPING

441

442

DRABKIN

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3p Probes

P

1

24.3

SG115 MS445 MS1272 MJ1464 MJ1469 MJ1486 MW-Mbo103 MS30 MS434A MU192 h4J1199 MS1213 Ml1250 u11369 MJ1372 MJ1411 MJ1441 MJ1482A MJ1482B MJ1502 MJ1516 MJ1535 MJ1559 MW-Mb0189 MW-Not18 MW-Not30 m-Not47 P&Not55 MW-Not63 MW-Not65 MW-Not81 MW-Not97 MW-Not99 MW-Not102 MW-Not115 MW-Not120 MW-Not122 MW-Not124 MW-Not126 MW-Not131 MW-Not149 MW-Not170 MW-Not164 MW-Not165 MW-Not176 MW-Not188 MW-Not190 MW-Not202

f;:f

‘1

26 25

a.1 a

MS21

23 1 22

MS36 MS400 MS451 MS510 MS1182 MJ1437 MJ1526 MJ1534 MW-Mbol6e-2 MW-Mb0203 MW-Mb&?26 Mb0232 PGNot6 MW-Not10 P&Not12 MW-Not14 MW-Not17 MW-Not19 MW-Not21 MW-Not25 MW-No48 MW-Not29 MW-Not32 MW-Not38 PE-Not50 MW-Not54 MW-Not60 MW-Not61 MW-Not71 MW-Not72 MW-Not73 MW-Not75 MW-Not78 MW-Not80 MW-Not83 MW-Not89 MW-Not96 MW-Not105 MW-Not108 MW-Not109 MW-Not111 MW-Not129 MW-Not138 MW-Not142 MW-Not147 MW-Not153 MW-Not158 MW-Not160 MW-Not167 MW-Not168 MW-Not186 MW-Not203 MW-Not-214 MW-Not216 MW-Not218 MW-Not228 MW-Not-245 MW-Not249

21,3

21.2

m :

21,l 14.3 14.2 14.1

MS125 MS156 MS430 MS453 MS1111 MS1118 MS1120 MS1135 MJ1206 MS1221 MJ1388B MJ1417 MJ1422 MJ1430 Ml1438 MJ1454 MJ1497 MJ1498 Ml1499 MJ1517 hU1525 hU1557 MJ1570 MW-Mb084 MW-Mbo-249 MW-Mb0157 MW-Mb0225 MW-Mb0258 MW-Not13 MW-Not36 PENdO MW-Not43 MW-Not41 MW-Not49 MW-Not59 MW-Not87 MW-Not90 MW-Not121 MW-Not132 MW-Not137 MW-Not-140 MW-Not143 MW-Not145 MW-Not146 MW-Not157 MW-Not159 MW-Not162 MW-Not163 MW-Not174 MW-Not178 MW-Not184 MW-Not191 MW-Not192 MW-Not195 MW-Not197 MW-Not198 MW-Not200 hfW-Not215 MW-Not220 MW-Not229 MW-Not239 MW-Not240 MW-Not251

MW-Mbo2g2 MW-Mba40-1 MW-Mb080 MWMbo96.2 MW-Not123 MW-Mb0157 MW-Mb0171 MW-Mbol78 MW-Mbol94 MW-Not196 MW-Mb0215 MW-Mb0216 MW-Mbo256 MW-Mbo257 MW-Mbo276 MW-Not158 MW-Not173 MW-Not186 MJ1201 MJ1502 MJ1504

3

12

/

W-Not76 S1271 W-Not94 W-Not166 SS08D 11399

1I:f

i

3P FIG. 3. Localization of chromosome random (MboI) clones were derived from are not represented in these libraries.

3 probes. Flow-sorted probes are indicated by an MJ, MS, or SG prefix. The Not1 boundary and hybrid H3-4 (3pter+q21::q26.2+qter) and therefore DNA sequences from the 3q21eq26.2 region

have also occurred. Alternatively, we have not excluded the possibility that R227-3A contains an unrecognized noncontiguous fragment. Additional experiments such as in situ hybridization, pulsed-field mapping, or genetic linkage studies should resolve this question, and for now we have collectively referred to the probes from this region as being derived from 3q25-3q26.2. Fourteen probes define the q26.2+q27(28) region. These are characterized by their absence in hybrids R227-3A and 273-2-3B4 (which excludes localization to the cen+q21 region) and presence in H3-4 and R148-3. No inconsistencies have been observed in this set of probes. Finally, 3 probes are absent in R148-3 but present in H3-4, which localizes them to 3q27(28)+qter.

NotI

and Random

(Partial

MboI)

Probes

A NotI-partial Mb01 boundary library was constructed in the vector EMBLG from hybrid H3-4, which contains the single human chromosome 3pter+3q21: : q26.2+qter. This hybrid provides a relative enrichment in probes from 3p because of the absence of 3q21-*q26 material. Two hundred fortythree independent clones have been isolated and regionally mapped to varying degrees (Fig. 3 and Table 1). One hundred thirteen Not1 probes are located on 3p, and 130 are on 3q. The Not1 probes do detect accessible Not1 sites in genomic DNA, as shown in Fig. 4 using single and double digests with EcoRI plus Not1 or EcoRI and EogI. Additional pulsed-field mapping experiments (data not shown) have also demon-

HUMAN

CHROMOSOME

3 PHYSICAL

443

MAPPING

3q Probes

11.2 12

13,l 13.2 13.3

dS134 MS143 MS329 MS409 MS410 MS413 MS420 MS431 ulS432 MS434 MS513C MS524 MS525A MS1156 MS1177 till% MS1212 MS1228 MS1242 MJ1257 MJ1373 MJ1376 uIJ1398 MJ1442 MJ1456 IUJ1457 MJ1472 IUJ1484 hU1487 u111488 hU1533B MJ1544 MW-Not16 MW-Not26 MW-Not27 UW-Not34 MW-Not46 MW-Not68 MW-Not79 MW-Not82 rlW-Not84 MW-Not88 MW-Not91 MW-Not97 MW-Not100 tiW-Not101 MW-Not106 MW-Not107 MW-Not119 MW-Not130 UW-Not134 MW-Not150 MW-Not151 MW-Not166 MW-Not187 UW-Not188 MW-Not201 MW-Not205 MW-Not210 MW-Not217 UW-Not225 MW-Not235 MW-Not238 MW-Not243 MW-Not244 UW-Not246 MW-Not247 MW-Not257

21 22 23

IS85 MS183 MS337 IS452 MS1188 MS1223 IS1239 MJ1351 MJ1416 Ul501 MJ1508 MJ1519 I11530 MJ1531 MJ1536 :33

24 25.1 2;

US60 MS89 MS416 US1222 MJ1523 MJ1567

,

26.1 26.2 26.3 27

MS57A MS195 MS328 MS414 MS450 MS455 MS461 MS1138 hU1207 ,M11249 lUJ1463A MJ1463 ‘MJ1475 MJ1492

28 VI367

MJ1431

MJ1465

29

P&Not5 MW-Not8 MW-Not9 MW-Not11 MW-Not23 MW-Not24 MW-Not31 MW-Not33 MW-Not39 MW-Not42 MW-Not51 MW-Not92 MW-Not77 MW-Not93 MW-Not95 MW-Not98 MW-Not110 MW-Not112 MW-Not113 MW-No1114 MW-Not116 MW-Not118 MW-Not125 MW-Not127 MW-Not133 MW-Not135 MW-Not144 MW-Not148 MW-Not226 MW-Not139 MW-Not152 MW-Not154 MW-Not155 MW-Not164 MW-Not189 MW-Not191 MW-Not193 MW-Not194 MW-Not199 MW-Not206 MW-Not207 MW-Not208 MW-Not209 MW-Not211 MW-Not212 MW-Not213 MW-Not219 MW-Not224 MW-Not227 MW-Not231 MW-Not234 MW-Not241 MW-Not242 MW-Not248 MWNot256 MW-Mbc63 MW-Mbo95-2

FIG.

Nonrandom

Distribution

of Probes

As can be seen from Fig. 3 and Table 1, the distribution of probes from either the flow-sorted or Not1

I

3-Continued

strated that these clones detect Not1 sites in genomic DNA. These should be of considerable value in identifying genes associated with HTF islands and in the generation of a pulsed-field map. Nearly 300 partial Mb01 clones from chromosome 3 were isolated in EMBL3 (and EMBL3S), although only 58 have been mapped (Fig. 3 and Table 1). Twenty-four are from 3p and 34 are from 3q, bringing the total number of mapped probes to 432.

Apparent

PE-Not1 MW-Nor2 PLNot3 PISNot MW-Not15 MW-Not20 MW-Not22 MW-Not35 MW-Not37 P&Not44 PE-Nod5 F’KNot53 PISNot PE-Not57 WNot58 MW-Not60 MW-Not62 MW-Not64 MW-Not66 MW-Not67 MW-Not74 MW-Not85 MW-Not103 MW-Not136 MW-Not141 MW-Not156 MW-Not161 MW-Not169 MW-Not171 MW-Not172 MW-Notl7SMW-Not177 MW-Not179 MW-Not180 MW-Not181 MW-Not182 MW-Not183 MW-Not185 MW-Not250 MW-Mb&O MW-Mb071 MW-Mb089 MW-Mb&t-2 MW-Mbo93-2 MW-Mbo%-2 MW-Mbo98-2 MW-Mbo99-2 MW-Mb01002 MW-MbolO.2 MW-Mb0107 MW-Mb0126 MW-Mb0158 MW-Mb0162 MW-Mb0164 MW-Mb0167 MW-Mb0170 MW-Mb0172 MW-Mlwl75 MW-Mb0193 MW-Mb0195 MW-Mb0204 MW-Mb0206 MW-Mb0218 MW-Mba2M MW-Mb0236 MW-Mb0242 MW-Mb0245 MW-Mb0267 MW-Mb0281 MW-Mb0282 MW-Mb0287

libraries does not appear to be uniform. This is especially apparent in a comparison of the distribution of probes between the flow-sorted and the Not1 libraries. For example, so far only probes from the flow-sorted library have been mapped to region 3p25+pter. Region 3p21.2+p23 has 8 of the 60 flow-sorted probes (13%) but 46 of the 108 subregionally localized Not1 clones (43%). In contrast, a region of apparently similar size, 3p14.1(13)-*~21.1, has 22 (37%) flow-sorted and 35 (32%) Not1 probes. The cen+p14.1(13) region is relatively devoid of probes from both libraries. Differences are also suggested in the distribution of the flow-sorted probes for the long arm. While the cen-*q21 and q21-*q25 regions appear to be similar

444

DRABKIN

TABLE 1 Flow-sorted

Not1

Partial Mb01

Total

3P 3~25 + ~26 3~24.2 + p25 3~24.1 3~21.2 + p23 3p14.1 + p21.1 3cen + p13 Not sublocalized Total

6 16 1 8 23 3 3 60

0 24 0 46 35 3 5 113

1 1 0 4 5 0 13 24

7 41 1 58 63 6 21 197

3cen + q21 3q21 + q25 3q21 + q26.2 3q25 + q26.2 3q26.2 + q27(28) 3q27(28) + qter 3q26.2 --* qter 3cen + q21 or 3q26.2 + qter Total

32 15 1 6 14 3

36 ma. n.a. n.a. n.t. n.t. 55

0 n.a. n.a. n.a. n.t. n.t. 2

68 15 1 6 14 3 57

71

39 130

32 34

71 235

131

243

58

432

Total Note.

n.a., not applicable;

n.t.,

ET

AL.

ordering of probes within the 11 regions described in this report. The flow-sorted probes were selected for the absence of repetitive sequences which have been reported to be nonrandomly distributed (Ah and Kpn repeats are preferentially located in Giemsa light and dark bands, respectively) in the human genome (Korenberg and Rykowski, 1988). It might be expected, therefore, that these probes would be distributed in proportion to the amount of DNA contained in each region. At first glance, this appears not to be the case, at least for some regions of the chromosome. For example, region 3p21.2-*~23, which represents approximately one-third of the short arm, contains only 13% (8/60) of the flow-sorted probes. The cen-*q21 region, which cytogenetically represents approximately one-fourth to one-third of the long arm, contains 45% (32/71) of the probes, while the adjacent and similarly sized q21+q25 region contains only 20% (15/71).

not tested.

in size, more than twice the number of flow-sorted probes is derived from the more proximal cen-*q21 region. While ambiguities in the placement of cytogenetic breakpoints should temper any conclusions regarding the apparent nonrandom distribution of the various probes, for certain regions such differences appear striking. DISCUSSION This report describes the development of a somatic cell hybrid mapping panel and the isolation and localization of 432 probes from chromosome 3. The chromosomal regions defined by these hybrids range in size from perhaps only a few million base pairs separating the R227-3A and 273-2-3B4 breakpoints to approximately 20-30 megabases. We have developed several additional hybrids that subdivide and extend the 3p14.1+~21. region and further characterization of these probes is in progress. This region contains the site of the hereditary renal cell carcinoma 3; 8 translocation (Drabkin et al., 1985), the 3~14.2 fragile site (Glover and Stein, 1988; Smeets et al., 1986), the Greig polysynda@yly 3; 7 translocation (Tommerup and Nielsen, 1989), and other translocations, some of which may also be of biologic interest. We have also constructed 26 hybrids selected for breakage in the cen+q21 region; as well as a large set of radiation reduction hybrids. These should greatly facilitate the

A B c FIG. 4. Single and double digests of DNA from hybrid UCTP 2A3 (intact 3) hybridized with probe PE-Not3, demonstrating that the Not1 clones detect accessible Not1 (and EagI) sites in genomic DNA. The solid arrow indicates the EcoRI band (approximately 5 kb) which is reduced by double digestion with Not1 or EagI. The open arrow indicates the product (approximately 3 kb) after the double digest. Lanes: (A) EcoRI; (B) EcoRI plus NotI; (C) EcoRI plus EagI. (The enzymes EagI and Not1 recognize the same internal 6-bp sequence.) This probe did not detect CHO bands in separate experiments (data not shown).

HUMAN

CHROMOSOME

These differences might be partially due to ambiguities in the cytogenetic breakpoints that define the regions. However, this seems unlikely to account for all the differences observed. For example, while the 3p21.2-*p23 region contains only 13% of the 3p flow-sorted probes, 43% (46/108) of the 3p sublocalized Not1 clones are located here. The restriction patterns of these Not1 clones are all unique, and therefore the differences are not spurious. Variables that might help to explain these results could be cloning artifacts, the nonrandom distribution of genes, or differences in the condensation of DNA. The flow-sorted libraries were not constructed using an mcrA-Bhost (Whittaker et al., 1988) and therefore a bias against methylcytosine containing DNA or other types of DNA sequences probably occurred. The high frequency of Not1 probes localizing to the 3p21.2+p23 region is not unexpected because the area contains a large amount of Giemsa light band material which is believed to be gene rich (Bernardi, 1989). This clustering of genes within Giemsa light bands at least appears to be the case for human chromosome 21, where the majority of known genes have mapped to band 21q22 (Gardiner et aZ., 1990). We have constructed and mapped a chromosome 21 Not1 library and, like gene sequences, the overwhelming majority of clones are also located in the 21q22 light band (manuscript submitted). There are independent data for differences in DNA condensation between Giemsa light and dark bands. GC-rich isochores, which are gene-rich, are preferentially associated with Giemsa light and dark bands (Bernardi, 1989). In the human genome, GC-poor isochores predominate over GC-rich isochores by about 2:l despite the fact that the amount of Giemsa light and dark band material is nearly equal (Thiery et al., 1976; Cuny et al., 1981). This would indicate that the DNA is more condensed in the Giemsa dark bands. The clustering of Not1 sites in Giemsa light bands along with an increased amount of DNA in Giemsa dark bands would explain at least some of the discrepancies we have noted, especially given some variability in the location of the cytogenetic breakpoints. Also, overall differences in the density of repetitive elements would influence our results. Whatever the mechanism, there do seem to be regions where probes from one type of library are either over- or underrepresented. Given the complexity of 210 million base pairs of DNA for this chromosome, the construction of aphysical or genetic map for it is not trivial. However, the compartmentalization provided by these and other somatic cell hybrids greatly simplifies this task. Several physical linkage relationships have already been determined for probes in the 3p14+p21 region, which would have been considerably more difficult without the regional mapping information. These probes should facilitate the construction of both physical and

3 PHYSICAL

445

MAPPING

genetic linkage maps and permit identification many of the disease loci that reside on chromosome

of 3.

ACKNOWLEDGMENTS This work was supported by NIH Grants HD23826 and GM 40873. We acknowledge support from the CU Research Foundation and assistance from the Colorado Cancer Center Cytogenetics Core.

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