Am. J. Hum. Genet. 49:1189-1196, 1991

A Radiation Hybrid Map of the Proximal Long Arm of Human I I Containing the Multiple Endocrine Neoplasia Type I (MEN-I) and bcl-l Disease Loci

Charles W. Richard Ill, * Donald A. WithersT Timothy C. Meeker,t Susanne Maurer, Glen A. Evans,11 Richard M. Myers,t§ and David R. Cox*I§

Departments of *Psychiatry, tMedicine, tPhysiology, and §Biochemistry and Biophysics, University of California, San Francisco; and IIMolecular Genetics Laboratory, The Salk Institute, La Jolla, CA

Summary We describe a high-resolution radiation hybrid map of the proximal long arm of human containing the bcl-1 and multiple endocrine neoplasia type 1 (MEN-1) disease loci. We used X-ray irradiation and cell fusion to generate a panel of 102 hamster-human somatic cell hybrids containing fragments of human chromosome 11. Sixteen human loci in the 11q12-13 region were mapped by statistical analysis of the cosegregation of markers in these radiation hybrids. The most likely order for these loci is CiNH-OSBP- (CD5 /CD20)-PGA-FTH1-COX8-PYGM-SEA-KRN1-(MTC/P1EH/HSTFI /INT2)-GST3-PPP1A. Our localization of the human protooncogene SEA between PYGM and INT2, two markers that flank MEN-1, suggests SEA as a potential candidate for the MEN-1 locus. We map two mitogenic fibroblast growth factor , HSTF1 and INT2, close to bcl-1, a mapping that is consistent with previously published data. Our map places the human leukocyte antigen genes CD5 and CD20 far from the bcl-1 locus, indicating that CD5 and CD20 expression is unlikely to be altered by bcl-1 rearrangements. PPP1A, which has been postulated as a MEN-1 candidate tumor suppressor gene, and GST3, a gene transcriptionally active in many human cancers, both map distal to the bcl-1 translocation cluster and the region containing MEN-1, and therefore are unlikely to be directly involved in bcl-1 or MEN-1.

Introduction translocation, encompasses over 63 kb ofDNA (Tsuji- Much progress has been made in recent years in estab- moto et al. 1985; Rabbitts et al. 1988; Meeker et al. lishing the chromosomal location of cancer-causing 1989). MEN-1 is an autosomal dominant trait that genes, either by examination of chromosome re- predisposes individuals to neoplasms of the parathy- arrangements in leukemias or by linkage analysis and roid, pancreatic islets, and anterior pituitary. The tumor deletion analysis in familial cancers. The proxi- gene responsible for this syndrome has been localized mal long arm of human chromosome 11 (region by genetic linkage analysis to the area between 1 1q12- 11q12-13) contains at least two separate cancer dis- 13 flanking markers PGA and INT2 (Larsson et al. ease loci: bcl-1 and multiple endocrine neoplasia type 1988; Bale et al. 1989; Nakamura et al. 1989). Tumor 1 (MEN-1). The bcl-1 locus, defined as the chromo- deletion analysis of the smallest region of overlap has some 11 translocation breakpoint region in leukemia further narrowed the region to between markers and lymphoma cells carrying the t(11;14)(ql3;32) PYGM and Dl 15146 (Bystrom et al. 1990). By a com- bination of several mapping methods including in situ hybridization, meiotic mapping, and somatic cell hy- Received April 30, 1991; revision received July 22, 1991. brid mapping panels (Junien and McBride 1989), a Address for correspondence and reprints: Charles W. Richard number of genetic markers have been mapped to the III, M.D., Ph.D., Box 0554, HSE 1556, University of California, 1 1q12-13 region in the vicinity of bcl-1 and MEN-1. San Francisco, CA 94143-0554. i 1991 by The American Society of Human Genetics. All rights reserved. In many cases, however, the order of these markers 0002-9297/91 /4906-0006$02.00 and the distances between them are poorly defined. In 1189 1190 Richard et al. an effort to improve the map ofchromosome 11 in the published genomic or cDNA sequences. In caseswhere region of MEN-1 and bcl-1 we used radiation hybrid only cDNA sequences were available, primer se- (RH) mapping (Cox et al. 1990) to derive the in- quences were chosen from either the 3' or 5' untrans- termarker distances and order of 16 loci previously lated regions. Primer sequences for SEA were chosen assigned to human chromosome 11q12-13. The loci after sequencing a DNA fragment subcloned from a mapped include CD5 (antigen CD5), CD20 (antigen genomic SEA probe. MTC PCR primers were chosen CD20), C1NH (complement component 1 inhibitor), from sequences in the major translocation cluster of COX8 (cytochrome c oxidase subunit VIII), FTH1 the bcl-1 region (Tsujimoto et al. 1984, 1985). P1 1EH (ferritin heavy polypeptide-like 6), GST3 (glutathi- primers were chosen from a second translocation one-S-transferase 3, pi), HSTF1 (heparin secretory breakpoint sequence located 63 kb telomeric to the transforming 1), INT2 (murine mammary tu- MTC cluster (Meeker et al. 1989). Two sets ofHSTF1 mor virus integration site, v-int oncogene homolog), primers were used. Each 100-gl PCR reaction tube KRN1 (ultra-high sulfur keratin 1), OSBP (oxysterol- contained 250 ng genomic hybrid DNA, 67 mM Tris binding protein), PGA (pepsinogen A), PYGM (mus- HCl pH 8.8, 6.7 mM MgCl2, 16.6 mM (NH4)2SO4, cle glycogen phosphorylase), PPP1A (protein phos- 10 mM ,B-mercaptoethanol, 4% dimethyl sulfoxide phorylase 1), and two DNA probes (PilEH and (DMSO), 600 gM of each dNTP, 1 igM of each MTC) from the 63-kb bd-1 breakpoint region. primer, and 2 units of Taq polymerase (Perkin-Elmer- In the RH mapping procedure, a human-hamster Cetus). Thirty-five cycles of PCR were performed on hybrid cell line containing an intact single human a Perkin-Elmer-Cetus thermal cycler at 96°C for 30 s chromosome as the only human material is subjected of denaturation, various temperatures and times of to lethal X-irradiation breaking the chromosome into annealing (table 1), and 72°C for 90 s of extension. fragments and resulting in cell death. These irradiated Twenty microliters ofeach RH PCR amplification was cells are then fused to a nonirradiated recipient ham- electrophoresed through mixed agarose gels (2% Nu- ster cell line to rescue the human chromosomal frag- Sieve GTG agarose and 1% GTG agarose [FMC Bio- ments in viable RH cells. The presence or absence of products]) containing ethidium bromide (0.5 gg/ml). specific human DNA sequences in RHs is determined Each radiation hybrid was scored for the presence or either by Southern blot hybridization or by PCR-based absence of a specific human chromosome 11 marker assays. The relative order and distance between mark- by visual inspection of PCR-amplified DNA in ethid- ers is determined by a statistical algorithm that esti- ium stained gels. Formost loci, DNA from each hybrid mates the frequency of breakage between markers (0). was amplified and scored in two separate experiments. The greater the value of 0, the greater the distance between markers. Distance, expressed in centiRays Cell Lines and Culture Conditions (cRsooo), is calculated from 0 using a mapping func- Human-hamster, HPRT +, somatic cell hybrid J1 tion. (Kao et al., 1976) was maintained in Hams F-12 Using this RH mapping technique, we have derived medium supplemented by 10% FCS, 10,000 U a high-resolution map of 11q12-13 that places SEA penicillin/ml, and 1 tig streptomycin/ml (pen/strep). within the flanking markers for MEN-1 and confirms A Chinese hamster cell line deficient for hypoxanthine- the previously reported location of genes HSTF1 and guanine phosphoribosyltransferase (HPRT) activity, INT2 near the bcl-1 locus. 380-6, was maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FCS, and pen/strep. Material and Methods Generation of RH Cell Lines PCR Assays Human-hamster RH somatic cell lines were gener- PCR assays were developed to determine the pres- ated as described elsewhere (Cox et al. 1989). In brief, ence or absence of human chromosome 11 loci in RHs donor HPRT + J1 cells (containing chromosome 11 by designing oligonucleotide sequences that can be as the only human material) were lethally irradiated used to selectively amplify human but not hamster with 9,000 rads of X-rays and fused to HPRT- sequences. Oligonucleotide sequences specific for each deficient 380-6 hamster cells using polyethylene gly- of the 16 loci are summarized in table 1. Primer se- col. J1 x 380-6 RHs were selected by growth in HAT quences for most genes except SEA were chosen from medium (DMEM, 10% FCS, 100 gM hypoxanthine, Radiation Hybrid Map of 1lql2-13 1191

Table I PCR Primers and Amplification Conditions

ANNEALING FRAG- CONDITIONS MENT SIZE Time Temperature Locus NAME (bp) PRIMER SEQUENCE (min) (OC) bcl-1 MTC ...... 82 GAGCTCCCTGAACACCTGGC 30 58 GAGGCTTTGTCCTACCATCC bcl-1 P11EH ...... 506 GGTGGGATCCAAGGGAAGTTGGTGC 60 55 TCCCGGATCCACAGCCTATTTCGGG CD5 ...... 431 AGAAGGATCCGCAGGGGTGGATGCT 30 64 ACTGGGATCCATGAGCAAAAAGCCG CD20 ...... 144 CTGAGGATCCAAGGCCTCAAATCTC 60 55 ACCGGATCCCTATCAGCGATTTCAT COX8 ...... 176 TCCCTCACACTGTGACCTGACCAGC 30 65 GGGGACCCCACCAAGCAGGGTCAGT C1NH ...... 428 CTGTGTGGGCTGACAGAGG 45 65 AGTGGTTTGGAGAGCATGG FTH1 ...... 173 GTCAAAAGACGCGGACAGAA 60 55 AATGGCGGGTGACAGGTGAA GST3 ...... 509 GAGGTTCAGTAAACACAGCC 60 63 TTAGCAAAGCAGAGCAGACC HSTF1 (set 1) ...... 549 CACCGATGAGTGCACGTTCAAGGAG 30 65 CAGACAGAGATGCTCCACGCCATAC HSTF1 (set 2) ...... 430 GCCCTTCCCTTCTCTAGTCC 30 60 GTTCCCCTTCTTGGTCTTCC INT2 ...... 251 CAGAGCTCCACGCCCAGTGATCTTT 30 65 AAGAGCTCTGGCTGCCCCAAGGAAC KRN1 ...... 228 AGTGGGAAACCTCAGGTAGCTCCCGA 30 65 CAGTTTGGCTCAGACATATGGGGGCA OSBP ...... 386 CAGAGCTTGCTCTGACTCTC 60 55 CATCCTCTGTCCTCTTCTCC PGA ...... 343 CCGAATCCCTGGACACTAAGCCAGG 30 65 AAAGTGGGCACAGGACAGAGGCCAG PPP1A ...... 109 CACACCACCCTGTGCCCCAGATGAT 30 65 AAGGTCCATGTTCCCCGTGACAGGT PYGM ...... 704 TGCTCATGCACCATGACCGGTGAGC 30 65 CCAAGAGAGTGTGACAGACTCAAGG SEA ...... 130 CTCAAGGCCAGGCATCACT 90 60 GGACTCTTCCATGCCAGTG

1 pM aminopterin, 12 gM thymidine, and pen/strep). One HPRT revertant was observed in 107 control The surviving cells received a functional HPRT gene 380-6 cells. Of 205 RHs, 171 were expanded as single from J1, together with nonselectively retained frag- colonies in HAT medium. Ofthese, 102 ofthe fastest- ments of human chromosome 11. The only change to growing cell lines were expanded for DNA extraction the published protocol (Cox et al. 1989) was to in- and subsequent PCR analysis. crease the proportion of recipient to donor cells from 1:1 to 2:1. An average of 1.3 J1 x 380-6 RH clones Analysis of RH Data per plate were evident 14-21 d after fusion, indicating The order and intermarker distances of 16 chromo- an efficiency of hybrid formation of approximately some 11q12-13 markers were determined by a statisti- one RH per 4 x 104 recipient 380-6 cells. No growth cal analysis of the cosegregation of markers in RHs was observed in 107 irradiated, nonfusedj1 cells, indi- using the methods of moments to estimate 0 (Cox et cating 9,000 rads was a lethal cell dose of irradiation. al. 1990). This approach makes use ofdata on all pairs 1192 Richard et al. ofmarkers in every hybrid for which both markers are Results scored. The method assumes that breakage occurs at We scored 101 RHs for the presence or absence of random along the chromosome and that fragments are 16 specific human chromosome 11 DNA markers by retained independently. Because the retention fre- visual inspection of PCR-amplified DNA in ethidium- quencies of the individual markers were not signifi- stained gels. For most loci, DNA from each hybrid cantly different in this data set, we used a single reten- was amplified and scored in separate experiments in tion frequency, R, in our analysis: two different laboratories. Concordant results were N N obtained approximately 98% of the time. CDS and R = >2Rm/ZF Tm, CD20 were retained or lost together in all RHs tested m=1 m=1 andwere analyzed as a single locus in further statistical analysis. HSTF1, INT-2, MTC, and P1 1EH also co- where Rm is defined as the number of hybrids that segregated together and were analyzed as a single lo- retain marker m, Tm is defined as the total number of cus. The R's of the 16 markers ranged from 21% to hybrids scored for marker m, and N is defined as the 39%, with an average R of 26% (table 2). Since the total number of markers. We estimate 0 between two marker R's were not statistically different from one A and the following equation: 0 = markers, B, by another, an average R was used in the RH algorithm [(A+Bj-) + (A-B+)] / [2(T)(R)(1 - R)] where (A+B) to estimate two-point distances (see Material and is the observed number of hybrid clones retaining Methods). Distances and lod scores of all pairwise A not (A-B+) is the observed marker but marker B, marker combinations with lod scores above 3 are clones B but not number of hybrid retaining marker shown in table 3. The most likely order of loci on marker A, T is the total number of hybrids analyzed 1 1q12-13, defined as that which maximized the sum for both markers A and B, and R is as defined above. of the lod scores of adjacent loci, was cen-ClNH- The mapping function, D = -In (1 -8), is used to OSBP-(CD5,CD20)-PGA-FTH1-COX8-PYGM-SEA- estimate D, the distance between two markers. D is KRN1-(MTC,P1 1EH,HSTF1 ,INT2,)-GST3-PPP1A- expressed in centiRays, where a distance of 1 cRsooo tel. Alternate orders were then compared to the best corresponds to a 1 % 0 of between two markers after order as described in Material and Methods. In this exposure to 9,000 rads of X-rays. The lod score for RH map, all markers except GST3 were ordered with a marker pair is defined as lod = log [L(O)/L(0 = 1)] where L(O) is the likelihood of obtaining the observed data for a given pair of markers assuming that the frequency of breakage between markers is 0, and Table 2 = is two marker L(O 1) the likelihood assuming that the RH Marker R's are not linked; that is, that 0 = 1 (Cox et al. 1990). The maximum lod score for 0<0<1 is used to identify Marker Locus R Hybrids Scored marker pairs for which there is significant evidence of C1NH ...... 39 99 linkage, in a manner analogous to meiotic mapping. OSBP ...... 33 101 Because we use a single R in this analysis, and since CD5 ...... 31 101 each hybrid is scored for almost every marker, the CD20 ...... 31 101 PGA ...... 30 101 likelihood for any order of the set of markers is esti- FTH1 ...... 31 101 mated as the sum of the pairwise lod scores between COX8 ...... 25 100 adjacent markers. The map with the highest likelihood PYGM ...... 22 101 is defined as that which includes the entire set ofmark- SEA ...... 21 101 ers in an order such that the sum of the lod scores KRN1 ...... 21 101 KRN1 ...... 21 101 This map is between adjacent loci is maximized. HSTF1 ...... 23 101 readily determined by inspection using a trial and error INT2 ...... 23 101 process (Cox et al. 1990). The likelihood of this best P1IEH ...... 23 101 order is then compared to the likelihoods of orders in MTC ...... 23 101 which two to six adjacent loci are inverted. Loci that GST3 ...... 21 100 PPP1A ...... 23 101 can be on the map with odds of to 1 over placed 1,000 Average ...... 26 alternative orders are considered as framework loci. Table 3 RH Two-Point Analysis

No. OF CLONES OBSERVED Marker A Marker B cRsooo lod + + - - Total

C1NH ...... OSBP 50 10.81 28 11 4 56 99 CINH ...... CD5a 92 5.50 23 16 7 53 99 C1NH ...... PGA 80 6.57 24 15 6 54 99 C1NH ...... FTH1 86 6.16 24 15 7 53 99 C1NH ...... COX8 101 4.28 20 19 S 54 98 OSBP ...... CD52 23 15.82 28 5 3 65 101

OSBP ...... PGA 33 12.77 26 7 4 64 101

OSBP ...... FTH1 53 9.06 24 9 7 61 101

OSBP ...... COX8 105 3.10 16 16 9 59 100 CD5 ...... PGA 14 18.59 28 3 2 68 101 CD5a ...... FTH1 23 15.41 27 4 4 66 101 CD5 ...... COX8 68 5.63 18 12 7 63 100 CD5a ...... PYGM 89 3.45 15 6 7 63 101 CD5a ...... SEA 72 4.70 16 15 S 65 101 CD5 ...... KRN1 91 3.12 14 16 7 63 100 CD5a ...... INT2a 83 4.03 16 15 7 63 101 CD5 ...... GST3 85 3.69 15 6 6 63 100 PGA ...... FTH1 14 18.59 28 2 3 68 101 PGA ...... COX8 45 8.61 20 9 66 100 PGA ...... PYGM 62 5.78 17 13 66 101 PGA ...... SEA 67 5.07 16 14 66 101 PGA ...... KRN1 85 3.40 14 15 7 64 100 PGA ...... INT2a 89 3.45 15 15 8 63 101 PGA ...... GST3 79 4.00 15 15 6 64 100 FTH1 ...... COX8 41 9.53 21 9 4 66 100 FTH1 ...... PYGM 57 6.54 18 13 4 66 101 FTH1 ...... SEA 62 5.78 17 14 4 66 101 FTH1 ...... KRN1 79 4.00 15 15 6 64 100 FTH1 ...... INT2a 83 4.03 16 15 7 63 101

COX8 ...... PYGM 23 12.07 19 6 2 73 100

COX8 ...... SEA 34 9.21 17 8 3 72 100

COX8 ...... KRN1 45 7.17 16 9 S 70 100 COX8 ...... INT2a 68 4.37 14 11 8 67 100

COX8 ...... GST3 80 3.06 12 13 8 66 99

COX8 ...... PPP1A 85 3.12 13 12 10 65 100 PYGM ...... SEA 20 12.56 18 4 3 76 101 PYGM ...... KRN1 30 9.46 15 7 8 71 101 PYGM ...... INT2a 49 6.41 15 7 8 71 101 PYGM ...... GST3 49 5.98 14 8 7 71 100 PYGM ...... PPP1A 67 4.12 13 9 10 69 101 SEA ...... KRN1 27 10.13 16 4 S 75 100 SEA ...... INT2a 30 9.95 17 4 6 74 101 SEA ...... GST3 30 9.46 16 S S 74 100 SEA ...... PPP1A 45 6.89 15 6 8 72 101 KRN1 ...... INT2a 14 14.66 19 2 3 76 100 KRN1 ...... GST3 50 5.55 13 8 7 71 99 KRN1 ...... PPP1A 63 4.41 13 8 10 69 100 INT2a ...... GST3 37 8.24 16 7 S 72 100 INT2a ...... PPP1A 53 5.96 15 8 8 70 101

GST3 ...... PPP1A 14 14.66 19 2 3 76 100 NOTE. -Distances between 1 1q12-13 loci were determined by RH mapping. All pairwise combinations of markers with lod scores above 3.0 are listed under markers A and B. "No. of Clones Observed" shows the number of hybrids that retain both marker A and B ( + + ), marker A but not marker B ( + - ), marker B but not marker A (- + ), neither marker A nor B ( - - ) and the total number of hybrids analyzed. The lod score for each marker is a measure of the likelihood that the two markers are linked. a CD5 and CD20 are treated as a single locus, as are INT2, HSTF1, MTC, and PllEH. 1194 Richard et al.

RADIATION HYBRID MAP 1 la 12-13 in situ hybridization, while HSTF1, PPP1A, bcl-1, and GST3 have been mapped to 11q13.3. The 11q12- HUMAN JIX3806 CANCER CHROMOSOME RADIATION DISEASE 11q13.3 region makes up approximately 10% of the 11 HYBRID GENES total length of chromosome 11 by quantitative mea- MAP surement of band size (Francke and Oliver 1978). 1 1q CEN Given an estimate of 150 Mb for the total length of chromosome 11, the 11q12-11q13.3 region encom- CINH passes approximately 15 Mb. Since our RH map mea- sures 277 cRgooo between C1NH and GST3, 1 cRsooo OSBP equals approximately 55 kb. This estimate is compa- CD5, CD20 rable to cRgooo/kb ratios obtained on ilpiS and PGA 1 1q22-23 with this RH panel and ratios obtained in FTH6L other RH panels of other human con- structed with the same dose ofX-rays (Cox et al. 1990; I COX8 D. R. Cox, unpublished data). I PYGM PYGM Wada et al. (1988) have shown that HSTF1 lies 35 SEA MEN- I kb downstream of INT2 in the same transcriptional , KRN1 orientation. By pulsed-field gel electrophoresis (PFGE), HSTF 1,INT2 bcl- I I INT2 MTC,P1 IEH Nguyen et al. (1988) have discovered colocalization [GST3]* of bcl-1 and INT2 to the same 1,050-kb NruI PFGE 1 PPla fragment, and Bale et al. (1990) have reported colocal- ization of bcl-1 and INT2 to the same 480-kb Sall PFGE fragment. Our RH data are in agreement with these results, as no breaks occur between MTC, P1 1EH, HSTF1, or INT2. In addition, the gene order Figure I Graphic representation of RH map ofhuman chro- on the RH is mosome 1lq12-13. cRgooo distances are shown to the left of the PGA-PGYM-INT2 map consistent with figure. Locations of MEN-1 and bcl-1 disease gene loci are shown the gene order determined by meiotic linkage analysis to the right ofthe RH map. With the exception ofGST3, all markers (Julier et al. 1990). are placed on the RH map at odds greater than 1,000 to 1 compared Amplification of several 11q12-13 markers (HSTF1, to orders in which up to six adjacent loci are inverted. The pound INT2, bcd-1, SEA, and GST3) has been noted in breast sign (#) indicates that GST3 is only 200 times more likely to be centromeric rather than telomeric to PPla. cancer and other aggressive solid tumors in humans (Tsuda et al. 1988, 1989a, 1989b; Yoshida et al. 1988; Berenson et al. 1989; Theillet et al. 1989,1990; Bale et al. 1990; Saint-Ruf et al. 1991). In a survey odds of greater than 1,000 to 1 against inversion of of 11q12-13 amplicon size in 297 mammary tumors, up to six adjacent loci. GST3 was 200 times more Theillet et al. (1990) found six carcinomas where bcl-1 likely to be located centromeric to PPP1A than te- alone was amplified, 50 tumors with co-amplification lomeric. The total region spanned by the 11q12-13 of bcl-1, HSTF1, and INT2, and three tumors where loci was 277 cRgooo. A graphic representation of this amplification extended to SEA. This amplicon distri- RH map with cRgooo distances is shown in figure 1. bution suggests a linear order of these loci that is con- sistent with our RH map. Human genes on 11q12-13 map to several different Discussion mouse chromosomes. The genes FTH1, PYGM, We demonstrate here the utility ofusing PCR-based OSBP, CD5, and CD20 form a syntenic group with RH mapping to provide new order information for mouse chromosome 19 (Levanon et al. 1990; Nadeau 16 chromosome 11q12-13 markers. Comparison of 1990). On mouse chromosome 19, however, the syn- cRgooo distance to actual physical distance is difficult teny group is interrupted by Ahd-2, a human chromo- since precise physical measurement of distances be- some 12 gene. The order of genes located on either tween these genes on chromosome 11 is not currently side ofthis interruption is consistent with the RH order known. C1NH, the most centromeric marker, was of these markers on human chromosome 11. Other previously localized to the 1 1q12-1 1q13.1 region by genes on our chromosome 11 qi 2-13 map are homolo- Radiation Hybrid Map of llql2-13 1195 gous to genes on other mouse chromosomes. Human telomeric to INT2, this gene appears unlikely to be HSTF1 and INT2 are syntenic with mouse 7, while directly involved in bcl-1 or MEN-1. the more telomeric locus GST3 is syntenic with mouse chromosome 9. Note added in proof: A candidate bcl-1 gene has Our findings have a number ofimplications relevant recently been identified approximately 110 kb telo- to the identification of the cancer-associated genes meric to the MTC (Withers et al. 1991). bcl-1 and MEN-1. Figure 1 shows the RH map in relation to MEN-1 flanking markers and the bcl-1 Acknowledgments translocation cluster. We have confirmed the previous assignment of the human protooncogene SEA to C.W.R. is supported by a Pfizer postdoctoral fellowship 1 1q13 and have localized SEA to between the MEN-1 and NIMH Physician Scientist Award MH00802. T.M. is supported by an American Cancer Society research award. flanking markers, PYGM and INT2. Prior linkage D.R.C. and R.M.M. are supported by NIH grants. Cell data (Larsson et al. 1989) in MEN-1 families showed lines J1 and 380-6 were generous gifts of Carol Jones and SEA linked to MEN-1 with a lod score of 1.13 atO = 0. Uta Francke, respectively. We thank Phil Byrd for the se- Although this lod was lower than that of INT2 or quence ofHSTF1 primer set 2 and Doug Smith ofCollabora- PYGM, the SEA probe was less informative than the tive Research for the genomic SEA probe. other markers studied. Multipoint linkage analysis (Bale et al. 1989) places the MEN-1 locus 2.8 cM References centromeric to INT2, in a region we now estimate to be very close to SEA. We suggest further investigation Bale SJ, Bale AE, Stewart K, Dachowski L, McBride OW, of this protooncogene in MEN-1 families. At a mini- Glaser T, Green J, et al (1989) Linkage analysis of multi- mum, a more informative SEA probe could prove use- ple endocrine neoplasia type 1 with INT2 and other mark- ful in further narrowing down the MEN-1 region ers on chromosome 11. Genomics 4:320-322 by Bale AE, Wong E, Arnold A (1990) The parathyroid ade- linkage analysis or tumor deletion analysis. We have noma breakpoint sequence in 1q13 maps close to BLC1 also assigned KRN1 to between the MEN-1 flanking and is not candidate for MEN1. Am J Hum Genet 47:A1 markers PYGM and INT2. Although it is unlikely that Barker HM, Jones TA, da Cruz e Silva EF, Spurr NK, Sheer this keratin gene ofthe hair cuticle plays any causative D, Cohen PT (1990) Localization of the gene encoding a role in MEN-1, a polymorphic KRN1 probe could type I protein phosphatase catalytic subunit to human also assist in defining the MEN-1 gene by linkage anal- chromosome band 11q13. Genomics 7:159-166 ysis or tumor deletion analysis. BerensonJR, YangJ, Mickel RA (1989) Frequent amplifica- CD20 and CD5 encode B lymphocyte-specific, tion of the bcl-1 locus in head and neck squamous cell cell-surface molecules that are involved in B cell activa- carcinomas. Oncogene 4:1111-1116 tion and differentiation. Hecht et al. (1989) and Ted- Boyer TD (1989) The glutathione S-transferases: an update. al. Hepatology 9:486-496 der et (1989) have speculated that alterations in the Bystrom C, Larsson C, Blomberg C, Sandelin K, Falkmer expression ofthe CD20 or CDS genes may result after U, Skogseid B, Oberg K, et al (1990) Localization of the a t(i 1;14) chromosomal alteration. Our data suggest MEN1 gene to a small region within chromosome 11q13 CD20 and CDS to be 153 cRsooo (an estimated 8.4 by deletion mapping in tumors. Proc Natl Acad Sci USA Mb) centromeric to bcl-1, and therefore unlikely to be 87:1968-1972 directly involved in bcl-1. Cox DR, Burmeister M, Price ER, Kim S, Myers RM (1990) The potent tumor suppressor okadaic acid selec- Radiation hybrid mapping: a somatic cell genetic method tively inhibits PPP1A and PPP2A, leading Barker for constructing high resolution maps of mammalian (1990) to speculate that PPP1A may be a candidate chromosomes. Science 250:245-250 tumor-suppressor gene, the mutation of which could Cox DR, Pritchard CA, Uglum E, Casher D, Kobori J, My- result in MEN-1. Our data place PPP1A 51 cRgooo (an ers RM (1989) Segregation of the Huntington disease re- 2.8 gion of human chromosome 4 in a somatic cell hybrid. estimated Mb) distal to the MEN-1 region, and it Genomics 4:397-407 is therefore unlikely to be the MEN-1 gene. Francke U, Oliver N (1978) Quantitative analysis of high GST3 (GST-7t) is overexpressed in many cancers, resolution trypsin-Giemsa bands on human prometa- and it has been postulated that it plays a role in many phase chromosomes. Hum Genet 45:137-165 drug-refractory malignancies (for review see Boyer Hecht B. Kipps T, Johnston N, Cannizaro L (1989) Leu 1 [1989] or Morrow and Cowan [1990]). As our data (CD5) cell surface antigen mapped to 11q13 by in situ place the GST3 gene 37 cRgooo (an estimated 2.0 Mb) hybridization. Cytogenet Cell Genet 51:1012 1196 Richard et al.

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