Proc. Natl. Acad. Sci. USAVol. 80, pp. 4794-4797, August 1983Genetics

Localization of c-ras oncogene family on humangerm-line chromosomes

(c-onc gene mapping/chromosome change/cancer)

SURESH C. JHANWAR*, BENJAMIN G. NEELt, WILLIAM S. HAYWARD*, AND R. S. K. CHAGANTI**Memonal Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021; and tCornell University Medical College, 1300 York Avenue, New York,New York 10021

Communicated by Paul A. Marks, April 19, 1983

ABSTRACT The c-ras family is a set of c-onc genes that arehighly conserved in vertebrates. The genes in this family are ho-mologous to the transforming genes of Harvey and Kirsten mu-rine sarcoma viruses (v-Ha-ras and v-Ki-ras, respectively). Usingan in situ molecular hybridization method, we detected three siteson the human pachytene chromosomes that exhibited significanthybridization to v-Ki-ras and v-Ha-ras probes. These were chro-momere positions that corresponded to bands lIpl4.1, 12pl2.1,and 12q24.2 of somatic chromosomes. The relationship betweenthese chromosomal sites and previously defined members of thehuman c-ras gene family is discussed. These chromosomal sitesare known to be involved in specific chromosome changes in a va-riety of tumors and in several congenital disorders that predisposeto neoplastic disease.

c-onc genes are normal cellular genes that are homologous tothe transforming genes (v-onc genes) of RNA tumor viruses (1).Although the normal cellular functions of c-onc genes are un-known, it seems likely that they play a role in the control of cellproliferation, development, or differentiation. Several so-called"human transforming genes" identified by transfection of NIH3T3 mouse cells with DNA from human tumors have been shownto be c-onc genes (2-5). Abnormal expression or changes in thecoding sequences of c-onc genes play important roles in theinduction of tumors by both slowly transforming RNA viruses(6) and nonviral agents (7).The occurrence of specific chromosome abnormalities, es-

pecially translocations, has been recognized for some time as afeature of most tumor types (8-10). Recent data on the assign-ment of c-onc genes to human chromosomes from somatic cellhybridization (11-18) or in situ molecular hybridization studies(19, 20) have shown that a number of c-onc genes are situatedon the same chromosomes that are involved in specific abnor-malities in tumor cells. In several cases, the positions of c-oncgenes correspond to the breakpoints in chromosomes that gen-erate specific translocations in tumor cells (18-21). An exampleof the latter is the chromosomal position of c-myc and its pos-sible activation by translocation to regions containing the im-munoglobulin genes in human Burkitt lymphoma. c-myc is lo-cated on human chromosome 8 at band 8q24 (19, 20), the siteof its breakage in the formation of a set of translocations thatcharacterize Burkitt lymphoma t(2;8) (pl3;q24), t(8;14) (q24;q32),and t(8;22) (q24;qll) (22-24). The human immunoglobulin Klight chain, A light chain, and heavy chain genes have been lo-calized to 2pl3>cen, 22q, and 14q32, respectively (25-27). Inthe 8/14 translocation c-myc is translocated into the immu-noglobulin locus (18, 20). An analogous translocation involving

the c-myc and immunoglobulin C, loci occurs in BALB/c plas-macytomas (20, 28, 29). c-myc was previously shown to be ac-tivated by insertion of proviral regulatory sequences in B-celllymphoma induced by avian leukosis viruses (30). Recently, Er-ikson et al. (31) have reported that c-myc expression is increasedin Burkitt lymphoma cells in comparison to lymphoblastoid cellstransformed by Epstein-Barr virus.

The c-ras genes are a highly conserved and complex familyof c-onc genes present in vertebrates (32, 33). These genes arehomologous to the v-onc genes of the Harvey and Kirsten mu-rine sarcoma viruses (v-Ha-ras and v-Ki-ras, respectively). Tu-mor DNA transfection experiments in several laboratories haveimplicated the c-ras gene family in human bladder (2, 3) andcolon carcinomas (4) and neuroblastoma (5). Because of the pos-sible role of c-ras genes in human neoplasia and the furtherpossibility that these genes might be involved in specific neo-plasia-related chromosome abnormalities, we investigated thechromosomal positions of the c-ras genes. We present here ourresults of mapping the germ-line positions of these genes by insitu molecular hybridization. We find three significant sites ofhybridization on meiotic pachytene chromosomes correspond-ing to somatic metaphase bands 11pl4.1, 12p12. 1, and 12q24.2.A fourth site, which exhibited consistent but weak hybridiza-tion, was observed at 3p21.3.

MATERIALS AND METHODS

Molecular Probes. Clones BS-9 and HiHi 3 consist, respec-tively, of a 450-nucleotide insert containing v-Ha-ras and a 1.0-kilobase-pair fragment containing v-Ki-ras; both were cloned inthe EcoRI site of pBR322 and lack sequences homologous to rat30S RNA (32, 34). The clones were generously supplied by R.Ellis and E. Scolnick. For use as probes in in situ hybridizationexperiments, these plasmids were labeled with 3H to high spe-cific activity (5-10 x 107 dpm/,ug) by nick-translation as de-scribed (19).

In Situ Hybridization. Chromosome preparation, hybridiza-tion, autoradiography, and grain counting were performed ac-cording to methods described previously (19, 35, 36). How-ever, in the present study, we considered each chromosomalsite in each cell that exhibited autoradiographic grains to be one"hybridization event" at that site. We thus analyzed hybrid-ization events instead of grains. We feel that this is a more ac-curate representation of the hybridization specificity than ananalysis based on total grain count because grain numbers mayvary from experiment to experiment due to technical factorssuch as differences in the specific activities of the probes, hy-bridization time, and the length of autoradiographic exposure.In the present experiments, grains at a given site ranged innumber between 1 and 5, although single grains predominated.

4794

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Genetics: Jhanwar et al. Proc. Natl. Acad. Sci. USA 80 (1983) 4795

RESULTSForty-three informative autoradiographs of pv-Ki-ras probe hybridization and 51 from Xbridization were available for analysis. Theshybridization events involving v-Ki-ras andras. Of the 94 v-Ki-ras hybridization events,

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tributed at three specific chromosome regions, which corre-achytene cells from sponded to the somatic metaphase bands (36), namely, 11p14.1v-Ha-ras probe hy- (9%), 12pl2.1 (6%), and 12q24.2 (14%) (Fig. 1 d-l). The de-e cells exhibited 94 viation from expected number of hybridizations was calculated97 involving v-Ha- on the basis of random distribution proportional to the length27 (29%) were dis- of the chromosome arms and was found to be highly significant

at these three sites (P < 0.001) (Fig. 2a). Of the 97 v-Ha-rashybridization events, 39 (40%) occurred at the same three sitesas above with frequencies of 19%, 5%, and 16%, respectively(Fig. 1 d-l). The deviation from expected hybridizations on eachof these chromosome arms was again highly significant (P <

p. 0.001) (Fig. 2b).--* Both probes hybridized to one other site-namely, the chro-

momere region corresponding to somatic metaphase band 3p21.3*3 ^ (36) (Fig. 1 a-c). The frequency of hybridization events at this

Is site (v-Ki-ras: 3%; v-Ha-ras: 3%) was not statistically significantwith either probe. However, no other site was detected to which

|q g both probes hybridized; therefore, we feel that sequences withweak homology to the c-ras family are possibly located at 3p2l.3.

At the sites of specific hybridization, the grains in generalC were seen in the region extending from one chromomere distal

to one chromomere proximal to the chromomeres correspond-ing to the bands noted above. Pachytene chromomere and high-resolution somatic metaphase band idiograms of chromosomes3, 11, and 12 showing all the hybridization events and their sitesobserved are illustrated in Fig. 3.

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Chromosome Number b

FIG. 1. Autoradiographs of human pachytene bivalents showingsilver grains (arrows) at sites of specific hybridization with v-Ha-rasand v-Ki-ras probes. Three representative bivalents are illustrated foreach site of hybridization. p and q are short and long arms of chro-mosomes. (a-c) Chromosome 3 (a, v-Ha-ras; b, v-Ha-ras; c, v-Ki-ras).(d-f) Chromosome 11 (d, v-Ha-ras; e, v-Ha-ras; f, v-Ki-ras). (g-l) Chro-mosome 12 (g, v-Ha-ras; h, v-Ha-ras; i, v-Ki-ras; j, v-Ki-ras; k, v-Ha-ras; 1, v-Ha-ras). Note that in g hybridization occurred at sites on botharms.

FIG. 2. Histograms of expected and observed hybridization eventswith v-Ki-ras (a) and v-Ha-ras (b) probes. v-Ha-ras hybridized signif-icantly more frequently than v-Ki-ras at llpl4.1 (P < 0.001). This probealso hybridized more frequently at llpl4.1 and 12q24.2 compared to3p21.3 and 12pl2.1 (llpl4.1 vs. 3p21.3,P < 0.001; llpl4.1 vs. 12pl2.1,P < 0.01; 12q24.2 vs. 3p2l.3, P < 0.01; 12q24.2 vs. 12pl2.1, P < 0.02).v-Ki-ras hybridized significantly more frequently at 12q24.2 than at3p21.3 (P < 0.02); the differences in hybridization frequency of v-Ki-ras between other sites were statistically insignificant.

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Proc. Natl. Acad. Sci. USA 80 (1983)

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3 11 12FIG. 3. High-resolution metaphase band (M) and pachytene chro-

momere (P) idiograms of chromosomes 3, 11, and 12 showing hybrid-ization events. Bands corresponding to chromomeres at which specifichybridizations occurred are indicated by arrows on the left of each Midiogram. Numbers of hybridization events observed along the entirechromosome are indicated on either side of the P idiogram (left, v-Ha-ras hybridization; right, v-Ki-ras hybridization). C is centromere andp and q are short and long arms of chromosomes.

DISCUSSIONThe exact number of c-ras-related genes in the human genomeis at present unclear. Chang et at (33) identified four human c-

ras genes. Two of these (c-Ha-ras 1 and c-Ha-ras 2) are closelyrelated to v-Ha-ras and the other two (c-Ki-ras 1 and c-Ki-ras2) are closely related to v-Ki-ras. However, Shimizu et al. (5)identified a c-ras related human gene that is less closely relatedto either v-Ki-ras or v-Ha-ras and that appears to be differentfrom the four previously identified c-ras genes (E. Stavnezer,personal communication). The number of c-ras-related se-

quences detected in the human genome in Southern blot hy-bridizations strongly depends on the conditions of stringency(E. Stavnezer, personal communication; unpublished data). Theconditions used in our experiments allow cross-hybridizationbetween the different members of the c-ras gene family. Thishas the advantage of increasing the sensitivity of detection ofgenes by in situ chromosomal hybridization.

In our study, significantly more hybridization was found withv-Ha-ras than with v-Ki-ras at band llpl4.1 (P < 0.001). Thisis consistent with the results of somatic cell hybridization ex-

periments that assigned c-Ha-ras 1 to chromosome 11 (15) on

the short arm (16). Thus, we conclude that c-Ha-ras 1 is locatedat llpl4.1. Sakaguchi et al (17) reported that c-Ki-ras 2 is lo-cated on chromosome 12. Presumably, this gene is located ateither 12p12.1 or 12q24.2. The weak signal at 3p2l.3 may rep-resent one of the c-ras-related sequences that has less homol-ogy to the v-ras probes used in our hybridizations. Experimentsusing higher stringency conditions of hybridization and specificc-ras gene probes may help resolve these issues.

The sites to which we have mapped c-ras-related sequencesare involved in gain, loss, deletion, duplication, and translo-cation (with breakpoints in lipl4, 12pll, 12q24, and 3p2l) ina number of myeloid and lymphoid neoplasms (9, 37, 38). Aninherited translocation, t(3;8) (p21;q24), has been reported tobe associated with hereditary renal cell carcinoma in one family(39). In a patient with familial renal cell carcinoma but withoutcongenital chromosome abnormality, the tumor cells carried a

t(3;11) (pl3;plS) (40). Whang-Peng et al. (41) observed a spe-cific deletion encompassing the region 3pl4.3p23 in small cellcarcinoma of the lung.

Germ-line deletion of the region 11p13.11pl4.1, to which

we have mapped c-Ha-ras 1, has been associated with the an-iridia-Wilms tumor syndrome, a rare congenital anomaly ofchildren characterized by aniridia, multiple developmental de-fects, mental retardation, and Wilms tumor (42, 43). In one pa-tient with Wilms tumor unassociated with the aniridia-Wilmstumor syndrome and with normal constitutional chromosomalcomplement, an interstitial deletion involving the regionllpl3-41p14 was detected in tumor cells (44). Such a deletionmight alterc-ras expression (e.g., by removing a negative mod-ulator or by joining the coding sequences to an active positivemodulator) or cause change in the primary structure of the on-cogene product. Alternatively, germ-line deletion of a c-ras gene,which may play a role in normal development, could lead todevelopmental abnormalities and predisposition to neoplasia.

Note Added in Proof. While this manuscript was in press O'Brien etal (45) reported somatic cell hybridization studies showing ras-relatedgenes to be present on chromosomes 6 (c-Ki-ras-1), 11 (c-Ha-ras-1), 12(c-Ki-ras-2), and X (c-Ha-ras-2). We have studied a total of 73 infor-mative pachytene cells from three individuals (43 from the study re-ported above and 30 additional cells from a new study) hybridized insitu with the v-Ki-ras probe and found no specific hybridization onchromosome 6. Therefore, our studies do not reveal a germ-line site forc-Ki-ras on chromosome 6. Because the sex chromosomes form the sexvesicle at pachytene, hybridization on the X chromosome could not beevaluated.

We thank Winston Hew and Anne Manwell for excellent technicalassistance and Seeta Chaganti for performing the statistical analyses.This work was supported by Grants CA 20194, CA 34502, and CA 34775from the National Institutes of Health and by the Flora E. Griffin Me-morial Fund. B.G.N. is a biomedical fellow in the Medical ScientistTraining Program of the National Institutes of Health.

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