Molecular cloning and chromosomal mapping of a human locus related to the transforming gene of Moloney murine sarcoma virus.

Abstract

Human DNA was analyzed for the presence of sequences homologous to the transforming gene (v-mos) of Moloney murine sarcoma virus. A single 2.5-kilobase pair (kbp) EcoRIgenerated fragment of human DNA was identified by using cloned v-mo8 as probe. This DNA was molecularly cloned in a bacteriophage vector. By heteroduplex and restriction enzyme analyses, this human DNA fragment, designated c-mos (human), contained a 0.65-kbp region ofcontinuous homology with v-mos and was present as a single copy in human DNA. By testing for the presence of c-mos (human) in somatic cell hybrids possessing various numbers of human chromosomes, as well as in subelones of such hybrids, it was possible to assign c-mos (human) Cit/10% dextran sulfate (10). Filters were washed at 450C in 0.1 X NaCi/Cit.Enrichment and Molecular Cloning ofc-mos (Human). High molecular weight DNA was extracted from human placenta tissue as reported (11). EcoRI digested DNA was subjected to RPC-5 column chromatography (12) and agarose gel electrophoresis followed by blotting analysis (13). Hybridization was performed under relaxed conditions. A nick-translated, molecularly cloned v-mos fragment (14) composed of sequences extending from the Bgl I site to the HindIII site was used to probe the fractions. This probe contained 80% of the 1. 15-kbp v-mos gene. The appropriate RPC-5 fractions were pooled and subjected to further fractionation in a 1.0% preparative agarose gel. Those fractions hybridizing to v-mos were ligated to EcoRIcut A Charon 16A DNA, packaged in vitro (15), and plated onto Escherichia coli LE 392. Plaques containing recombinant phage were detected (16) and purified.Restriction Enzyme Mapping. Restriction endonuclease mapping was carried out by single and double digestion analysis and the partial digestion method of Smith and Birnstiel (17).Electron Microscopy. Heteroduplex analysis was performed according to the method of Davis et al. (18). Equal amounts of the appropriate DNAs (1-2 pRg) were mixed, denatured with 0.1 M NaOH/0.02 M EDTA (pH 8.5), neutralized with 0.2 M Tris HCI (pH 7.2), and hybridized in 50% formamide. After dialysis against 10 mM Tris.HCI/1 mM EDTA (pH 8.5) at 40C overnight, a portion was mixed with 100 mM Tris-HCl (pH 7.8)! 10 mM EDTA and cytochrome c at 50 Ag/ml and spread onto a water hypophase. The heteroduplexes were transferred to parlodion-coated grids, stained with 0.1 mM uranyl acetate, and shadowed with platinum/palladium. By using a Tektronix graphics system, DNA contour lengths were measured, with single-and double-stranded 4X174 DNA serving as internal length standards.Cell Hybridization. Human and rodent cells were cocultivated (1: 1 mixture) for 24 hr before induction ofcell fusion with 52.5% (vol/vol) polyethylene glycol 1000 (19)

Two human c-onc genes are located on the long arm of chromosome 8.

“…The c-sis gene (simian sarcoma virus) was localized to chromosome 22 (61), the c-fes (Snyder-Theilen strain of feline sarcoma virus) to chromosome 15 (60), and c-myb (avian myeloblastosis virus) to chromosome 6 (62). In addition, in agreement with our results, Prakash et ad have reported that c-mos is on chromosome 8 (63). It is intriguing that each of these chromosomes has been implicated in neoplasia-associated chromosome abnormalities.…”

Isolation and analysis of the 21q+ chromosome in the acute myelogenous leukemia 8;21 translocation: evidence that c-mos is not translocated.

“…Analysis of cells from two Ph'-positive patients has revealed that the breakpoint in chromosome 9 is near c-abl (9 (11), has been mapped by using in situ hybridization to band 8q22, the band involved in the 8;21 translocation (12). Moreover, band 21q22, the site of the breakpoint on the chromosome 21 involved in this translocation, is a region which, when trisomic, leads to the development of Down syndrome, the most common chromosomal cause of mental retardation in humans and a chromosomal disease with an increased risk of leukemia (13 …”

“…(The human chromosome assignment of each isozyme marker is indicated in brackets.) The isozymes used were: glutathione reductase (GSR) [8]; lactate dehydrogenase A (LDHA) [11]; hexosaminidase A (HEXA) [15]; and soluble superoxide dismutase (SOD1) [21].…”

Localization of c-ras oncogene family on human germ-line chromosomes.

“…Recent data on the assignment of c-onc genes to human chromosomes from somatic cell hybridization (11)(12)(13)(14)(15)(16)(17)(18) or in situ molecular hybridization studies (19,20) (18,20). An analogous translocation involving the c-myc and immunoglobulin C, loci occurs in BALB/c plasmacytomas (20,28,29).…”