Linkage in cultured Chinese hamster cells of two genes, emtB and leuS, involved in protein synthesis and isolation of cell lines with mutations in three linked genes.

Abstract

We have determined via segregation analyses from appropriate hybrids that two genes involved in protein synthesis, one encoding for a ribosomal protein (emtB) and one encoding for leucyl-tRNA synthetase (IeuS), cosegregate at a very high frequency and are linked in both Chinese hamster ovary and lung cells. In contrast, the emtA locus, defined by a second complementation group of emetine-resistant mutants which also have alterations affecting protein synthesis and probably the ribosome, is not linked to IeuS . In addition, we have determined that a third gene, one that can be altered to give rise to chromate resistance, is syntenic with emtB and IeuS . We have selected cell lines with mutations in each of these three linked genes and have shown that the three loci cosegregate at a high frequency. Because the mutations in these three linked genes provide easily distinguishable phenotypes, these cell lines should provide a powerful tool for examining several important questions concerning mitotic recombination in somatic cells.The genetic and biochemical dissection of protein synthesis in mammalian cells has benefited greatly in recent years from the use of mutant methodology. A large number of cultured somatic cell mutants have been isolated with alterations in different components of the protein synthetic machinery (9,10,12,16,18,19). One goal of this laboratory is to expand the number of such mutants in cultured Chinese hamster cells and to characterize them at the molecular level, especially mutants with alterations affecting various components of the ribosome . A second goal is to use such mutants to begin constructing a genetic map of this group of functionally related genes.We have isolated and partially characterized three distinct types of Chinese hamster cell mutants that were selected as being resistant to the protein synthesis inhibitor, emetine (19; J Hill, L. Vock, and J. Wasmuth, manuscript in preparation) . Although the phenotypes of all three types of mutants are very similar, and all have alterations affecting protein synthesis directly, they clearly define three distinct complementation groups . For all three classes, the emetine-resistant phenotypes are recessive. We have designated the loci defined by these different complementation groups as emtA, emtB, and emtC . Two laboratories (4, 8) have obtained evidence that Chinese hamster ovary (CHO), but not Chinese hamster lung (CHL), cells are functionally hemizygous for a locus that can be altered

Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases.

“…CHO cell line Asn-5 has a mutation in the asnS gene, which renders asparaginyl-tRNA synthetase thermolabile and the cells nonviable at 39°C (2). UCW 56 is derived from the CHO cell line tsH-1 and has a mutation in the leuS gene which results in leucyl-tRNA synthetase being thermolabile (27,32). This cell line is also nonviable at 39°C.…”

Construction and characterization of Chinese hamster cell EmtA EmtB double mutants.

“…The mutation in the chr locus renders the cells resistant to normally toxic concentrations of sodium chromate. The emtA locus is not linked to chromosome 2 (4,24). These linkage relationships and the genetic makeup of these hybrids is shown in Fig.…”

“…As shown in Fig. 2, EmtBr segregants arising as a result of loss of the chromosome(s) 2 from the CHL parent, which carries the wild-type emtB allele, will simultaneously become temperature sensitive [LeuS(Ts)] and Chrr, since the wild-type leuS and chr alleles will segregate along with the wildtype emtB allele (24). In contrast, EmtAr segregants arising as a result of loss of the chromosome(s) from the CHO parent carrying the wildtype emtA allele will remain temperature resistant (LeuS+) and Chrs.…”

“…Thus, the emtB locus, which is altered in EmtB mutants, is undoubtedly the structural gene for ribosomal protein S14. Two-dimensional gel electrophoretic analyses have not yet defined alterations in specific ribosomal proteins that can be correlated with mutations in the emtA and emtC loci (4 (24). This cell line has mutations in three genes, emtB, leuS, and chr, all of which are linked on the long arm of chromosome 2 (2, 24).…”

“…The emtB, leuS, and chr genes are localized to a region on the long arm of chromosome 2 that has been rendered physically haploid as a result of an interstitial deletion of one chromosome 2 homolog in CHO cells (7,27). Thus, the (24). All cell lines were grown as monolayer cultures as described previously (25).…”