Search

Interspecific somatic cell hybrids were generated by fusion of Chinese hamster spleen cells or primary fibroblasts with cells from an adenine phosphoribosyltransferase (APRT)-deficient mouse subline derived from LMTK- Cl.1D. Subclones which had been selected for either retention or loss ofAPRT were subjected to combined isozyme and chromosome segregation analysis. Concordant expression or segregation of Chinese hamster APRT, lactate dehydrogenase A (LDHA), isocitrate dehydrogenase 2 (IDH2), and a-glucosidase (GAA) with Chinese hamster chromosome 3 allowed provisional assignment of all four loci to that chromosome. Exceptional subclones, in which coordinate segregation of these syntenic markers was disrupted by chromosome breakage or deletions, allowed further localization of these genes to specific regions of the 3 chromosome.

Fourteen independent somatic cell hybrid clones made between diphtheria toxin (DT)-resistant mouse Cl1D cells and DT-sensitive Chinese hamster ovary (CHO) cells slowly segregated CHO chromosome. Concordant segregation analysis of electrophoretically resolvable Chinese hamster chromosome 2 gene products and CHO chromosomes 2 and Z2 (having q1–q24 deletion) in DT-selected and control hybrid subclones was conducted. Analysis revealed that loci for DT sensitivityand galactose-1-phosphate uridyltransferasecould be regionally assigned to Chinese hamster chromosome 2q1–24 and were physically hemizygous in CHO cells. Enolase (ENO1), 6 phosphogluconate dehydrogenase (PGD),and phosphoglucomutase 1 (PGM1)were located outside the q1–24 region on Chinese hamster chromosome 2 and were dizygous in CHO cells. Functional dizygosity of ENO1, PGD,and PGM1in CHO cells, as determined by the isolation of diploid heterozygous electrophoretic shift mutants following UV and EMS exposure, confirmed their location outside the Z2 deletion and indicated that the deletion did not result in the inactivation of adjacent loci. These results are discussed in relation to current theories on the basis for high frequency of drug-resistant autosomal recessive mutants in CHO cells and conservation of mammalian autosomal linkage groups.

Aminoacyl-tRNA synthetase (aaRS) activities in extracts of mutant strains of the Chinese hamster ovary line (CHO) were examined for alterations in thermal stability. Mutants having low activity for MetRS, AsnRS, or GlnRS contained aaRSs that were inactivated much more rapidly upon heating than those from wild-type cells. Revertant lines, isolated from cultures of these mutants (Asn-5, Met-2, and Gln-2) after treatment with nitrosoguanidine or ethyl methanesulfonate, had thermolabilities intermediate between mutant and wild-type, and consistently had higher activities than the mutants. With a modified in vivo aminoacylation procedure, two previously exceptional mutants, Arg-1 and His-1, showed pronounced reductions in the amount of arginyl-tRNA or histidyl-tRNA, respectively, under restrictive conditions, compared to wild type. Revertants of Arg-1 (like the mutant itself) had no measurable ArgRS in vitro activity (<0.4% of wild type) although in vivo aminoacylation in the one revertant tested was partially restored. These data provide evidence that the forward mutations have occurred in the structural genes of the aaRSs and that most of the reversions are probably the result of second-site point mutations in the aaRS genes.

Using a tritiated amino acid suicide procedure designed specifically to select conditional protein synthesis mutants, we have isolated and characterized a large number of such mutants of Chinese hamster ovary cells. All of the mutants are genetically stable and behave as recessives in somatic cell hybrids. Most of the new mutants are phenotypically dependent on the concentration of a specific amino acid as well as on temperature. In addition to identifying many additional leucyl- and asparagyl-tRNA synthetase mutants, complementation analysis has distinguished four new genetic classes representing methionine-, glutamine-, histidine-, and arginine-dependent mutants. Biochemical characterization of representative mutants from each of these six classes has identified the primary lesions as being defective aminoacyl-tRNA synthetases. Our selection results further demonstrate the high specificity of the3H-amino acid procedure for isolating protein synthesis mutants. Reconstruction experiments performed with two representative mutants indicated a selection efficiency of approximately 10% under standard conditions.

Further application of the tritiated amino acid suicide selection procedure, designed to select conditional protein synthesis mutants, has yielded over 300 aminoacyl-tRNA synthetase mutants of Chinese hamster ovary cells, representing seven complementation groups. A number of these mutants and their revertants were used to examine the extent of X-chromosome linkage or other functional hemizygosity in CHO cells. Our results indicated that none of the seven different aaRSloci tested was X linked, and the the glnRSlocus, unlike the asnRSand leuRSloci, does not appear to be functionally hemizygous in CHO cells.