Structural and functional hemi- and dizygous Chinese hamster chromosome 2 gene loci in CHO cells

Abstract

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 sensitivity and galactose-1-phosphate uridyltransferase could 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 PGM1 in 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.

Genetic effects of chromosomal rearrangements in Chinese hamster ovary cells: expression and chromosomal assignment of TK, GALK, ACP1, ADA, and ITPA loci.

“…However, CHO cells appear to be dizygous for 14 of the 17 randomly selected enzyme loci for which electrophoretic mobility shift mutations have been obtained (17,18,26; see above), suggesting that functional hemizygosity may be much less extensive than once thought. Furthermore, in every case thus far in which putatively functionally hemizygous loci have been assigned to specific chromosomal regions in CHO cells, their hemizygosity has been attributable to physical deletion or monosomy involving these chromosomes (4,18,26,27). Our segregation data for ADA, ITPA, ACPI, TK, and GALK in interspecific somatic cell hybrids has provided confirmation that each of these gene loci is functionally diploid in CHO cells.…”

“…Among the 527 clones picked and analyzed for electrophoretic shift variations at over 40 enzyme loci after ethyl methanesulfonate exposure, two (EMS2-36 and AEM-37) were variant for ADA, and one (2HEME-12) was variant for ITPA. Mutations detected for other loci have either been previously described (17)(18)(19)26) or will be presented elsewhere after subcloning for verification. The ADA and ITPA variants were verified as mutations by recovery of the variant patterns in all subclones (e.g., Fig.…”

“…During the past few years, over 35 gene loci have been assigned to the chromosomes of euploid Chinese hamster cells (3,4,8,12,13,18,(24)(25)(26)(27). The location and functional behavior of alleles of these loci in CHO cells, the most widely used Chinese hamster line for somatic cell genetic and mutation research, are not as well defined since CHO cells have undergone stable cytogenetic alterations resulting from deletions, translocations, and other types of chromosomal rearrangements.…”

“…The functional ploidy of loci associated with these chromosomes in CHO cells has been shown, both by concordant segregation and mutational analysis, to be constitutive with respect to their chromosomal assignments (4,18,26,27). It remains to be determined whether loci present on CHO chromosomes resulting from rearrangements are constitutively expressed or perhaps modified in their expression owing to position effects.…”

Multidrug-resistant phenotype cosegregates with an amplified gene in somatic cell hybrids of drug-resistant Chinese hamster ovary cells and drug-sensitive murine cells.

“…We have successfully used this strategy to select for segregants of CHO chromosome 2, which harbors a diptheria toxin sensitivity locus (25). Segregants of chromosome 2 cosegregate chromosome 1 with high frequency (25 histochemical examination of these loci have been described previously (9,22,24).…”