Acquisition of Chick Cytosol Thymidine Kinase Activity by Thymidine Kinase-Deficient Mouse Fibroblast Cells After Fusion With Chick Erythrocytes

Abstract

When chick erythrocyte nuclei are introduced into the cytoplasm of tissue culture cells of the same or a different species, they are activated to resume DNA synthesis (Cook, 1970;Harris, 1970;Harris and Cook, 1969). Numerous studies have shown that initiation of DNA synthesis is associated with an enhanced activity of the salvage pathway enzyme, thymidine (dT) kinase, in synchronized cell cultures, regenerating rat liver, compensatory hypertrophy of rat kidney, hormone-stimulated tissues, phytohemagglutinin-stimulated lymphocytes, and in virus-infected cells [see Kit et al., 1974 b for review]. It was therefore of interest to learn whether activation of chick erythrocyte dT kinase occurs after chick erythrocyte nuclei are introduced into the cytoplasm of mouse fibroblasts. Chick erythrocytes have little, if any, dT kinase activity. The chick erythrocytes were fused in the presence of UV-inactivated Sendai virus with LM(TK-) cells, a mutant mouse fibroblast line

Cotransfer of two linked human genes into cultured mouse cells.

“…When LM(TK-) cells were incubated in selective medium no clones could be identified. This observation confirms previous reports that revertants of LM(TK-) have not been found, i.e., the frequency of reversion must be lower than 10-8 (13). The doubling rate of the parental line B82 in Dulbecco's modified Eagle's medium was found to be about 18 hr.…”

Chromosome-mediated gene transfer results in two classes of unstable transformants.

“…The TK-deficient established mouse cell line LM(TK-) (14) was used as the recipient for CMGT. The human epithelial-like cell line HeLa S3 (TK+) (15) was used as the chromosome donor.…”

Thymidine Kinases Induced by Avian and Human Herpesviruses

“…The electrophoretic mobilities relative to the tracking dye ( Rm) of the chick cytosol and mitochondrial-specific dT kinases are about 0.20 and 0.6-0.7, respectively. The disc PAGE mobilities of chick cytosol and mitochondrial dT kinases can readily be distinguished from those of mouse cytosol (Rm = 0.3) and mouse mitochondrial (Rm=0.8) dT kinases [22].…”

“…In this property, the avian herpesvirusinduced dT kinases resemble the HSV-1-and HSV-2-induced dT kinases ( fig. 5b,e) [23] and the vertebrate mitochondrial dT kinase isozymes, but differ from vertebrate cytosol dT kinases [9,10,[22][23][24],…”

“…The disc PAGE analyses of dT kinase isozymes of uninfected CEF cells and of cells infected by avian herpesviruses are shown in figures 2 and 3. Primary cultures of CEF cells and chick embryo CAMs contain distinctive cytosol and mitochondrial isozymes of dT kinase [22] ( fig. 2a, 3a.b).…”