Abstract
Thymidylate synthase-negative mutants of mouse FM3A cells were transformed to thymidine prototrophs by human DNA. The stable transformants had only human thymidylate synthase and segments of human DNA. They grew normally but had unusually high levels of the human enzyme. In two transformants examined, however, neither was the dTTP pool elevated nor the dCTP pool decreased. DNA synthesis in permeabilized cells of a transformant was more efficient than that in the wild type with dATP, dGTP, dCTP, and dUMP as substrates, but this was not so when dUMP was replaced by dTTP. Unlike the mouse enzyme, the human enzyme in the transformants did not co-sediment with DNA polymerase alpha and thymidine kinase in a sucrose gradient, suggesting that the human enzyme is not incorporated into a multienzyme complex for DNA replication. The high levels of the human enzyme in the transformants were suppressed to various degrees by fusion with a wild type mouse line. No active hybrid dimer enzyme was found between the human and mouse enzymes, which each consist of two identical subunits. Thus, the human enzyme in the transformants seems to behave differently from the mouse enzyme and its overproduction seems to be necessary for supporting the normal growth of the transformants.
Highlights
In this work, we employed interspecific gene transfer between human and mouse cells to facilitate assessment of the fates and behaviors of the transferred gene and its product
We describe to what extent human thymidylate synthase was able to replace the mouse enzyme when this was absent in mutant cells, in view of the following unique features of thymidylate synthase: (i) an alteration in thymidylate synthase activity should affect the dTTP pool and in turn other dNTP pools, because dTMPoritsderivative is known to hybrid dimer enzyme was found between the human regulate the productionof ribonucleotide reductase [2, 14,15,16,17]
The humanenzyme in the transformants and activates GDP reduction [18]; (ii) thymidylate synthase seems to behave differently from the mouse enzyme is known to be a component of a multi-enzyme complex with and its overproduction seems to be necessary for sup- other enzymes for DNA replication, such as DNA polymerporting the normal growth of the transformants
Summary
1.9 f 0.2 [110] 12 separation of dCyd from Cyd, both obtained from the corresponding diphosphates, was achieved by borate anion-exchange column chromatography instead of thin layer chromatography as described [30]. _perDmNeAabSiylinztahteisoinsinoPf ecremllesawbialsizceadrCrieeldlso-Luyt saosldeceistchriinb-emdeedlsiaetwedhere [28, 30, 31]. For measurement of DNA synthesis, 2 X 10" permeabilized cells were incubated at 37 "C in 0.1 mlof reaction mixture consisting of150 mM sucrose, mM KCI, 35 mM Na-4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid (pH 7.4), mM potassium phosphate (pH 7.4), 5 mM MgCI,, 5 mM ATP, and 0.25 mM each of various combinations of DNA precursors Sedimentation Analysis of Enzymes-Cells were harvested and " T h e valuesareexpressedasnanomoles of "Hreleased/mg of suspended in sucrose buffer (0.2M5sucrose, 50 mM Tris-HCI (pH 7.6), protein/h f standard deviation in two to four separate determina-. FM3A cells were routinely grown in suspension in normal medium in plastic petri dishes. The karyotypesof the hybrids were determined as described [1, 3] to confirm that the cells were hybrids and the cells were maintained in normal growth medium.
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