Deregulation of DNA polymerase beta by sense and antisense RNA expression in mouse 3T3 cells alters cell growth.
DNA polymerase beta (beta-pol) and its mRNA are maintained at constitutive levels during the cell cycle and during stages of cell growth in culture. To study biological consequences of variations in the level of this DNA repair enzyme and/or its mRNA, we prepared expression vectors in which cDNA for human beta-pol is inserted under the control of a metallothionein promoter (pMT) in the sense and antisense orientation, respectively, and these vectors then were used for stable transformation of mouse 3T3 cells. Vectors also contained the mouse DHFR gene, such that culture of transformants in medium with increasing concentrations of methotrexate resulted in amplification of inserted DNA. The levels of sense and antisense transcripts are strongly increased by culture of transformants in medium with 65 microM Zn2+, although some expression is detected even without Zn2+ induction. After five days of induction, the beta-pol level was about threefold higher in sense cells and about 10-fold lower in antisense cells than in parallel cultures without induction. The antisense line has a threefold increased cell doubling time in the presence of 65 microM Zn2+ compared with the absence of Zn2+. Zn2+ (65 microM) induction for the sense line results in normal growth for the first three days and, thereafter, a complete cessation of growth. Yet, these blocked cells remain fully viable. The results indicate that sudden deregulation of beta-pol expression alters cell growth in mouse 3T3 cells.
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
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Zmudzka
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Wilson
D000818:Animals; D015139:Blotting, Southern; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D002712:Chlorides; D004256:DNA Polymerase I / Q000502:physiology*; D005784:Gene Amplification; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D005822:Genetic Vectors; D015698:Genomic Library; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D012313:RNA / Q000096:biosynthesis; D016372:RNA, Antisense / Q000096:biosynthesis; D014162:Transfection; D015032:Zinc; D017967:Zinc Compounds
D002712:Chlorides; D016372:RNA, Antisense; D017967:Zinc Compounds; D012313:RNA; C016837:zinc chloride; D008668:Metallothionein; D004256:DNA Polymerase I; D015032:Zinc