Abstract
Isolated circular molecules of the yeast plasmid 2-micron DNA were converted to linear molecules with restriction endonuclease PstI and ligated with T4 DNA ligase to PstI restriction fragments of total yeast DNA. A haploid strain of Saccharomyces cerevisiae carrying a deletion in thehis4 locus was transformed with the ligated DNA mixture to histidine prototrophy. One unstable histidine prototrophic transformant was obtained. Grown in the absence of histidine, 70–75% of the cells were auxotrophic and this number increased in non-selective medium. The histidine auxotrophic variants carried a deletion in thehis4 locus which had patterns of complementation and UV-induced mitotic recombination identical to the originalhis4 deletion. When the transformant was crossed to ahis4 strain and sporulated, the unstable histidine prototrophy segregated in a non-mendelian way: All five possible segregations from 0∶4 to 4∶0 were observed. When strains carrying the transformed character were crossed to ahis4 karl strain a low frequency of cytoduction of the unstable histidine prototrophy was observed. Nucleic acid from the transformant was able to transform a strain which carried another deletion in thehis4 locus. Treatment of the transformant with ethidium bromide caused an extensive induction of petites without any observable change in the frequency of histidine prototrophic cells.
It is concluded that theHIS4 gene function in the transformant is not stably associated with any chromosome. We take the instability as indication that only one or a few copies of the gene conferring the prototrophy are present in the prototrophic cells. The data are consistent with the assumption that the transformant contains a 2-micron DNA in which is inserted a chromosomal DNA region containing theHIS4 gene. A derivative of the transformant with increased stability has been isolated.
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Beggs, J. D.: Transformation of yeast by a replicating hybrid plasmid. Nature 275, 104–109 (1978)
Boram, W. R. &H. Roman: Recombination in Saccharomyces cerevisiae: A DNA repair mutation associated with elevated mitotic gene conversion. Proc. Natl. Acad. Sci. U.S. 73, 2828–2832 (1976)
Borst, P.: Mitochondrial nuclei acids. Ann. Rev. Biochem. 41, 333–376 (1972)
Clark-Walker, G. D.: Isolation of circular DNA from a mitochondrial fraction from yeast. Proc. Natl. Acad. Sci. U.S. 69, 388–392 (1972)
Conde, J. &G. R. Fink: A mutant of Saccharomyces cerevisiae defective for nuclear fusion. Proc. Natl. Acad. Sci. U.S. 73, 3651–3655 (1976)
Cryer, D. R., R. Eccleshall &J. Marmur: Isolation of yeast DNA. Meth. Cell Biol. 12, 39–44 (1975)
Fink, G. R. &C. A. Styles. Gene conversion of deletions in theHIS4 region of yeast. Genetics 77, 231–244 (1974)
Griffiths D. E., W. E. Lancashire &E. D. Zanders: Evidence for an extra-chromosomal element involved in mitochondrial function: A mitochondrial episome? FEBS Lett. 53, 126–130 (1975)
Guidelines for Research Involving Recombinant DNA Molecules. Federal Register U.S., 22 DEC 1978, pp. 60108-60131
Hawthorne, D. C. &R. K. Mortimer: Chromosome mapping in Saccharomyces: Centromere-linked genes. Geneties 45, 1085–1110 (1960)
Hinnen, A., J. B. Hicks &G. R. Fink: Transformation of yeast. Proc. Natl. Acad. Sci. U.S. 75, 1929–1933 (1978)
Hirt, B.: Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365–369 (1967)
Kapuściński, J. &B. Skoczylas: Simple and rapid fluorimetric method for DNA microassay. Anal. Biochem. 83, 252–257 (1977)
Livingston, D. M. &H. L. Klein: Deoxyribonucleic acid sequence organization of a yeast plasmid. J. Bacteriol. 129, 472–481 (1977)
Livingston, D. M. &D. M. Kupfer: Control of Saccharomyces cerevisiae 2 μm DNA replication by cell division cycle genes that control nuclear DNA replication. J. Mol. Biol. 116, 249–260 (1977)
Newlon, C. S. &W. L. Fangman: Mitochondrial DNA synthesis in cell cycle mutants of Saccharomyces cerevisiae. Cell 5, 423–428 (1975)
Newlon, C. S., T. D. Petes, L. M. Hereford &W. L. Fangman: Replication of yeast chromosomal DNA. Nature 247, 32–35 (1974)
Ratzkin, B. &J. Carbon: Functional expression of cloned yeast DNA in Escherichia coli. Proc. Natl. Acad. Sci. U.S. 74, 487–491 (1977)
Shaffer, B., J. Rytka &G. R. Fink: Nonsense mutations affecting theHIS4 enzyme complex of yeast. Proc. Natl. Acad. Sci. U.S., 63, 1198–1205 (1969)
Sinclair, J. H., B. J. Stevens, P. Sanghavi &M. Rabinowitz: Mitochondrial-satellite and circular DNA filaments in yeast. Science 156, 1234–1237 (1967)
Zakharov, I. A. &B. Ph. Yarovoy: Cytoduction as a new tool in studying the cytoplasmic heredity in yeast. Mol. Cellul. Biochem. 14, 15–18 (1977)
Zimmermann, F. K.: Detection of genetically active chemicals using various yeast systems. In: Chemical Mutagens. Principles and Methods for their Detection. A. Hollaender ed., Plenum Press. New York-London, Vol. III pp. 209–239 (1973)
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Kielland-Brandt, M.C., Nilsson-Tillgren, T., Holmberg, S. et al. Transformation of yeast without the use of foreign DNA. Carlsberg Res. Commun. 44, 77 (1979). https://doi.org/10.1007/BF02906523
DOI: https://doi.org/10.1007/BF02906523