Regulated expression of endonuclease EcoRI in Saccharomyces cerevisiae: nuclear entry and biological consequences (original) (raw)

Abstract

In an investigation to determine how proteins are localized within the nucleus of a cell, we demonstrate that the restriction endonuclease EcoRI is able to enter and function within the nucleus of Saccharomyces cerevisiae when this prokaryotic protein is synthesized in vivo. The EcoRI endonuclease was produced in yeast under the transcriptional control of a regulated yeast promoter by ligating a DNA fragment containing only coding sequences for the endonuclease to the promoter element of the yeast GAL1 gene (the structural gene for galactokinase, EC 2.7.1.6). Yeast cells harboring a plasmid containing this promoter-gene fusion are able to grow under conditions that repress transcription from the GAL1 promoter. However, under inducing conditions, these yeast cells are unable to grow. Moreover, rad52 mutants, which are deficient in the repair of double-strand breaks, are more sensitive to the presence of the promoter-gene fusion plasmid than are wild-type cells. We demonstrate that the EcoRI endonuclease activity is present in lysates prepared from yeast transformants grown under conditions that induce transcription of GAL1, but this activity is not detectable in cells grown under conditions that repress transcription from the promoter. Furthermore, analysis of yeast chromosomal DNA shows that the endonuclease enters the yeast nucleus and cleaves DNA specifically at EcoRI recognition sites.

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  1. Adams B. G. Induction of galactokinase in Saccharomyces cerevisiae: kinetics of induction and glucose effects. J Bacteriol. 1972 Aug;111(2):308–315. doi: 10.1128/jb.111.2.308-315.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barnes G., Hansen W. J., Holcomb C. L., Rine J. Asparagine-linked glycosylation in Saccharomyces cerevisiae: genetic analysis of an early step. Mol Cell Biol. 1984 Nov;4(11):2381–2388. doi: 10.1128/mcb.4.11.2381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  5. De Robertis E. M., Longthorne R. F., Gurdon J. B. Intracellular migration of nuclear proteins in Xenopus oocytes. Nature. 1978 Mar 16;272(5650):254–256. doi: 10.1038/272254a0. [DOI] [PubMed] [Google Scholar]
  6. Dingwall C., Sharnick S. V., Laskey R. A. A polypeptide domain that specifies migration of nucleoplasmin into the nucleus. Cell. 1982 Sep;30(2):449–458. doi: 10.1016/0092-8674(82)90242-2. [DOI] [PubMed] [Google Scholar]
  7. Goldstein L., Ko C. Distribution of proteins between nucleus and cytoplasm of Amoeba proteus. J Cell Biol. 1981 Mar;88(3):516–525. doi: 10.1083/jcb.88.3.516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Greene P. J., Gupta M., Boyer H. W., Brown W. E., Rosenberg J. M. Sequence analysis of the DNA encoding the Eco RI endonuclease and methylase. J Biol Chem. 1981 Mar 10;256(5):2143–2153. [PubMed] [Google Scholar]
  9. Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ho K. S., Mortimer R. K. Induction of dominant lethality by x-rays in radiosensitive strain of yeast. Mutat Res. 1973 Oct;20(1):45–51. doi: 10.1016/0027-5107(73)90096-1. [DOI] [PubMed] [Google Scholar]
  11. Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Johnston S. A., Hopper J. E. Isolation of the yeast regulatory gene GAL4 and analysis of its dosage effects on the galactose/melibiose regulon. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6971–6975. doi: 10.1073/pnas.79.22.6971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Malone R. E., Esposito R. E. The RAD52 gene is required for homothallic interconversion of mating types and spontaneous mitotic recombination in yeast. Proc Natl Acad Sci U S A. 1980 Jan;77(1):503–507. doi: 10.1073/pnas.77.1.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Miller D. S., Lau Y. T., Horowitz S. B. Artifacts caused by cell microinjection. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1426–1430. doi: 10.1073/pnas.81.5.1426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Newman A. K., Rubin R. A., Kim S. H., Modrich P. DNA sequences of structural genes for Eco RI DNA restriction and modification enzymes. J Biol Chem. 1981 Mar 10;256(5):2131–2139. [PubMed] [Google Scholar]
  16. Paine P. L., Moore L. C., Horowitz S. B. Nuclear envelope permeability. Nature. 1975 Mar 13;254(5496):109–114. doi: 10.1038/254109a0. [DOI] [PubMed] [Google Scholar]
  17. Peters R. Nuclear envelope permeability measured by fluorescence microphotolysis of single liver cell nuclei. J Biol Chem. 1983 Oct 10;258(19):11427–11429. [PubMed] [Google Scholar]
  18. Polisky B., Greene P., Garfin D. E., McCarthy B. J., Goodman H. M., Boyer H. W. Specificity of substrate recognition by the EcoRI restriction endonuclease. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3310–3314. doi: 10.1073/pnas.72.9.3310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Resnick M. A. Genetic control of radiation sensitivity in Saccharomyces cerevisiae. Genetics. 1969 Jul;62(3):519–531. doi: 10.1093/genetics/62.3.519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sabatini D. D., Kreibich G., Morimoto T., Adesnik M. Mechanisms for the incorporation of proteins in membranes and organelles. J Cell Biol. 1982 Jan;92(1):1–22. doi: 10.1083/jcb.92.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Scott J. H., Schekman R. Lyticase: endoglucanase and protease activities that act together in yeast cell lysis. J Bacteriol. 1980 May;142(2):414–423. doi: 10.1128/jb.142.2.414-423.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. St John T. P., Davis R. W. Isolation of galactose-inducible DNA sequences from Saccharomyces cerevisiae by differential plaque filter hybridization. Cell. 1979 Feb;16(2):443–452. doi: 10.1016/0092-8674(79)90020-5. [DOI] [PubMed] [Google Scholar]
  23. St John T. P., Scherer S., McDonell M. W., Davis R. W. Deletion analysis of the Saccharomyces GAL gene cluster. Transcription from three promoters. J Mol Biol. 1981 Oct 25;152(2):317–334. doi: 10.1016/0022-2836(81)90245-x. [DOI] [PubMed] [Google Scholar]
  24. Stinchcomb D. T., Mann C., Davis R. W. Centromeric DNA from Saccharomyces cerevisiae. J Mol Biol. 1982 Jun 25;158(2):157–190. doi: 10.1016/0022-2836(82)90427-2. [DOI] [PubMed] [Google Scholar]
  25. Stinchcomb D. T., Struhl K., Davis R. W. Isolation and characterisation of a yeast chromosomal replicator. Nature. 1979 Nov 1;282(5734):39–43. doi: 10.1038/282039a0. [DOI] [PubMed] [Google Scholar]
  26. Strathern J. N., Klar A. J., Hicks J. B., Abraham J. A., Ivy J. M., Nasmyth K. A., McGill C. Homothallic switching of yeast mating type cassettes is initiated by a double-stranded cut in the MAT locus. Cell. 1982 Nov;31(1):183–192. doi: 10.1016/0092-8674(82)90418-4. [DOI] [PubMed] [Google Scholar]
  27. Struhl K., Cameron J. R., Davis R. W. Functional genetic expression of eukaryotic DNA in Escherichia coli. Proc Natl Acad Sci U S A. 1976 May;73(5):1471–1475. doi: 10.1073/pnas.73.5.1471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Unwin P. N., Milligan R. A. A large particle associated with the perimeter of the nuclear pore complex. J Cell Biol. 1982 Apr;93(1):63–75. doi: 10.1083/jcb.93.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]