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. 1987 Dec;117(4):645–655. doi: 10.1093/genetics/117.4.645

Identifying Mutations in Duplicated Functions in Saccharomyces cerevisiae: Recessive Mutations in Hmg-Coa Reductase Genes

Michael E Basson 1, Robert L Moore 1, Jules O'Rear 1, Jasper Rine 1
PMCID: PMC1203238  PMID: 2828155

Abstract

The two yeast genes for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, HMG1 and HMG2, each encode a functional isozyme. Although cells bearing null mutations in both genes are inviable, cells bearing a null mutation in either gene are viable. This paper describes a method of screening for recessive mutations in the HMG1 gene, the gene encoding the majority of HMG-CoA reductase activity in the cell. This method should be applicable to the isolation of mutations in other cases of duplicated genes or duplicated functions in yeast. Four recessive point mutations were recovered in HMG1. These mutations exhibited intragenic complementation: one allele is in one complementation group and three alleles are in a second complementation group. Assays of HMG-CoA reductase activity indicated that the point mutations destroy most if not all of the activity encoded by HMG1. Intragenic complementation occurred with partial restoration of enzymatic activity. HMG1 was mapped to the left arm of chromosome XIII near SUP79, and HMG2 was mapped to the right arm of chromosome XII near SST2. A slight deleterious effect of a null mutation in either HMG-CoA reductase gene was detected by a co-cultivation experiment involving the wild-type strain and the two single mutants.

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Selected References

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  1. 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]
  2. Basson M. E., Thorsness M., Rine J. Saccharomyces cerevisiae contains two functional genes encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5563–5567. doi: 10.1073/pnas.83.15.5563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beg Z. H., Brewer H. B., Jr Modulation of rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity by reversible phosphorylation. Fed Proc. 1982 Aug;41(10):2634–2638. [PubMed] [Google Scholar]
  4. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Brown D. A., Simoni R. D. Biogenesis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an integral glycoprotein of the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1674–1678. doi: 10.1073/pnas.81.6.1674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown M. S., Faust J. R., Goldstein J. L., Kaneko I., Endo A. Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts incubated with compactin (ML-236B), a competitive inhibitor of the reductase. J Biol Chem. 1978 Feb 25;253(4):1121–1128. [PubMed] [Google Scholar]
  8. Brown M. S., Goldstein J. L. Multivalent feedback regulation of HMG CoA reductase, a control mechanism coordinating isoprenoid synthesis and cell growth. J Lipid Res. 1980 Jul;21(5):505–517. [PubMed] [Google Scholar]
  9. Carle G. F., Olson M. V. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res. 1984 Jul 25;12(14):5647–5664. doi: 10.1093/nar/12.14.5647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chattoo B. B., Sherman F., Azubalis D. A., Fjellstedt T. A., Mehnert D., Ogur M. Selection of lys2 Mutants of the Yeast SACCHAROMYCES CEREVISIAE by the Utilization of alpha-AMINOADIPATE. Genetics. 1979 Sep;93(1):51–65. doi: 10.1093/genetics/93.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Clarke C. F., Edwards P. A., Lan S. F., Tanaka R. D., Fogelman A. M. Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase mRNA levels in rat liver. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3305–3308. doi: 10.1073/pnas.80.11.3305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Clarke C. F., Fogelman A. M., Edwards P. A. Transcriptional regulation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene in rat liver. J Biol Chem. 1985 Nov 15;260(26):14363–14367. [PubMed] [Google Scholar]
  13. Cohen D. C., Massoglia S. L., Gospodarowicz D. Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in vascular endothelial cells. Separate sterol and non-sterol components. J Biol Chem. 1982 Sep 25;257(18):11106–11112. [PubMed] [Google Scholar]
  14. Edwards P. A., Lan S. F., Fogelman A. M. Alterations in the rates of synthesis and degradation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase produced by cholestyramine and mevinolin. J Biol Chem. 1983 Sep 10;258(17):10219–10222. [PubMed] [Google Scholar]
  15. Emr S. D., Vassarotti A., Garrett J., Geller B. L., Takeda M., Douglas M. G. The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal. J Cell Biol. 1986 Feb;102(2):523–533. doi: 10.1083/jcb.102.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gil G., Goldstein J. L., Slaughter C. A., Brown M. S. Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase from the hamster. I. Isolation and sequencing of a full-length cDNA. J Biol Chem. 1986 Mar 15;261(8):3710–3716. [PubMed] [Google Scholar]
  17. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  18. Harwood H. J., Jr, Brandt K. G., Rodwell V. W. Allosteric activation of rat liver cytosolic 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase by nucleoside diphosphates. J Biol Chem. 1984 Mar 10;259(5):2810–2815. [PubMed] [Google Scholar]
  19. Lacroute F., Piérard A., Grenson M., Wiame J. M. The biosynthesis of carbamoyl phosphate in Saccharomyces cerevisiae. J Gen Microbiol. 1965 Jul;40(1):127–142. doi: 10.1099/00221287-40-1-127. [DOI] [PubMed] [Google Scholar]
  20. Liscum L., Cummings R. D., Anderson R. G., DeMartino G. N., Goldstein J. L., Brown M. S. 3-Hydroxy-3-methylglutaryl-CoA reductase: a transmembrane glycoprotein of the endoplasmic reticulum with N-linked "high-mannose" oligosaccharides. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7165–7169. doi: 10.1073/pnas.80.23.7165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Liscum L., Finer-Moore J., Stroud R. M., Luskey K. L., Brown M. S., Goldstein J. L. Domain structure of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a glycoprotein of the endoplasmic reticulum. J Biol Chem. 1985 Jan 10;260(1):522–530. [PubMed] [Google Scholar]
  22. Luskey K. L., Faust J. R., Chin D. J., Brown M. S., Goldstein J. L. Amplification of the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, in UT-1 cells. J Biol Chem. 1983 Jul 10;258(13):8462–8469. [PubMed] [Google Scholar]
  23. Panini S. R., Sexton R. C., Rudney H. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by oxysterol by-products of cholesterol biosynthesis. Possible mediators of low density lipoprotein action. J Biol Chem. 1984 Jun 25;259(12):7767–7771. [PubMed] [Google Scholar]
  24. Parks L. W. Metabolism of sterols in yeast. CRC Crit Rev Microbiol. 1978;6(4):301–341. doi: 10.3109/10408417809090625. [DOI] [PubMed] [Google Scholar]
  25. Qureshi N., Dugan R. E., Cleland W. W., Porter J. W. Kinetic analysis of the individual reductive steps catalyzed by beta-hydroxy-beta-methylglutaryl-coenzyme A reductase obtained from yeast. Biochemistry. 1976 Sep 21;15(19):4191–4107. doi: 10.1021/bi00664a010. [DOI] [PubMed] [Google Scholar]
  26. Reynolds G. A., Basu S. K., Osborne T. F., Chin D. J., Gil G., Brown M. S., Goldstein J. L., Luskey K. L. HMG CoA reductase: a negatively regulated gene with unusual promoter and 5' untranslated regions. Cell. 1984 Aug;38(1):275–285. doi: 10.1016/0092-8674(84)90549-x. [DOI] [PubMed] [Google Scholar]
  27. Schwartz D. C., Cantor C. R. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell. 1984 May;37(1):67–75. doi: 10.1016/0092-8674(84)90301-5. [DOI] [PubMed] [Google Scholar]
  28. 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]

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