• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of geneticsGeneticsCurrent IssueInformation for AuthorsEditorial BoardSubscribeSubmit a Manuscript
Genetics. Nov 1992; 132(3): 665–673.
PMCID: PMC1205205

Construction of a Gal1-Regulated Yeast Cdna Expression Library and Its Application to the Identification of Genes Whose Overexpression Causes Lethality in Yeast

Abstract

We have constructed a galactose-inducible expression library by cloning yeast cDNAs unidirectionally under control of the GAL1 promoter in a centromeric shuttle vector. Eleven independent libraries were made each with an average size of about 1 X 10(6) clones, about 50 times larger than the reported mRNA population in a yeast cell. From this library, LEU2 and HIS3 cDNAs were recovered at a frequency of about 1 in 10(4) and in 12 out of 13 cases these were expressed in a galactose-dependent manner. Sequence analysis of leu2 and his3 complementing cDNAs indicates that they contain all the coding sequence and much of the 5' untranslated region. To test the utility of the library for the identification of genes whose overexpression confers a specific phenotype, we screened 25,000 yeast transformants for lethality on galactose. Among 15 clones that showed galactose inducible lethality were cDNAs encoding structural proteins, including ACT1 (actin), TUB2 (β-tubulin) and ABP1 (actin-binding protein 1), and genes in signal transduction pathways, including TPK1 (a cAMP-dependent protein kinase) and GLC7 (type 1 protein phosphatase). cDNAs overexpressing NHPB (nonhistone protein B) and NSR1 (nuclear sequence recognition protein) were also found to be lethal. Among these, ACT1 was isolated four times, and NSR1 three times. The useful features of this library for cDNA cloning in yeast by complementation, and for the identification of genes whose over-expression confers specific phenotypes, are discussed.

Full Text

The Full Text of this article is available as a PDF (970K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Abovich N, Rosbash M. Two genes for ribosomal protein 51 of Saccharomyces cerevisiae complement and contribute to the ribosomes. Mol Cell Biol. 1984 Sep;4(9):1871–1879. [PMC free article] [PubMed]
  • Andreadis A, Hsu YP, Hermodson M, Kohlhaw G, Schimmel P. Yeast LEU2. Repression of mRNA levels by leucine and primary structure of the gene product. J Biol Chem. 1984 Jul 10;259(13):8059–8062. [PubMed]
  • Boeke JD, LaCroute F, Fink GR. 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. [PubMed]
  • Botstein D, Fink GR. Yeast: an experimental organism for modern biology. Science. 1988 Jun 10;240(4858):1439–1443. [PubMed]
  • Burke D, Gasdaska P, Hartwell L. Dominant effects of tubulin overexpression in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Mar;9(3):1049–1059. [PMC free article] [PubMed]
  • Carlson M, Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. [PubMed]
  • Drubin DG, Miller KG, Botstein D. Yeast actin-binding proteins: evidence for a role in morphogenesis. J Cell Biol. 1988 Dec;107(6 Pt 2):2551–2561. [PMC free article] [PubMed]
  • Feng ZH, Wilson SE, Peng ZY, Schlender KK, Reimann EM, Trumbly RJ. The yeast GLC7 gene required for glycogen accumulation encodes a type 1 protein phosphatase. J Biol Chem. 1991 Dec 15;266(35):23796–23801. [PubMed]
  • Gallwitz D, Perrin F, Seidel R. The actin gene in yeast Saccharomyces cerevisiae: 5' and 3' end mapping, flanking and putative regulatory sequences. Nucleic Acids Res. 1981 Dec 11;9(23):6339–6350. [PMC free article] [PubMed]
  • Gallwitz D, Sures I. Structure of a split yeast gene: complete nucleotide sequence of the actin gene in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 May;77(5):2546–2550. [PMC free article] [PubMed]
  • Hereford LM, Rosbash M. Number and distribution of polyadenylated RNA sequences in yeast. Cell. 1977 Mar;10(3):453–462. [PubMed]
  • Herskowitz I. Functional inactivation of genes by dominant negative mutations. Nature. 1987 Sep 17;329(6136):219–222. [PubMed]
  • Hurt EC. A novel nucleoskeletal-like protein located at the nuclear periphery is required for the life cycle of Saccharomyces cerevisiae. EMBO J. 1988 Dec 20;7(13):4323–4334. [PMC free article] [PubMed]
  • Johnston M, Davis RW. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. [PMC free article] [PubMed]
  • Lee WC, Xue ZX, Mélèse T. The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs. J Cell Biol. 1991 Apr;113(1):1–12. [PMC free article] [PubMed]
  • Liu HP, Bretscher A. Disruption of the single tropomyosin gene in yeast results in the disappearance of actin cables from the cytoskeleton. Cell. 1989 Apr 21;57(2):233–242. [PubMed]
  • McKnight GL, McConaughy BL. Selection of functional cDNAs by complementation in yeast. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4412–4416. [PMC free article] [PubMed]
  • Meeks-Wagner D, Hartwell LH. Normal stoichiometry of histone dimer sets is necessary for high fidelity of mitotic chromosome transmission. Cell. 1986 Jan 17;44(1):43–52. [PubMed]
  • Meeks-Wagner D, Wood JS, Garvik B, Hartwell LH. Isolation of two genes that affect mitotic chromosome transmission in S. cerevisiae. Cell. 1986 Jan 17;44(1):53–63. [PubMed]
  • Nagashima K, Kasai M, Nagata S, Kaziro Y. Structure of the two genes coding for polypeptide chain elongation factor 1 alpha (EF-1 alpha) from Saccharomyces cerevisiae. Gene. 1986;45(3):265–273. [PubMed]
  • Nasmyth KA, Reed SI. Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2119–2123. [PMC free article] [PubMed]
  • Neff NF, Thomas JH, Grisafi P, Botstein D. Isolation of the beta-tubulin gene from yeast and demonstration of its essential function in vivo. Cell. 1983 May;33(1):211–219. [PubMed]
  • Ng R, Abelson J. Isolation and sequence of the gene for actin in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3912–3916. [PMC free article] [PubMed]
  • Ohkura H, Kinoshita N, Miyatani S, Toda T, Yanagida M. The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases. Cell. 1989 Jun 16;57(6):997–1007. [PubMed]
  • Qin SL, Xie AG, Bonato MC, McLaughlin CS. Sequence analysis of the translational elongation factor 3 from Saccharomyces cerevisiae. J Biol Chem. 1990 Feb 5;265(4):1903–1912. [PubMed]
  • Reed SI, Hadwiger JA, Richardson HE, Wittenberg C. Analysis of the Cdc28 protein kinase complex by dosage suppression. J Cell Sci Suppl. 1989;12:29–37. [PubMed]
  • Rose MD, Broach JR. Cloning genes by complementation in yeast. Methods Enzymol. 1991;194:195–230. [PubMed]
  • Rose MD, Fink GR. KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast. Cell. 1987 Mar 27;48(6):1047–1060. [PubMed]
  • Rose MD, Novick P, Thomas JH, Botstein D, Fink GR. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. [PubMed]
  • Schultheiss T, Lin ZX, Ishikawa H, Zamir I, Stoeckert CJ, Holtzer H. Desmin/vimentin intermediate filaments are dispensable for many aspects of myogenesis. J Cell Biol. 1991 Sep;114(5):953–966. [PMC free article] [PubMed]
  • Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. [PMC free article] [PubMed]
  • Strathern JN, Higgins DR. Recovery of plasmids from yeast into Escherichia coli: shuttle vectors. Methods Enzymol. 1991;194:319–329. [PubMed]
  • Struhl K. Nucleotide sequence and transcriptional mapping of the yeast pet56-his3-ded1 gene region. Nucleic Acids Res. 1985 Dec 9;13(23):8587–8601. [PMC free article] [PubMed]
  • Struhl K, Davis RW. Transcription of the his3 gene region in Saccharomyces cerevisiae. J Mol Biol. 1981 Nov 5;152(3):535–552. [PubMed]
  • Trueheart J, Boeke JD, Fink GR. Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein. Mol Cell Biol. 1987 Jul;7(7):2316–2328. [PMC free article] [PubMed]

Articles from Genetics are provided here courtesy of Genetics Society of America

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • Nucleotide
    Nucleotide
    Published Nucleotide sequences
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...