Skip to main content
SpringerLink
Log in

A functional analysis of the repeated methionine initiator tRNA genes (IMT) in yeast

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Summary

Standard laboratory yeast strains have from four to five genes encoding the methionine initiator tRNA (IMT). Strain S288C has four IMT genes with identical coding sequences that are colinear with the RNA sequence of tRNA MetI . Each of the four IMT genes from strain S288C is located on a different chromosome. A fifth IMT gene with the same coding sequence is present in strain A364A but not in S288C. By making combinations of null alleles in strain S288C, we show that each of the four IMT genes is functional and that tRNA MetI is not limiting in yeast strains with three or more intact genes. Strains containing a single IMT2, 3 or 4 gene grow only after amplification of the remaining IMT gene. Strains with only the IMT1 gene intact are viable but grow extremely slowly; normal growth is restored by the addition of another IMT gene by transformation, providing a direct test for IMT function.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

IMT and imt :

(imt=initiator methionine tRNA), designate the genotype of the wild-type and the mutant alleles respectively, of the initiator methionine transfer RNA gene

met-tRNA MetI :

methionylated initiator methionine transfer RNA

eIF-2:

eukaryotic initiation factor two

GTP:

guanosine 5′-triphosphate

References

  • Benton WD, Davis RW (1977) Screening λgt recombinant clones by hybridization to single plaques in situ. Science 196:180–182

    Google Scholar 

  • Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197:345–346

    Google Scholar 

  • Boeke JD, Garfinkel DJ, Styles CA, Fink GR (1985) Ty elements transpose through an RNA intermediate. Cell 40:491–500

    Google Scholar 

  • Botstein D, Falco SC, Stewart SE, Brennen M, Scherer S, Stinchomb DT, Struhl K, Davies RW (1979) Sterile host yeast (SHY): A eukaryotic system of biological containment for recombinant DNA experiments. Gene 8:17–24

    Google Scholar 

  • Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472

    Google Scholar 

  • Calagan JL, Pirtle RM, Pirtle IL, Kashdan MA, Vreman HJ, Dudock BS (1980) Homology between chloroplast and prokaryotic initiator tRNA. Nucleotide sequence of spinach chloroplast methionine initiator tRNA. J Biol Chem 255:9981–9984

    Google Scholar 

  • Carle GF, Olson MV (1984) Separation of chromosomal DNA molecules from yeast by orthogonal-field-alteration gel electrophoresis. Nucleic Acids Res 12:5647–5664

    Google Scholar 

  • Cigan MA, Donahue TF (1986) The methionine initiator tRNA genes of yeast. Gene 41:343–348

    Google Scholar 

  • Gaber RF, Mathison L, Edelman I, Culbertson MR (1983) Frameshift suppression in Saccharomyces cerevisiae. VI. Complete genetic map of twenty-five suppressor genes. Genetics 103:389–407

    Google Scholar 

  • Gill DR, Hatfull GF, Salmond GPC (1986) A new cell division operon in Escherichia coli. Mol Gen Genet 205:134–145

    Google Scholar 

  • Grunstein M, Hogness DS (1975) Colony hybridization: A method for isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci USA 72:3961–3965

    Google Scholar 

  • Hamlyn PH, Gait MJ, Milstein C (1981) Complete sequence of an immunoglobulin mRNA using specific priming and the dideoxynucleotide method of RNA sequencing. Nucleic Acids Res 9:4485–4494

    Google Scholar 

  • Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–197

    Google Scholar 

  • Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168

    Google Scholar 

  • Kozak M (1983) Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev 47:1–45

    Google Scholar 

  • Kuo CL, Campbell JL (1983) Cloning of Saccharomyces cerevisiae DNA replication genes: Isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation. Mol Cell Biol 3:1730–1737

    Google Scholar 

  • Maitra U, Stringer EA, Chaudhuri A (1982) Initiation factors in protein biosynthesis. Annu Rev Biochem 51:869–900

    Google Scholar 

  • Mandel M, Higa A (1970) Calcium dependent bacteriophage DNA infection. J Mol Biol 53:159–162

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • McKnight GL, Cardillo TS, Sherman F (1981) An extensive deletion causing overproduction of yeast iso-2-cytochrome c. Cell 25:409–419

    Google Scholar 

  • Messing J, Crea R, Seeburg PH (1981) A system for shotgun DNA sequencing. Nucleic Acids Res 9:309–321

    Google Scholar 

  • Moldave K (1985) Eukaryotic protein synthesis. Annu Rev Biochem 54:1109–1149

    Google Scholar 

  • Mortimer RK, Schild D (1985) Genetic map of Saccharomyces cerevisiae. Microbiol Rev 49: 181–213

    Google Scholar 

  • Nilsson-Tillgren T, Gjermansen C, Holmberg S, Litske Peterson JG, Kielland-Brandt MC (1986) Analysis of chromosome V and the ILV1 gene from Saccharomyces carlsbergensis. Carlsberg Res Commun 51:309–326

    Google Scholar 

  • Norrander J, Kempe T, Messing J (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101–106

    Google Scholar 

  • Olsen MV, Loughney K, Hall BD (1979) Identification of the yeast DNA sequences that correspond to specific tyrosine-inserting nonsense suppressor loci. J Mol Biol 132:387–410

    Google Scholar 

  • Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation: A model system for the study of recombination. Proc Natl Acad Sci USA 78:6354–6358

    Google Scholar 

  • Perkins D (1949) Biochemical mutants in the smut fungus Ustilago maydis. Genetics 34:607–626

    Google Scholar 

  • Rose MD, Novick P, Thomas JH, Botstein D, Fink GR (1987) A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 60:237–243

    Google Scholar 

  • Sanger F, Nicklen S, Coulsen AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Google Scholar 

  • Schatz PJ, Solomon F, Botstein D (1986) Genetically essential and non-essential α-tubulin genes specify functionally interchangeable proteins. Mol Cell Biol 6:3722–3733

    Google Scholar 

  • Scherer S, Davis RW (1979) Replacements of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci USA 76:4951–4955

    Google Scholar 

  • Schevitz RW, Podjarny AD, Krishnamachari N, Hughes JJ, Sigler PB (1979) Crystal structure of a eukaryotic initiator tRNA. Nature 278:188–190

    Google Scholar 

  • Sharp SJ, Schaack J, Cooley L, Burke DJ, Soll D (1985) Structure and transcription of eukaryotic tRNA genes. Crit Rev Biochem 19:107–144

    Google Scholar 

  • Sherman F, Fink GR, Hicks JB (1986) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Simsek M, RajBhandary UL (1972) The primary structure of yeast initiator transfer ribonucleic acid. Biochem Biophys Res Commun 49:508–515

    Google Scholar 

  • Sprinzl M, Hartman T, Meissner F, Moll J, Vorderwülbecke T (1987) Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res 15:r53-r188

    Google Scholar 

  • Struhl K, Stinchcomb DT, Scherer S, Davies RW (1979) High-frequency transformation of yeast: Autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci USA 76:1035–1039

    Google Scholar 

  • Tschumper G, Carbon J (1980) Sequence of a DNA fragment containing a chromosomal replicator and the TRP1 gene. Gene 10:157–166

    Google Scholar 

  • Venegas A, Gonzalez E, Bull P, Valenzuela P (1982) Isolation and structure of a yeast initiator tRNAMet gene. nucleic Acids Res 10:1093–1096

    Google Scholar 

  • Wallace RB, Johnson MJ, Hirose T, Miyake T, Kawashima EH, Itakura K (1981) The use of synthetic oligonucleotides as hybridization of oligonucleotides of mixed sequence to rabbit β-globin DNA. Nucleic Acids Res 9:879–894

    Google Scholar 

  • Wrede P, Woo NH, Rich A (1979) Initiator tRNAs have a unique anticodon loop formation. Proc Natl Acad Sci USA 76:3289–3293

    Google Scholar 

  • Young RA, Davis RW (1983) Yeast RNA polymerase II genes: isolation with antibody probes. Science 222:778–782

    Google Scholar 

Download references

Author information

Author notes

  1. Anders S. Byström

    Present address: Department of Microbiology, Umeå University, S-901 87, Umeå, Sweden

Authors and Affiliations

  1. Whitehead Institute, Nine Cambridge Center, 02142, Cambridge, MA, USA

    Anders S. Byström & Gerald R. Fink

Authors
  1. Anders S. Byström
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gerald R. Fink
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by C.P. Hollenberg

The calculation of Td values (the temperature at which half of the duplex is dissociated) for oligonucleotides used as probes in hybridizations was based on the assumption that the increase in Td value was 4° C for each G:C base pair and 2° C for each A:T base pair (Wallace et al. 1981)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Byström, A.S., Fink, G.R. A functional analysis of the repeated methionine initiator tRNA genes (IMT) in yeast. Mol Gen Genet 216, 276–286 (1989). https://doi.org/10.1007/BF00334366

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00334366

Key words

Search

Navigation