Molecular and General Genetics MGG

, Volume 216, Issue 2–3, pp 276–286 | Cite as

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

  • Anders S. Byström
  • Gerald R. Fink
Article

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 tRNAIMet. 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 tRNAIMetis 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.

Key words

Methionine Initiator tRNA tRNA(met) Yeast Multigene family 

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-tRNAIMet

methionylated initiator methionine transfer RNA

eIF-2

eukaryotic initiation factor two

GTP

guanosine 5′-triphosphate

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References

  1. Benton WD, Davis RW (1977) Screening λgt recombinant clones by hybridization to single plaques in situ. Science 196:180–182Google Scholar
  2. 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 197:345–346Google Scholar
  3. Boeke JD, Garfinkel DJ, Styles CA, Fink GR (1985) Ty elements transpose through an RNA intermediate. Cell 40:491–500Google Scholar
  4. 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 8:17–24Google Scholar
  5. Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472Google Scholar
  6. 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 255:9981–9984Google Scholar
  7. Carle GF, Olson MV (1984) Separation of chromosomal DNA molecules from yeast by orthogonal-field-alteration gel electrophoresis. Nucleic Acids Res 12:5647–5664Google Scholar
  8. Cigan MA, Donahue TF (1986) The methionine initiator tRNA genes of yeast. Gene 41:343–348Google Scholar
  9. Gaber RF, Mathison L, Edelman I, Culbertson MR (1983) Frameshift suppression in Saccharomyces cerevisiae. VI. Complete genetic map of twenty-five suppressor genes. Genetics 103:389–407Google Scholar
  10. Gill DR, Hatfull GF, Salmond GPC (1986) A new cell division operon in Escherichia coli. Mol Gen Genet 205:134–145Google Scholar
  11. Grunstein M, Hogness DS (1975) Colony hybridization: A method for isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci USA 72:3961–3965Google Scholar
  12. 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 9:4485–4494Google Scholar
  13. Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–197Google Scholar
  14. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168Google Scholar
  15. Kozak M (1983) Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev 47:1–45Google Scholar
  16. 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 3:1730–1737Google Scholar
  17. Maitra U, Stringer EA, Chaudhuri A (1982) Initiation factors in protein biosynthesis. Annu Rev Biochem 51:869–900Google Scholar
  18. Mandel M, Higa A (1970) Calcium dependent bacteriophage DNA infection. J Mol Biol 53:159–162Google Scholar
  19. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  20. McKnight GL, Cardillo TS, Sherman F (1981) An extensive deletion causing overproduction of yeast iso-2-cytochrome c. Cell 25:409–419Google Scholar
  21. Messing J, Crea R, Seeburg PH (1981) A system for shotgun DNA sequencing. Nucleic Acids Res 9:309–321Google Scholar
  22. Moldave K (1985) Eukaryotic protein synthesis. Annu Rev Biochem 54:1109–1149Google Scholar
  23. Mortimer RK, Schild D (1985) Genetic map of Saccharomyces cerevisiae. Microbiol Rev 49: 181–213Google Scholar
  24. 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 51:309–326Google Scholar
  25. Norrander J, Kempe T, Messing J (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101–106Google Scholar
  26. 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 132:387–410Google Scholar
  27. Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation: A model system for the study of recombination. Proc Natl Acad Sci USA 78:6354–6358Google Scholar
  28. Perkins D (1949) Biochemical mutants in the smut fungus Ustilago maydis. Genetics 34:607–626Google Scholar
  29. 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 60:237–243Google Scholar
  30. Sanger F, Nicklen S, Coulsen AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  31. Schatz PJ, Solomon F, Botstein D (1986) Genetically essential and non-essential α-tubulin genes specify functionally interchangeable proteins. Mol Cell Biol 6:3722–3733Google Scholar
  32. Scherer S, Davis RW (1979) Replacements of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci USA 76:4951–4955Google Scholar
  33. Schevitz RW, Podjarny AD, Krishnamachari N, Hughes JJ, Sigler PB (1979) Crystal structure of a eukaryotic initiator tRNA. Nature 278:188–190Google Scholar
  34. Sharp SJ, Schaack J, Cooley L, Burke DJ, Soll D (1985) Structure and transcription of eukaryotic tRNA genes. Crit Rev Biochem 19:107–144Google Scholar
  35. Sherman F, Fink GR, Hicks JB (1986) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  36. Simsek M, RajBhandary UL (1972) The primary structure of yeast initiator transfer ribonucleic acid. Biochem Biophys Res Commun 49:508–515Google Scholar
  37. 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-r188Google Scholar
  38. 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 76:1035–1039Google Scholar
  39. Tschumper G, Carbon J (1980) Sequence of a DNA fragment containing a chromosomal replicator and the TRP1 gene. Gene 10:157–166Google Scholar
  40. Venegas A, Gonzalez E, Bull P, Valenzuela P (1982) Isolation and structure of a yeast initiator tRNAMet gene. nucleic Acids Res 10:1093–1096Google Scholar
  41. 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 9:879–894Google Scholar
  42. Wrede P, Woo NH, Rich A (1979) Initiator tRNAs have a unique anticodon loop formation. Proc Natl Acad Sci USA 76:3289–3293Google Scholar
  43. Young RA, Davis RW (1983) Yeast RNA polymerase II genes: isolation with antibody probes. Science 222:778–782Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Anders S. Byström
    • 1
  • Gerald R. Fink
    • 1
  1. 1.Whitehead InstituteNine Cambridge CenterCambridgeUSA
  2. 2.Department of MicrobiologyUmeå UniversityUmeåSweden

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