Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1987 Mar;7(3):1208–1216. doi: 10.1128/mcb.7.3.1208

Cloning and characterization of LOS1, a Saccharomyces cerevisiae gene that affects tRNA splicing.

D J Hurt, S S Wang, Y H Lin, A K Hopper
PMCID: PMC365194  PMID: 3031485

Abstract

Saccharomyces cerevisiae strains carrying los1-1 mutations are defective in tRNA processing; at 37 degrees C, such strains accumulate tRNA precursors which have mature 5' and 3' ends but contain intervening sequences. Strains bearing los1-1 and an intron-containing ochre-suppressing tRNA gene, SUP4(0), also fail to suppress the ochre mutations ade2-1(0) and can1-100(0) at 34 degrees C. To understand the role of the LOS1 product in tRNA splicing, we initiated a molecular study of the LOS1 gene. Two plasmids, YEpLOS1 and YCpLOS1, that complement the los1-1 phenotype were isolated from the YEp24 and YCp50 libraries, respectively. YEpLOS1 and YCpLOS1 had overlapping restriction maps, indicating that the DNA in the overlapping segment could complement los1-1 when present in multiple or single copy. Integration of plasmid DNA at the LOS1 locus confirmed that these clones contained authentic LOS1 sequences. Southern analyses showed that LOS1 is a single copy gene. The locations of the LOS1 gene within YEpLOS1 and YCpLOS1 were determined by deletion and gamma-delta mapping. Two genomic disruptions of the LOS1 gene were constructed, i.e., an insertion of a 1.2-kilobase fragment carrying the yeast URA3 gene, los1::URA3, and a 2.4-kilobase deletion from the LOS1 gene, los1-delta V. Disruption or deletion of most of the LOS1 gene was not lethal; cells carrying the disrupted los1 alleles were viable and had phenotypes similar to those of cells carrying the los1-1 allele. Thus, it appears that the los1 gene product expedites tRNA splicing at elevated temperatures but is not essential for this process.

Full text

PDF
1208

Images in this article

1211Image
on p.1211
1212Image
on p.1212
1213Image
on p.1213
1213Image
on p.1213
1214Image
on p.1214

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brandriss M. C., Soll L., Botstein D. Recessive lethal amber suppressors in yeast. Genetics. 1975 Apr;79(4):551–560. doi: 10.1093/genetics/79.4.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  4. Etcheverry T., Colby D., Guthrie C. A precursor to a minor species of yeast tRNASer contains an intervening sequence. Cell. 1979 Sep;18(1):11–26. doi: 10.1016/0092-8674(79)90349-0. [DOI] [PubMed] [Google Scholar]
  5. Etcheverry T., Salvato M., Guthrie C. Recessive lethality of yeast strains carrying the SUP61 suppressor results from loss of a transfer RNA with a unique decoding function. J Mol Biol. 1982 Jul 15;158(4):599–618. doi: 10.1016/0022-2836(82)90251-0. [DOI] [PubMed] [Google Scholar]
  6. Goodman H. M., Olson M. V., Hall B. D. Nucleotide sequence of a mutant eukaryotic gene: the yeast tyrosine-inserting ochre suppressor SUP4-o. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5453–5457. doi: 10.1073/pnas.74.12.5453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Greer C. L., Peebles C. L., Gegenheimer P., Abelson J. Mechanism of action of a yeast RNA ligase in tRNA splicing. Cell. 1983 Feb;32(2):537–546. doi: 10.1016/0092-8674(83)90473-7. [DOI] [PubMed] [Google Scholar]
  8. Guyer M. S. Uses of the transposon gamma delta in the analysis of cloned genes. Methods Enzymol. 1983;101:362–369. doi: 10.1016/0076-6879(83)01027-7. [DOI] [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. Hopper A. K., Banks F. A yeast mutant which accumulates precursor tRNAs. Cell. 1978 Jun;14(2):211–219. doi: 10.1016/0092-8674(78)90108-3. [DOI] [PubMed] [Google Scholar]
  11. Hopper A. K., Schultz L. D., Shapiro R. A. Processing of intervening sequences: a new yeast mutant which fails to excise intervening sequences from precursor tRNAs. Cell. 1980 Mar;19(3):741–751. doi: 10.1016/s0092-8674(80)80050-x. [DOI] [PubMed] [Google Scholar]
  12. Hutchison H. T., Hartwell L. H., McLaughlin C. S. Temperature-sensitive yeast mutant defective in ribonucleic acid production. J Bacteriol. 1969 Sep;99(3):807–814. doi: 10.1128/jb.99.3.807-814.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Knapp G., Beckmann J. S., Johnson P. F., Fuhrman S. A., Abelson J. Transcription and processing of intervening sequences in yeast tRNA genes. Cell. 1978 Jun;14(2):221–236. doi: 10.1016/0092-8674(78)90109-5. [DOI] [PubMed] [Google Scholar]
  15. Knapp G., Ogden R. C., Peebles C. L., Abelson J. Splicing of yeast tRNA precursors: structure of the reaction intermediates. Cell. 1979 Sep;18(1):37–45. doi: 10.1016/0092-8674(79)90351-9. [DOI] [PubMed] [Google Scholar]
  16. Kuo C. L., Campbell J. L. 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. 1983 Oct;3(10):1730–1737. doi: 10.1128/mcb.3.10.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laten H., Gorman J., Bock R. M. Isopentenyladenosine deficient tRNA from an antisuppressor mutant of Saccharomyces cerevisiae. Nucleic Acids Res. 1978 Nov;5(11):4329–4342. doi: 10.1093/nar/5.11.4329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lathe R., Kieny M. P., Skory S., Lecocq J. P. Linker tailing: unphosphorylated linker oligonucleotides for joining DNA termini. DNA. 1984;3(2):173–182. doi: 10.1089/dna.1984.3.173. [DOI] [PubMed] [Google Scholar]
  19. Lee M. C., Knapp G. Transfer RNA splicing in Saccharomyces cerevisiae. Secondary and tertiary structures of the substrates. J Biol Chem. 1985 Mar 10;260(5):3108–3115. [PubMed] [Google Scholar]
  20. Najarian D., Dihanich M. E., Martin N. C., Hopper A. K. DNA sequence and transcript mapping of MOD5: features of the 5' region which suggest two translational starts. Mol Cell Biol. 1987 Jan;7(1):185–191. doi: 10.1128/mcb.7.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nasmyth K. A., Reed S. I. 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. doi: 10.1073/pnas.77.4.2119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. O'Farrell P. Z., Cordell B., Valenzuela P., Rutter W. J., Goodman H. M. Structure and processing of yeast precursor tRNAs containing intervening sequences. Nature. 1978 Aug 3;274(5670):438–445. doi: 10.1038/274438a0. [DOI] [PubMed] [Google Scholar]
  23. Ogden R. C., Lee M. C., Knapp G. Transfer RNA splicing in Saccharomyces cerevisiae: defining the substrates. Nucleic Acids Res. 1984 Dec 21;12(24):9367–9382. doi: 10.1093/nar/12.24.9367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Peebles C. L., Gegenheimer P., Abelson J. Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease. Cell. 1983 Feb;32(2):525–536. doi: 10.1016/0092-8674(83)90472-5. [DOI] [PubMed] [Google Scholar]
  25. Peebles C. L., Ogden R. C., Knapp G., Abelson J. Splicing of yeast tRNA precursors: a two-stage reaction. Cell. 1979 Sep;18(1):27–35. doi: 10.1016/0092-8674(79)90350-7. [DOI] [PubMed] [Google Scholar]
  26. Phizicky E. M., Schwartz R. C., Abelson J. Saccharomyces cerevisiae tRNA ligase. Purification of the protein and isolation of the structural gene. J Biol Chem. 1986 Feb 25;261(6):2978–2986. [PubMed] [Google Scholar]
  27. Post-Beittenmiller M. A., Hamilton R. W., Hopper J. E. Regulation of basal and induced levels of the MEL1 transcript in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Jul;4(7):1238–1245. doi: 10.1128/mcb.4.7.1238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Roman H, Phillips M M, Sands S M. Studies of Polyploid Saccharomyces. I. Tetraploid Segregation. Genetics. 1955 Jul;40(4):546–561. doi: 10.1093/genetics/40.4.546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rose M., Botstein D. Structure and function of the yeast URA3 gene. Differentially regulated expression of hybrid beta-galactosidase from overlapping coding sequences in yeast. J Mol Biol. 1983 Nov 15;170(4):883–904. doi: 10.1016/s0022-2836(83)80193-4. [DOI] [PubMed] [Google Scholar]
  30. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  31. Rudolph H., Koenig-Rauseo I., Hinnen A. One-step gene replacement in yeast by cotransformation. Gene. 1985;36(1-2):87–95. doi: 10.1016/0378-1119(85)90072-1. [DOI] [PubMed] [Google Scholar]
  32. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  33. St John T. P., Davis R. W. The organization and transcription of the galactose gene cluster of Saccharomyces. J Mol Biol. 1981 Oct 25;152(2):285–315. doi: 10.1016/0022-2836(81)90244-8. [DOI] [PubMed] [Google Scholar]
  34. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ullrich A., Shine J., Chirgwin J., Pictet R., Tischer E., Rutter W. J., Goodman H. M. Rat insulin genes: construction of plasmids containing the coding sequences. Science. 1977 Jun 17;196(4296):1313–1319. doi: 10.1126/science.325648. [DOI] [PubMed] [Google Scholar]
  37. Yost H. J., Lindquist S. RNA splicing is interrupted by heat shock and is rescued by heat shock protein synthesis. Cell. 1986 Apr 25;45(2):185–193. doi: 10.1016/0092-8674(86)90382-x. [DOI] [PubMed] [Google Scholar]
  38. Zakian V. A., Scott J. F. Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA. Mol Cell Biol. 1982 Mar;2(3):221–232. doi: 10.1128/mcb.2.3.221. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES