Molecular analysis of Saccharomyces cerevisiae chromosome I. On the number of genes and the identification of essential genes using temperature-sensitive-lethal mutations

Steven D. Harris, Judy Cheng, Tom A. Pugh, John R. Pringle

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Previous analyses of Saccharomyces cerevisiae chromosome I have suggested that the majority (>75%) of single-copy essential genes on this chromosome are difficult or impossible to identify using temperature-sensitive (Ts-) lethal mutations. To investigate whether this situation reflects intrinsic difficulties in generating temperature-sensitive proteins or constraints on mutagenesis in yeast, we subjected three cloned essential genes from chromosome I to mutagenesis in an Escherichia coli mutator strain and screened for Ts- lethal mutations in yeast using the "plasmid-shuffle" technique. We failed to obtain Ts- lethal mutations in two of the genes (FUN12 and FUN20), while the third gene yielded such mutations, but only at a low frequency. DNA sequence analysis of these mutant alleles and of the corresponding wild-type region revealed that each mutation was a single substitution not in the previously identified gene FUN19, but in the adjacent, newly identified essential gene FUN53. FUN19 itself proved to be non-essential. These results suggest that many essential proteins encoded by genes on chromosome I cannot be rendered thermolabile by single mutations. However, the results obtained with FUN53 suggest that there may also be significant constraints on mutagenesis in yeast. The 5046 base-pair interval sequenced contains the complete FUN19, FUN53 and FUN20 coding regions, as well as a portion of the adjacent non-essential FUN21 coding region. In all, 68 to 75% of this interval is open reading frame. None of the four predicted products shows significant homologies to known proteins in the available databases.

Original languageEnglish (US)
Pages (from-to)53-65
Number of pages13
JournalJournal of Molecular Biology
Volume225
Issue number1
DOIs
StatePublished - May 5 1992

Fingerprint

Essential Genes
Saccharomyces cerevisiae
Chromosomes
Mutation
Temperature
Mutagenesis
Genes
Yeasts
Proteins
DNA Sequence Analysis
Base Pairing
Open Reading Frames
Plasmids
Alleles
Databases
Escherichia coli

Keywords

  • Saccharomyces cerevisiae
  • chromosome
  • gene-number paradox
  • mutagenesis
  • temperature-sensitive mutants

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Cite this

Molecular analysis of Saccharomyces cerevisiae chromosome I. On the number of genes and the identification of essential genes using temperature-sensitive-lethal mutations. / Harris, Steven D.; Cheng, Judy; Pugh, Tom A.; Pringle, John R.

In: Journal of Molecular Biology, Vol. 225, No. 1, 05.05.1992, p. 53-65.

Research output: Contribution to journalArticle

@article{a581d46e2d024d6faf82ec43ca52c2e8,
title = "Molecular analysis of Saccharomyces cerevisiae chromosome I. On the number of genes and the identification of essential genes using temperature-sensitive-lethal mutations",
abstract = "Previous analyses of Saccharomyces cerevisiae chromosome I have suggested that the majority (>75{\%}) of single-copy essential genes on this chromosome are difficult or impossible to identify using temperature-sensitive (Ts-) lethal mutations. To investigate whether this situation reflects intrinsic difficulties in generating temperature-sensitive proteins or constraints on mutagenesis in yeast, we subjected three cloned essential genes from chromosome I to mutagenesis in an Escherichia coli mutator strain and screened for Ts- lethal mutations in yeast using the {"}plasmid-shuffle{"} technique. We failed to obtain Ts- lethal mutations in two of the genes (FUN12 and FUN20), while the third gene yielded such mutations, but only at a low frequency. DNA sequence analysis of these mutant alleles and of the corresponding wild-type region revealed that each mutation was a single substitution not in the previously identified gene FUN19, but in the adjacent, newly identified essential gene FUN53. FUN19 itself proved to be non-essential. These results suggest that many essential proteins encoded by genes on chromosome I cannot be rendered thermolabile by single mutations. However, the results obtained with FUN53 suggest that there may also be significant constraints on mutagenesis in yeast. The 5046 base-pair interval sequenced contains the complete FUN19, FUN53 and FUN20 coding regions, as well as a portion of the adjacent non-essential FUN21 coding region. In all, 68 to 75{\%} of this interval is open reading frame. None of the four predicted products shows significant homologies to known proteins in the available databases.",
keywords = "Saccharomyces cerevisiae, chromosome, gene-number paradox, mutagenesis, temperature-sensitive mutants",
author = "Harris, {Steven D.} and Judy Cheng and Pugh, {Tom A.} and Pringle, {John R.}",
year = "1992",
month = "5",
day = "5",
doi = "10.1016/0022-2836(92)91025-K",
language = "English (US)",
volume = "225",
pages = "53--65",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Molecular analysis of Saccharomyces cerevisiae chromosome I. On the number of genes and the identification of essential genes using temperature-sensitive-lethal mutations

AU - Harris, Steven D.

AU - Cheng, Judy

AU - Pugh, Tom A.

AU - Pringle, John R.

PY - 1992/5/5

Y1 - 1992/5/5

N2 - Previous analyses of Saccharomyces cerevisiae chromosome I have suggested that the majority (>75%) of single-copy essential genes on this chromosome are difficult or impossible to identify using temperature-sensitive (Ts-) lethal mutations. To investigate whether this situation reflects intrinsic difficulties in generating temperature-sensitive proteins or constraints on mutagenesis in yeast, we subjected three cloned essential genes from chromosome I to mutagenesis in an Escherichia coli mutator strain and screened for Ts- lethal mutations in yeast using the "plasmid-shuffle" technique. We failed to obtain Ts- lethal mutations in two of the genes (FUN12 and FUN20), while the third gene yielded such mutations, but only at a low frequency. DNA sequence analysis of these mutant alleles and of the corresponding wild-type region revealed that each mutation was a single substitution not in the previously identified gene FUN19, but in the adjacent, newly identified essential gene FUN53. FUN19 itself proved to be non-essential. These results suggest that many essential proteins encoded by genes on chromosome I cannot be rendered thermolabile by single mutations. However, the results obtained with FUN53 suggest that there may also be significant constraints on mutagenesis in yeast. The 5046 base-pair interval sequenced contains the complete FUN19, FUN53 and FUN20 coding regions, as well as a portion of the adjacent non-essential FUN21 coding region. In all, 68 to 75% of this interval is open reading frame. None of the four predicted products shows significant homologies to known proteins in the available databases.

AB - Previous analyses of Saccharomyces cerevisiae chromosome I have suggested that the majority (>75%) of single-copy essential genes on this chromosome are difficult or impossible to identify using temperature-sensitive (Ts-) lethal mutations. To investigate whether this situation reflects intrinsic difficulties in generating temperature-sensitive proteins or constraints on mutagenesis in yeast, we subjected three cloned essential genes from chromosome I to mutagenesis in an Escherichia coli mutator strain and screened for Ts- lethal mutations in yeast using the "plasmid-shuffle" technique. We failed to obtain Ts- lethal mutations in two of the genes (FUN12 and FUN20), while the third gene yielded such mutations, but only at a low frequency. DNA sequence analysis of these mutant alleles and of the corresponding wild-type region revealed that each mutation was a single substitution not in the previously identified gene FUN19, but in the adjacent, newly identified essential gene FUN53. FUN19 itself proved to be non-essential. These results suggest that many essential proteins encoded by genes on chromosome I cannot be rendered thermolabile by single mutations. However, the results obtained with FUN53 suggest that there may also be significant constraints on mutagenesis in yeast. The 5046 base-pair interval sequenced contains the complete FUN19, FUN53 and FUN20 coding regions, as well as a portion of the adjacent non-essential FUN21 coding region. In all, 68 to 75% of this interval is open reading frame. None of the four predicted products shows significant homologies to known proteins in the available databases.

KW - Saccharomyces cerevisiae

KW - chromosome

KW - gene-number paradox

KW - mutagenesis

KW - temperature-sensitive mutants

UR - http://www.scopus.com/inward/record.url?scp=0026589242&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026589242&partnerID=8YFLogxK

U2 - 10.1016/0022-2836(92)91025-K

DO - 10.1016/0022-2836(92)91025-K

M3 - Article

C2 - 1583694

AN - SCOPUS:0026589242

VL - 225

SP - 53

EP - 65

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 1

ER -