Correspondence of yeast UAA suppressors to cloned tRNAUCA Ser genes

James Broach, Linda Friedman, Fred Sherman

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

We have analyzed the genes encoding tRNAUCA Ser of yeast. By hybridization analysis we have established that only three genes encode tRNAUCA Ser, all of which have been cloned. The DNA sequences of the coding regions of all the three genes are identical, although considerable sequence divergence exists at the 3′ and 5′ flanking regions. The suppressor locus to which each of the three cloned tRNAUCA Ser genes corresponds was determined by integrative transformation of yeast. A leu2- sup+ yeast strain was transformed to Leu+ with plasmids which carried the LEU2 gene of yeast and one of the DNA fragments containing a tRNAUCA Ser gene. Integration of the plasmid at the homologous transfer RNA locus on the chromosome introduced the LEU2 marker at the site, which could then be followed in standard genetic crosses. By this method, we have shown that the three cloned fragments correspond to the serine-inserting nonsense suppressors SUP16, SUP17 and SUP19. Finally, the SUP16 allele was recovered directly from yeast by integrating a pBR322-LEU2-sup16+ hybrid plasmid at the SUP16 locus. Appropriate digestion of genomic DNA isolated from the transformed strain permitted recovery of either the wild-type allele or the SUP16 allele on plasmids that were otherwise identical to the original transforming plasmid. This method is generally applicable to the recovery of any mutant allele of yeast for which one has DNA containing the corresponding wildtype gene.

Original languageEnglish (US)
Pages (from-to)375-387
Number of pages13
JournalJournal of Molecular Biology
Volume150
Issue number3
DOIs
StatePublished - Aug 15 1981

Fingerprint

Yeasts
Plasmids
Genes
Alleles
DNA
Genetic Crosses
3' Flanking Region
5' Flanking Region
Transfer RNA
Serine
Digestion
Chromosomes

All Science Journal Classification (ASJC) codes

  • Virology

Cite this

Broach, James ; Friedman, Linda ; Sherman, Fred. / Correspondence of yeast UAA suppressors to cloned tRNAUCA Ser genes. In: Journal of Molecular Biology. 1981 ; Vol. 150, No. 3. pp. 375-387.
@article{b03111a317fd4e1696c815a6f8550667,
title = "Correspondence of yeast UAA suppressors to cloned tRNAUCA Ser genes",
abstract = "We have analyzed the genes encoding tRNAUCA Ser of yeast. By hybridization analysis we have established that only three genes encode tRNAUCA Ser, all of which have been cloned. The DNA sequences of the coding regions of all the three genes are identical, although considerable sequence divergence exists at the 3′ and 5′ flanking regions. The suppressor locus to which each of the three cloned tRNAUCA Ser genes corresponds was determined by integrative transformation of yeast. A leu2- sup+ yeast strain was transformed to Leu+ with plasmids which carried the LEU2 gene of yeast and one of the DNA fragments containing a tRNAUCA Ser gene. Integration of the plasmid at the homologous transfer RNA locus on the chromosome introduced the LEU2 marker at the site, which could then be followed in standard genetic crosses. By this method, we have shown that the three cloned fragments correspond to the serine-inserting nonsense suppressors SUP16, SUP17 and SUP19. Finally, the SUP16 allele was recovered directly from yeast by integrating a pBR322-LEU2-sup16+ hybrid plasmid at the SUP16 locus. Appropriate digestion of genomic DNA isolated from the transformed strain permitted recovery of either the wild-type allele or the SUP16 allele on plasmids that were otherwise identical to the original transforming plasmid. This method is generally applicable to the recovery of any mutant allele of yeast for which one has DNA containing the corresponding wildtype gene.",
author = "James Broach and Linda Friedman and Fred Sherman",
year = "1981",
month = "8",
day = "15",
doi = "10.1016/0022-2836(81)90553-2",
language = "English (US)",
volume = "150",
pages = "375--387",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "3",

}

Correspondence of yeast UAA suppressors to cloned tRNAUCA Ser genes. / Broach, James; Friedman, Linda; Sherman, Fred.

In: Journal of Molecular Biology, Vol. 150, No. 3, 15.08.1981, p. 375-387.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Correspondence of yeast UAA suppressors to cloned tRNAUCA Ser genes

AU - Broach, James

AU - Friedman, Linda

AU - Sherman, Fred

PY - 1981/8/15

Y1 - 1981/8/15

N2 - We have analyzed the genes encoding tRNAUCA Ser of yeast. By hybridization analysis we have established that only three genes encode tRNAUCA Ser, all of which have been cloned. The DNA sequences of the coding regions of all the three genes are identical, although considerable sequence divergence exists at the 3′ and 5′ flanking regions. The suppressor locus to which each of the three cloned tRNAUCA Ser genes corresponds was determined by integrative transformation of yeast. A leu2- sup+ yeast strain was transformed to Leu+ with plasmids which carried the LEU2 gene of yeast and one of the DNA fragments containing a tRNAUCA Ser gene. Integration of the plasmid at the homologous transfer RNA locus on the chromosome introduced the LEU2 marker at the site, which could then be followed in standard genetic crosses. By this method, we have shown that the three cloned fragments correspond to the serine-inserting nonsense suppressors SUP16, SUP17 and SUP19. Finally, the SUP16 allele was recovered directly from yeast by integrating a pBR322-LEU2-sup16+ hybrid plasmid at the SUP16 locus. Appropriate digestion of genomic DNA isolated from the transformed strain permitted recovery of either the wild-type allele or the SUP16 allele on plasmids that were otherwise identical to the original transforming plasmid. This method is generally applicable to the recovery of any mutant allele of yeast for which one has DNA containing the corresponding wildtype gene.

AB - We have analyzed the genes encoding tRNAUCA Ser of yeast. By hybridization analysis we have established that only three genes encode tRNAUCA Ser, all of which have been cloned. The DNA sequences of the coding regions of all the three genes are identical, although considerable sequence divergence exists at the 3′ and 5′ flanking regions. The suppressor locus to which each of the three cloned tRNAUCA Ser genes corresponds was determined by integrative transformation of yeast. A leu2- sup+ yeast strain was transformed to Leu+ with plasmids which carried the LEU2 gene of yeast and one of the DNA fragments containing a tRNAUCA Ser gene. Integration of the plasmid at the homologous transfer RNA locus on the chromosome introduced the LEU2 marker at the site, which could then be followed in standard genetic crosses. By this method, we have shown that the three cloned fragments correspond to the serine-inserting nonsense suppressors SUP16, SUP17 and SUP19. Finally, the SUP16 allele was recovered directly from yeast by integrating a pBR322-LEU2-sup16+ hybrid plasmid at the SUP16 locus. Appropriate digestion of genomic DNA isolated from the transformed strain permitted recovery of either the wild-type allele or the SUP16 allele on plasmids that were otherwise identical to the original transforming plasmid. This method is generally applicable to the recovery of any mutant allele of yeast for which one has DNA containing the corresponding wildtype gene.

UR - http://www.scopus.com/inward/record.url?scp=0019414012&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0019414012&partnerID=8YFLogxK

U2 - 10.1016/0022-2836(81)90553-2

DO - 10.1016/0022-2836(81)90553-2

M3 - Article

VL - 150

SP - 375

EP - 387

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 3

ER -