Expression of the <i>Drosophila</i> 70 000 Dalton heat shock protein is translationally controlled in yeast

Collatz, E.; Plesset, Judith; Foy, J J; McLaughlin, Calvin S.

doi:10.1002/yea.320010106

Cited by 6 publications

(1 citation statement)

References 35 publications

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“…Yeast total RNA was prepared (48), and poly(A)+ RNA was selected by passage over oligo(dT)-cellulose (2). Fractions of 20 ,ug of the poly(A)+ RNA were loaded into each lane of a 1.5% agarose gel which contained formaldehyde (29) and subjected to electrophoresis at 45 V for 18 h. The RNA was transferred onto diazobenzyloxymethyl (DBM)-paper (1) as described by Collatz et al (13) and probed with a nick-translated 2.2-kbp HindIII-BamHI fragment (Fig. 1C) to determine transcript size and relative abundance.…”

Section: Methodsmentioning

confidence: 99%

Isolation and characterization of PRT1, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevisiae.

Keierleber¹,

Wittekind²,

Qin³

et al. 1986

Mol. Cell. Biol.

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We isolated a cloned DNA fragment containing PRTI, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevisiae, by complementation of the temperature-sensitive prtl-l mutation. The entire PRT1 gene is contained within a 3.2-kilobase-pair segment of the cloned DNA in YEpl3 H1.2. Southern blot analysis demonstrated that PRTI is a single copy gene which is transcribed into a 2.3-kilobase RNA. We determined the direction of transcription and mapped the 5' and 3' ends of the gene.The initiation of protein biosynthesis is a complex process, involving numerous factors that must interact in a precisely ordered fashion. In the yeast Saccharomyces cerevisiae the isolation and characterization of temperaturesensitive mutants (23,24) has facilitated the study of the initiation of protein biosynthesis. ts187, containing the temperature-sensitive prtl-J allele, is one such mutant. The incorporation of radiolabeled amino acids into protein is greatly reduced in ts187 at the restrictive temperature (23), and an examination of the translational apparatus shows that polysomes shift to monosomes within minutes at the restrictive temperature. This result indicates that ts187 suffers a defect in initiation (36). In addition, in vitro translation systems prepared from ts187 cells grown at the restrictive temperature are inhibited in the formation of the 43S preinitiation complex (eIF2-Met tRNAf-GTP plus 40S ribosomal subunits) relative to in vitro systems prepared from wildtype cells or ts187 cells grown at the permissive temperature (17). Other aspects of protein biosynthesis such as methionine activation and methionylation of tRNAMet, formation of the ternary complex, mRNA binding, binding of the 60S subunit to form the 80S initiation complex, elongation, and termination are unaffected in in vitro translation systems prepared from heated ts187 cells (17). This evidence suggests that PRTJ codes for an initiation factor that is involved in the formation of the 43S preinitiation complex, probably a subunit of eIF-3 (33).Initiation factor eIF-3 has been purified from a number of systems including reticulocytes, wheat germ, liver and ascites (5, 38-40, 44, 45). It has a molecular weight between 500,000 and 700,000 and is composed of 7 to 10 polypeptides (33). Use of purified eIF-3 preparations has resulted in the assignment of several roles for this factor: a ribosome dissociation activity (4,38,40,47), stimulation of the formation of the 43S preinitiation complex (6,15,21,25,47), and mRNA binding (4,27,46,47).As a next step in the understanding of the process of the initiation of protein biosynthesis, we report the isolation and preliminary characterization of PRTI, a gene encoding one of the polypeptides of a eucaryotic initiation factor. MATERIALS AND METHODSStrains and growth conditions. S. cerevisiae MNB2-6D (a prtl ade leu2 tyri) was derived from a cross between ts187 (a * Corresponding author.gall adel ade2 ural his7 lys2 tyri) and LL20 (a leu2 his3). A364a (22) (a adel ade2 ural tyri his7 lys2 gall) an...

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Section: Methodsmentioning

confidence: 99%

Isolation and characterization of PRT1, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevisiae.

Keierleber¹,

Wittekind²,

Qin³

et al. 1986

Mol. Cell. Biol.

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Two‐dimensional gel analysis of yeast proteins: Application to the study of changes in the levels of major polypeptides of Saccharomyces cerevisiae depending on the fermentable or nonfermentable nature of the carbon source

1988

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Taking advantage of the recent identification of polypeptides of the carbon metabolism machinery on the yeast protein map [1], we applied two-dimensional gel electrophoresis to a study of changes in protein composition of Saccharomyces cerevisiae depending on the fermentable or nonfermentable nature of the carbon source. The levels of the 250 most abundant polypeptides were compared. Thirty-three were found to display markedly increased levels during growth on nonfermentable carbon sources. These 33 polypeptides include 11 mitochondrial polypeptides and polypeptides corresponding to alcohol dehydrogenase II, acetyl-CoA synthetase, phosphoenol pyruvate kinase and hexokinase PI. Sixteen other polypeptides, in contrast, reached their higher levels during growth on fermentable carbon sources. Among these were identified the monomeric subunits of 6 glycolytic enzymes. Collectively the 33 polypeptides of the first class comprised over 30% of the total soluble proteins of cells grown on nonfermentable carbon source and 3% during growth on fermentable carbon source. The protein fraction of the 16 polypeptides of the second class corresponded to 10% and 38%, respectively. Together these results show that two-dimensional gel electrophoresis, when coupled with the identification of polypeptides of the carbon metabolism apparatus, provides a valuable tool for approaching questions concerning carbon metabolism in S. cerevisiae.

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The Analysis of Temperature-Sensitive Mutants of Saccharomyces Cerevisiae Altered in Components Required for Protein Synthesis

Moldave¹,

McLaughlin²

1988

Genetics of Translation

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Expression of the Drosophila 70 000 Dalton heat shock protein is translationally controlled in yeast

Cited by 6 publications

References 35 publications

Isolation and characterization of PRT1, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevisiae.

Isolation and characterization of PRT1, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevisiae.

Two‐dimensional gel analysis of yeast proteins: Application to the study of changes in the levels of major polypeptides of Saccharomyces cerevisiae depending on the fermentable or nonfermentable nature of the carbon source

The Analysis of Temperature-Sensitive Mutants of Saccharomyces Cerevisiae Altered in Components Required for Protein Synthesis

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