Deletion of a single-copy tRNA affects microtubule function in Saccharomyces cerevisiae.

Reijo, Renee A.; Cho, Dan-Sung; Huffaker, Tim C.

doi:10.1093/genetics/135.4.955

Cited by 9 publications

(2 citation statements)

References 26 publications

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“…A third isoacceptor is tRNA Arg1 CCU , which reads the AGG codon. It is encoded by a single gene copy and is dispensable, as shown by the viable phenotype of the knockout strain (23). Indeed, AGG codons can be read by tRNA Arg2 ICG .…”

Section: Discussionmentioning

confidence: 99%

A yeast knockout strain to discriminate between active and inactive tRNA molecules

Geslain

Martin²,

Camasses³

et al. 2003

Nucleic Acids Research

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Here we report the construction of a yeast genetic screen designed to identify essential residues in tRNA(Arg). The system consists of a tRNA(Arg) knockout strain and a set of vectors designed to rescue and select for variants of tRNA(Arg). By plasmid shuffling we selected inactive tRNA mutants that were further analyzed by northern blotting. The mutational analysis focused on the tRNA D and anticodon loops that contact the aminoacyl-tRNA synthetase. The anticodon triplet was excluded from the analysis because of its role in decoding the Arg codons. Most of the inactivating mutations are residues involved in tertiary interactions. These mutations had dramatic effects on tRNA(Arg) abundance. Other inactivating mutations were located in the anticodon loop, where they did not affect transcription and aminoacylation but probably altered interaction with the translation machinery. No lethal effects were observed when residues 16, 20 and 38 were individually mutated, despite the fact that they are involved in sequence-specific interactions with the aminoacyl-tRNA synthetase. However, the steady-state levels of the aminoacylated forms of U20A and U20G were decreased by a factor of 3.5-fold in vivo. This suggests that, unlike in the Escherichia coli tRNA(Arg):ArgRS system where residue 20 (A) is a major identity element, in yeast this position is of limited consequence.

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Section: Discussionmentioning

confidence: 99%

A yeast knockout strain to discriminate between active and inactive tRNA molecules

Geslain

Martin²,

Camasses³

et al. 2003

Nucleic Acids Research

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“…This gene rts1 was identified as an extragenic suppressor of tub2-104. Tub2-104 is a cold-sensitive allele of the gene encoding βtubulin in S. cerevisiae (Reijo et al 1993). Individual growth validation of the homozygous diploid strain that lacks this gene showed that this strain had only 33% growth (67% inhibition) in the presence of 10 µM of PelA after 20 generations ( Fiedler et al 2009).…”

Section: Pela Hop Microarray Hitsmentioning

confidence: 99%

Finding the Binding Site of Peloruside A and its Secondary Effects in Saccharomyces Cerevisiae using a Chemical Genetics Approach

Hanna¹

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<p>Peloruside A, a natural product isolated from the marine sponge Mycale hentscheli, is a microtubule-stabilising agent that has a similar mechanism of action to the anticancer drug paclitaxel and is cytotoxic to cultured mammalian cells. Peloruside appears to bind to a distinct site on mammalian tubulin that is different from that of the taxoid-site drugs. Because of the high sequence homology between yeast and mammalian tubulin, Saccharomyces cerevisiae (S. cerevisiae) was used as a model organism to characterise the peloruside-binding site with the aim of advancing our understanding about this site on mammalian tubulin. Wild type S. cerevisiae (BY4741) was sensitive to peloruside at uM concentrations; however, a strain that lacks the mad2 (Mitotic Arrest Deficient 2) gene showed increased sensitivity to the drug at much lower uM concentrations. This gene is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. The main aims of this project were to define the binding site of peloruside A using yeast tubulin to see if microtubule function and/or morphology is altered in yeast by peloruside, and to identify any secondary drug targets "friends of the target" through chemical genetic interactions profiling (Homozygous deletion profiling microarray). Site-directed mutagenesis was used to mutate two conserved amino acids (A296T; R306H) known to confer resistance to peloruside in mammalian cells. Based on a published computer model of the peloruside binding site on mammalian tubulin, we also mutated three other amino acids, two that were predicted to affect peloruside binding (Q291M and N337L), and one that was predicted to affect laulimalide binding but have little affect on peloruside binding (V333W). We also included a negative control that was predicted to have no effect on peloruside binding (R282Q) and would affect epothilone binding. We found that of the six point mutations, only Q291M failed to confer resistance in yeast and instead it increased the inhibition to the drug. Using a bud index assay, confocal microscopy, and flow cytometry, 40-50 uM peloruside was shown to block cells in G2/M of the cell cycle, confirming a direct action of the drug on microtubule function. Homozygous profiling (HOP) microarray analysis of a deletion mutant set of yeast genes was also carried out to identify gene products that interact with peloruside in order to link the drug to specific networks or biochemical pathways in the cells. From site-directed mutagenesis, we concluded that peloruside binds to yeast B-tubulin in the region predicted by the published model of the binding site, and therefore mapping the site on yeast tubulin could provide useful information about the mammalian binding site for peloruside. The bud index, flow cytometry, and confocal microscopy experiments provided further evidence that peloruside interacts with yeast tubulin. From HOP we found that peloruside has roles in the cell cycle, as expected, and has effects on protein transport, secretion, cell wall synthesis, and steroid biosynthesis pathways.</p>

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A member of a novel family of yeast ‘zn-finger’ proteins mediates the transition from stationary phase to cell proliferation.

et al. 1994

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The cloning and molecular characterization of the GCS1 gene from the budding yeast Saccharomyces cerevisiae show that stationary phase is in fact a unique developmental state, with requirements to resume cell proliferation that can be distinct from those for maintenance of proliferation. Deletion of the GCS1 gene products a novel phenotype: stationary‐phase mutant cells do not resume proliferation at a restrictive temperature of 15 degrees C, but mutant cells lacking Gcs1p that are proliferating at the permissive temperature of 29 degrees C continue to proliferate after transfer to 15 degrees C as long as nutrients are available. The GCS1 gene sequence predicts a 39 kDa polypeptide with a novel ‘Zn‐finger’ motif. A point mutation within the finger motif produces a phenotype that mimics that of deletion of the GCS1 gene, showing that the finger motif is essential for full Gcs1p activity. Gcs1p and the products of two newly identified genes, SPS18 and GLO3, constitute a family of novel Zn‐finger proteins.

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Deletion of a single-copy tRNA affects microtubule function in Saccharomyces cerevisiae.

Cited by 9 publications

References 26 publications

A yeast knockout strain to discriminate between active and inactive tRNA molecules

A yeast knockout strain to discriminate between active and inactive tRNA molecules

Finding the Binding Site of Peloruside A and its Secondary Effects in Saccharomyces Cerevisiae using a Chemical Genetics Approach

A member of a novel family of yeast ‘zn-finger’ proteins mediates the transition from stationary phase to cell proliferation.

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