Cycloheximide resistance in yeast: the gene and its portein

Kaàufer, Norbert F.; Fried, Howard M.; Schwindinger, William F.; Jasin, Maria; Warner, Jonathan R.

doi:10.1093/nar/11.10.3123

Cited by 188 publications

(100 citation statements)

References 25 publications

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“…Kaiifer et al subsequently cloned a cycloheximide-resistance allele of cyh2 and determined that its gene product was ribosomal protein L29 (Kaiifer et al, 1983). Yao and Yao have also implicated this same protein as the cycloheximide-resistance determinant in Tetrahymena (Yao and Yao, 1991).…”

Section: Discussionmentioning

confidence: 99%

“…If this is indeed the case, the effect is particularly dramatic with L41 alterations and may reflect the nature of the amino acid change involved. The mutation to cycloheximide resistance in the cyh2 allele involves a Gln+Glu change (Stocklein and Piepersberg, 1980;Kaiifer et al, 1983). An additional L29 mutation (Stocklein et al, 1981) involves a Gin-Lys change at the same relative position.…”

Section: Discussionmentioning

confidence: 99%

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Natural cycloheximide resistance in yeast

Dehoux¹,

Davies²,

Cannon

1993

European Journal of Biochemistry

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The yest Kluyveromyces lactis is resistant to high concentrations (1 mg/ml) of the antibiotic cycloheximide. Using in vitro translation studies it was confirmed that this extreme resistance is a property of ribosomes. The resistance determinant from K. lactis was cloned into Saccharomyces cerevisiae. Nucleotide sequence analysis of the determinant demonstrated that resistance was conferred by the K. Lactis ribosomal protein L41. K. lactis was shown to contain only one copy of the gene that encodes this protein and the gene was located to chromosome III. In contrast, S. cerevisiae was found to contain multiple copies of the gene for the corresponding ribosomal protein L41 which mapped to two of the three chromosomes V, XIV and VIII. Since the cycloheximide‐resistance gene of K. lactis causes essentially complete protection against inhibition by the drug, it is likely to be particularly useful as a selective marker in eukaryotic gene transfer studies.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Natural cycloheximide resistance in yeast

Dehoux¹,

Davies²,

Cannon

1993

European Journal of Biochemistry

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“…Oligonucleotide 1 (Table 2) was used to monitor mRNA decay of the PGK1 alleles listed in Table 1. The 506-bp AccI-EcoRI fragment in M13mp9 was used to prepare a radioactive probe to monitor the decay of the CYH2 mRNA (Kaufer et al 1983).…”

Section: Preparation Of Radioactive Probesmentioning

confidence: 99%

mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor.

Peltz¹,

Brown²,

Jacobson³

1993

Genes Dev.

285

349

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Nonsense mutations in a gene can accelerate the decay rate of the mRNA transcribed from that gene, a phenomenon we describe as nonsense-mediated mRNA decay. Using amber (UAG) mutants of the yeast PGK1 gene as a model system, we find that nonsense-mediated mRNA decay is position dependent, that is, nonsense mutations within the initial two-thirds of the PGKl-coding region accelerate the decay rate of the PGK1 transcript ~<12-fold, whereas nonsense mutations within the carboxy-terminal third of the coding region have no effect on mRNA decay. Moreover, we find that this position effect reflects (1) a requirement for sequences 3' to the nonsense mutation that may be necessary for translational reinitiation or pausing, and (2) the presence of an additional sequence that, when translated, inactivates the nonsense-mediated mRNA decay pathway. This stabilizing element is positioned within the coding region such that it constitutes the boundary between nonsense mutations that do or do not affect mRNA decay. Rapid decay of PGK1 nonsense-containing transcripts is also dependent on the status of the UPF1 gene. Regardless of the position of an amber codon in the PGK1 gene, deletion of the UPF1 gene restores wild-type decay rates to nonsense-containing PGK1 transcripts.

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“…The labeled fragments were cleaved, and the appropriate singly end-labeled fragment was isolated. The SI protection reactions were performed essentially as described previously (20). After the doublestranded DNA probes were denatured at 85°C for 15 min, DNA fragments were annealed to total RNA in 80% formamide-40 mM PIPES [piperazine-N-N'-bis (2-ethanesulfonic acid)] (pH 6.5)-400 mM NaCl-1 mM EDTA for 3 to 4 h at 52°C, lowering the incubation temperature by 1°C every hour.…”

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confidence: 99%

Introduction of functional artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe.

Gatermann¹,

Hoffmann²,

Rosenberg³

et al. 1989

Mol. Cell. Biol.

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Insertion of a 36-base-pair (bp) synthetic oligonucleotide comprising the sequence 5'-GTAGGT(19N)CTAAT (4N)AG-3' into several different positions within the coding region of the naturally intronless ura4 gene of Schizosaccharomyces pombe leads to an efficiently spliced gene producing a functional product. This suggests that the proper signals within an intron are sufficient to initiate and complete a splicing event independent of the location of the intron in the gene. Point mutations in the 5' junction (5'-GTAGGT-3') and in the putative branch sequence (5'-CTAAT-3') affect splicing efficiency significantly. A G-to-A transition at the first nucleotide at the 5' splice junction (5'-ATAGGT-3') abolishes the use of the authentic splice junction and leads to the increased use of an alternative splice site. No functional product is produced from this transcript. An A-to-G transition of the second A in the putative branch sequence (5'-CTAGT-3') lowers the splicing efficiency drastically, but still results in a functional gene product. Furthermore, extension of the 36-bp intron to introns more than 180 bp in size abolishes splicing, suggesting that the splicing apparatus might be restricted to very short introns. We discuss the possibility that S. pombe introns represent a simple type of eucaryotic intron.Intervening sequences appear to be spliced from nuclear pre-mRNA by a common mechanism. The precursor RNA of eucaryotic cells is assembled into a spliceosome (splicing complex) in which the actual splicing event takes place (5,7,15,23). Splicing can be viewed as a stepwise dynamic process which includes recognition of the intron, assembly of the spliceosome, and the actual splicing events (3, 13, 39). During this process, the precursor RNA is converted into an intermediate lariat structure by linking the first guanosine residue of the intron to an adenosine residue within the intron via a 2'-5' phosphodiester bond; the exon 1-intron junction is simultaneously cleaved. In the following steps, the 3' splice site is cleaved and exon 1 and exon 2 are linked together. The intron is released as a free lariat (9,37,42,44).There is evidence that at least five different small ribonucleoprotein particles (snRNPs) are involved in this process (29). These trans-acting factors are involved in the recognition of introns by interacting with specific cis-acting signals such as the 5' splice site, branch sequence, and 3' splice site of the precursor RNA (3, 4, 7). In addition, specific snRNPsnRNP interactions also seem to play a role in the spliceosome assembly and in the splicing process itself (4, 24). However, the precise functions of each of the snRNPs are not yet fully understood (6,7,27,36). Single proteins are also involved in the splicing process, although little is known about their functions (28).It is interesting that the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe appear to have introns which differ from one another and from those in higher eucaryotes. In contrast to the introns i...

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Cycloheximide resistance in yeast: the gene and its portein

Cited by 188 publications

References 25 publications

Natural cycloheximide resistance in yeast

Natural cycloheximide resistance in yeast

mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor.

Introduction of functional artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe.

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