PubMed Health⌕ Search

Biomedical subjects

S W Liebman

Publications and source records attributed to S W Liebman.

At least 37 records · Page 2Linked to original sources
PubMed1997

The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae.

It has been previously shown that genes transcribed by RNA polymerase II (RNAP II) are subject to position effect variegation when located near yeast telomeres. This telomere position effect requires a number of gene products that are also required for silencing at the HML and HMR loci. Here, we show that a null mutation of the DNA repair gene RAD6 reduces silencing of the HM loci and lowers the mating efficiency of MATa strains. Likewise, rad6-delta reduces silencing of the telomere-located RNAP II-transcribed genes URA3 and ADE2. We also show that the RNAP III-transcribed tyrosyl tRNA gene, SUP4-o, is subject to position effect variegation when located near a telomere and that this silencing requires the RAD6 and SIR genes. Neither of the two known Rad6 binding factors, Rad18 and Ubr1, is required for telomeric silencing. Since Ubrl is the recognition component of the N-end rule-dependent protein degradation pathway, this suggests that N-end rule-dependent protein degradation is not involved in telomeric silencing. Telomeric silencing requires the amino terminus of Rad6. Two rad6 point mutations, rad6(C88A) and rad6(C88S), which are defective in ubiquitin-conjugating activity fail to complement the silencing defect, indicating that the ubiquitin-conjugating activity of RAD6 is essential for full telomeric silencing.

DNA-Binding Proteins↗
PubMed1997

Does Saccharomyces need an organized nucleolus?

The ribosomal RNA (rRNA) genes of most eukaryotic organisms are arranged in one or more tandem arrays, often termed nucleolar organizer regions. The biological implications of this tandem organization are not known. We have tested the requirement for such a chromosomal organization by directly comparing the transcription and processing of rRNA in isogenic strains of Saccharomyces cerevisiae that differ only in the organization of their rRNA genes. Strain L-1489 carries the RDN locus, consisting of 100-150 copies of the rRNA genes in a tandem array on chromosome XII. Strain L-1521 has a complete deletion of the RDN array, but carries many copies of a plasmid that includes a single rRNA gene. While this strain grows reasonably well, the average transcriptional activity of the plasmid genes is substantially less than that of the chromosomal copies. However, there is little difference in the processing of the 35S pre-rRNA to the mature 25S:5.8S and 18S products. Thus, neither a chromosomal location nor a tandem repeat of the rRNA genes is important for the assembly and function of the many protein and RNA molecules necessary for the processing of the rRNA transcripts. We investigated the consequence of a dispersed gene arrangement on nucleolar structure. Immunofluorescence microscopy revealed that in strain L-1521 the yeast fibrillarin, Nop1p, rather than being confined to a defined nucleolus at the edge of the nucleus as it is in cells with the normal arrangement of rRNA genes, is spread throughout the nucleus. This observation implies that each plasmid rRNA gene can serve as a nucleolar organizer. Finally, data from pulse-labeling experiments show that the repression of rRNA transcription due to failure of the secretory pathway is independent of whether the rRNA genes are at the RDN locus on chromosome XII or on plasmids. This result suggests that the regulation of rRNA transcription occurs at the level of soluble factors rather than chromatin structure.

Cell Nucleolus↗
PubMed1996

The translational function of nucleotide C1054 in the small subunit rRNA is conserved throughout evolution: genetic evidence in yeast.

Mutations at position C1054 of 16S rRNA have previously been shown to cause translational suppression in Escherichia coli. To examine the effects of similar mutations in a eukaryote, all three possible base substitutions and a base deletion were generated at the position of Saccharomyces cerevisiae 18S rRNA corresponding to E. coli C1054. In yeast, as in E. coli, both C1054A (rdn-1A) and C1054G (rdn-1G) caused dominant nonsense suppression. Yeast C1054U (rdn-1T) was a recessive antisuppressor, while yeast C1054-delta (rdn-1delta) led to recessive lethality. Both C1054U and two previously described yeast 18S rRNA antisuppressor mutations, G517A (rdn-2) and U912C (rdn-4), inhibited codon-nonspecific suppression caused by mutations in eukaryotic release factors, sup45 and sup35. However, among these only C1054U inhibited UAA-specific suppressions caused by a UAA-decoding mutant tRNA-Gln (SLT3). Our data implicate eukaryotic C1054 in translational termination, thus suggesting that its function is conserved throughout evolution despite the divergence of nearby nucleotide sequences.

Base Sequence↗
PubMed1996

Genesis and variability of [PSI] prion factors in Saccharomyces cerevisiae.

We have previously shown that multicopy plasmids containing the complete SUP35 gene are able to induce the appearance of the non-Mendelian factor [PSI]. This result was later interpreted by others as a crucial piece of evidence for a model postulating that [PSI] is a self-modified, prion-like conformational derivative of the Sup35 protein. Here we support this interpretation by proving that it is the overproduction of Sup35 protein, and not the excess of SUP35 DNA or mRNA that causes the appearance of [PSI]. We also show that the "prion-inducing domain" of Sup35p is in the N-terminal region, which, like the "prion-inducing domain" of another yeast prion, Ure2p, was previously shown to be distinct from the functional domain of the protein. This suggests that such a chimeric organization may be a common pattern of some prion elements. Finally, we find that [PSI] factors of different efficiencies and different mitotic stabilities are induced in the same yeast strain by overproduction of the identical Sup35 protein. We suggest that the different [PSI]-containing derivatives are analogous to the mysterious mammalian prion strains and result from different conformational variants of Sup35p.

DNA, Fungal↗
PubMed1996

A translational fidelity mutation in the universally conserved sarcin/ricin domain of 25S yeast ribosomal RNA.

Recent evidence suggests that ribosomal RNAs have functional roles in translation. We describe here a new ribosomal RNA mutation that causes translational suppression and antibiotic resistance in eukaryotic cells. Using random mutagenesis of the cloned ribosomal RNA gene and in vivo selection, we isolated a C --> U mutation in the universally conserved sarcin/ricin domain in Saccharomyces cerevisiae 25S ribosomal RNA. This mutation changes the putative CG pair, which closes the GAGA tetraloop in the sarcin/ricin domain, into a weaker UG pair without eliminating ribosomal sensitivity to ricin. We show that suppression of several UGA, UAG, and frameshift mutations is evident when a portion of the cellular ribosomal RNA contains the C --> U mutation. Cells that contain essentially all mutant ribosomal RNA grow only 10% slower than the wild-type, but show increased suppression as well as resistance to paramomycin, G418, and hygromycin, and sensitivity to cycloheximide. Our results provide genetic evidence for the participation of the sarcin/ricin loop in maintaining translational accuracy and are discussed in terms of a hypothesis that this ribosomal RNA region normally undergoes a conformational change during translation.

Anti-Bacterial Agents↗
PubMed1995

Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [psi+].

The yeast non-Mendelian factor [psi+] has been suggested to be a self-modified protein analogous to mammalian prions. Here it is reported that an intermediate amount of the chaperone protein Hsp104 was required for the propagation of the [psi+] factor. Over-production or inactivation of Hsp104 caused the loss of [psi+]. These results suggest that chaperone proteins play a role in prion-like phenomena, and that a certain level of chaperone expression can cure cells of prions without affecting viability. This may lead to antiprion treatments that involve the alteration of chaperone amounts or activity.

Fungal Proteins↗
PubMed1995

Alterations in ribosomal protein RPS28 can diversely affect translational accuracy in Saccharomyces cerevisiae.

Three small-subunit ribosomal proteins shown to influence translational accuracy in Saccharomyces cerevisiae are conserved in structure and function with their procaryotic counterparts. One of these, encoded by RPS28A and RPS28B (RPS28), is comparable to bacterial S12. The others, encoded by sup44 (RPS4) or, sup46 and YS11A (RPS13), are homologues of procaryotic S5 and S4, respectively. In Escherichia coli, certain alterations in S12 cause hyperaccurate translation or antibiotic resistance that can be counteracted by other changes in S5 or S4 that reduce translational accuracy. Using site-directed and random mutagenesis, we show that different changes in RPS28 can have diametrical influences on translational accuracy or antibiotic sensitivity in yeast. Certain substitutions in the amino-terminal portion of the protein, which is diverged from the procaryotic homologues, cause varying levels of nonsense suppression or antibiotic sensitivity. Other alterations, found in the more conserved carboxyl-terminal portion, counteract SUP44- or SUP46-associated antibiotic sensitivity, mimicking E. coli results. Although mutations in these different parts of RPS28 have opposite affects on translational accuracy or antibiotic sensitivity, additive phenotypes can be observed when opposing mutations are combined in the same protein.

Amino Acid Sequence↗
PubMed1994

Mutations in eukaryotic 18S ribosomal RNA affect translational fidelity and resistance to aminoglycoside antibiotics.

Mutations have been created in the Saccharomyces cerevisiae 18S rRNA gene that correspond to those known to be involved in the control of translational fidelity or antibiotic resistance in prokaryotes. Yeast strains, in which essentially all chromosomal rDNA repeats are deleted and all cellular rRNAs are encoded by plasmid, have been constructed that contain only mutant 18S rRNA. In Escherichia coli, a C-->U substitution at position 912 of the small subunit rRNA causes streptomycin resistance. Eukaryotes normally carry U at the corresponding position and are naturally resistant to streptomycin. We show that a U-->C transition (rdn-4) at this position of the yeast 18S rRNA gene decreases resistance to streptomycin. The rdn-4 mutation also increases resistance to paromomycin and G-418, and inhibits nonsense suppression induced by paromomycin. The same phenotypes, as well as a slow growth phenotype, are also associated with rdn-2, whose prokaryotic counterpart, 517 G-->A, manifests itself as a suppressor rather than an antisuppressor. Neither rdn-2- nor rdn-4-related phenotypes could be detected in the presence of the normal level of wild-type rDNA repeats. Our data demonstrate that eukaryotic rRNA is involved in the control of translational fidelity, and indicate that rRNA features important for interactions with aminoglycosides have been conserved throughout evolution.

Base Sequence↗
PubMed1994

The yeast translational allosuppressor, SAL6: a new member of the PP1-like phosphatase family with a long serine-rich N-terminal extension.

The allosuppressor mutation, sal6-1, enhances the efficiency of all tested translational suppressors, including codon-specific tRNA suppressors as well as codon-nonspecific omnipotent suppressors. The SAL6 gene has now been cloned by complementation of the increased suppression efficiency and cold sensitivity caused by sal6-1 in the presence of the omnipotent suppressor sup45. Physical analysis maps SAL6 to chromosome XVI between TPK2 and spt14. The SAL6 gene encodes a very basic 549-amino acid protein whose C-terminal catalytic region of 265 residues is 63% identical to serine/threonine PP1 phosphatases, and 66% identical to yeast PPZ1 and PPZ2 phosphatases. The unusual 235 residue N-terminal extension found in SAL6, like those in the PPZ proteins, is serine-rich. The sal6-1 mutation is a frameshift at amino acid position 271 which destroys the presumed phosphatase catalytic domain of the protein. Disruptions of the entire SAL6 gene are viable, cause a slight growth defect on glycerol medium, and produce allosuppressor phenotypes in suppressor strain backgrounds. The role of the serine-rich N terminus is unclear, since sal6 phenotypes are fully complemented by a SAL6 allele that contains an in-frame deletion of most of this region. High copy number plasmids containing wild-type SAL6 cause antisuppressor phenotypes in suppressor strains. These results suggest that the accuracy of protein synthesis is affected by the levels of phosphorylation of the target(s) of SAL6.

Amino Acid Sequence↗
PubMed1993

An accuracy center in the ribosome conserved over 2 billion years.

The accuracy of translation in Escherichia coli is profoundly influenced by three interacting ribosomal proteins, S12, S4, and S5. Mutations at lysine-42 of S12, originally isolated as causing resistance to streptomycin, increase accuracy. Countervailing "ribosomal ambiguity mutations" (ram) in S4 or S5 decrease accuracy. In the eukaryotic ribosome of Saccharomyces cerevisiae, mutations in SUP46 and SUP44, encoding the proteins equivalent to S4 and S5, lead to omnipotent suppression--i.e., to less accurate translation. The evolution of ribosomal protein S12 can be traced, by comparison with archaebacteria and Tetrahymena, to S28 of S. cerevisiae, even though the two proteins share only very limited regions of homology. However, one region that has been conserved contains a lysine residue whose mutation leads to increased accuracy in E. coli. We have introduced into S28 of yeast the same amino acid substitutions that led to the original streptomycin-resistant mutations in E. coli. We find that they have a profound effect on the accuracy of translation and interact with SUP44 and SUP46, just as predicted from the E. coli model. Thus, the interplay of these three proteins to provide the optimal level of accuracy of translation has been conserved during the 2 billion years of evolution that separate E. coli from S. cerevisiae.

DNA↗
PubMed1993

A ubiquitin-conjugating enzyme, RAD6, affects the distribution of Ty1 retrotransposon integration positions.

A galactose-inducible Ty1 element was used to generate 59 independent Ty1 inserts that inactivate the CAN1 gene. As found in previous studies, the distribution of these elements shows a gradient of insertion frequency from highest to lowest between the 5' and 3' ends of the gene. However, 53 independent Ty1 and Ty2 insertions isolated by an identical procedure in an isogenic rad6 deletion strain do not show this bias. In this strain, the Ty elements insert randomly throughout CAN1. These results show that the ubiquitin-conjugating enzyme, RAD6, alters the integration site preferences of Ty1 retrotransposons.

Alleles↗
PubMed1992

Rearrangements occurring adjacent to a single Ty1 yeast retrotransposon in the presence and absence of full-length Ty1 transcription.

The structures of two unusual deletions from the yeast Saccharomyces cerevisiae are described. These deletions extend from a single Ty1 retrotransposon to an endpoint near a repetitive tRNA(Gly) gene. The deletions suggest that unique sequences flanked by two nonidentical repetitive sequences, or bordered on only one side by a transposable element, have the potential to be mobilized in the yeast genome. Models for the formation of these two unusual deletions were tested by isolating and analyzing 32 additional unusual deletions of the CYC1 region that extend from a single Ty1 retrotransposon. Unlike the most common class of deletions recovered in this region, these deletions are not attributable solely to homologous recombination among repetitive Ty1 or delta elements. They arose by two distinct mechanisms. In an SPT8 genetic background, most unusual deletions arose by transposition of a Ty1 element to a position adjacent to a tRNA(Gly) gene followed by Ty1-Ty1 recombination. In an spt8 strain, where full-length Ty1 transcription and, therefore, transposition are reduced, most deletions were due to gene conversion of a 7-kb chromosomal interval flanked by a Ty1 element and a tRNA(Gly) gene.

Base Sequence↗
PubMed1992

The yeast omnipotent suppressor SUP46 encodes a ribosomal protein which is a functional and structural homolog of the Escherichia coli S4 ram protein.

The accurate synthesis of proteins is crucial to the existence of a cell. In yeast, several genes that affect the fidelity of translation have been identified (e.g., omnipotent suppressors, antisuppressors and allosuppressors). We have found that the dominant omnipotent suppressor SUP46 encodes the yeast ribosomal protein S13. S13 is encoded by two similar genes, but only the sup46 copy of the gene is able to fully complement the recessive phenotypes of SUP46 mutations. Both copies of the S13 genes contain introns. Unlike the introns of other duplicated ribosomal protein genes which are highly diverged, the duplicated S13 genes have two nearly identical DNA sequences of 25 and 31 bp in length within their introns. The SUP46 protein has significant homology to the S4 ribosomal protein in prokaryotic-type ribosomes. S4 is encoded by one of the ram (ribosomal ambiguity) genes in Escherichia coli which are the functional equivalent of omnipotent suppressors in yeast. Thus, SUP46 and S4 demonstrate functional as well as sequence conservation between prokaryotic and eukaryotic ribosomal proteins. SUP46 and S4 are most similar in their central amino acid sequences. Interestingly, the alterations resulting from the SUP46 mutations and the segment of the S4 protein involved in binding to the 16S rRNA are within this most conserved region.

Alleles↗
PubMed1990

Isolation of omnipotent suppressors in an [eta+] yeast strain.

Omnipotent suppressors decrease translational fidelity and cause misreading of nonsense codons. In the presence of the non-Mendelian factor [eta+], some alleles of previously isolated omnipotent suppressors are lethal. Thus the current search was conducted in an [eta+] strain in an effort to identify new suppressor loci. A new omnipotent suppressor, SUP39, and alleles of sup35, sup45, SUP44 and SUP46 were identified. Efficiencies of the dominant suppressors were dramatically reduced in strains that were cured of non-Mendelian factors by growth on guanidine hydrochloride. Wild-type alleles of SUP44 and SUP46 were cloned and these clones were used to facilitate the genetic analyses. SUP44 was shown to be on chromosome VII linked to cyh2, and SUP46 was clearly identified as distinct from the linked sup45.

Alleles↗
PubMed1990

Sequence and functional similarity between a yeast ribosomal protein and the Escherichia coli S5 ram protein.

The accurate and efficient translation of proteins is of fundamental importance to both bacteria and higher organisms. Most of our knowledge about the control of translational fidelity comes from studies of Escherichia coli. In particular, ram (ribosomal ambiguity) mutations in structural genes of E. coli ribosomal proteins S4 and S5 have been shown to increase translational error frequencies. We describe the first sequence of a ribosomal protein gene that affects translational ambiguity in a eucaryote. We show that the yeast omnipotent suppressor SUP44 encodes the yeast ribosomal protein S4. The gene exists as a single copy without an intron. The SUP44 protein is 26% identical (54% similar) to the well-characterized E. coli S5 ram protein. SUP44 is also 59% identical (78% similar) to mouse protein LLrep3, whose function was previously unknown (D.L. Heller, K.M. Gianda, and L. Leinwand, Mol. Cell. Biol. 8:2797-2803, 1988). The SUP44 suppressor mutation occurs near a region of the protein that corresponds to the known positions of alterations in E. coli S5 ram mutations. This is the first ribosomal protein whose function and sequence have been shown to be conserved between procaryotes and eucaryotes.

Amino Acid Sequence↗
PubMed1990

Mutations in RAD6, a yeast gene encoding a ubiquitin-conjugating enzyme, stimulate retrotransposition.

The Saccharomyces cerevisiae DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme which polyubiquitinates histones in vitro. Here we show that mutations in rad6 increase the frequency of transposition of the retrotransposon Ty into the CAN1 and URA3 loci. Using isogenic RAD6 and rad6 strains, we measured a more than 100-fold increase in the spontaneous rate of retrotransposition due to rad6, although there was no increase in the Ty message level. This is the first time that a mutation in a host gene has been shown to result in an increased rate of retrotransposition.

Blotting, Northern↗
PubMed1989

Mutations in ADE3 reduce the efficiency of the omnipotent suppressor sup45-2.

Mutations in a known yeast gene, ADE3, were shown to act as an antisuppressor, reducing the efficiency of the omnipotent suppressor, sup45-2. The ADE3 locus encodes the trifunctional enzyme C1-tetrahydrofolate synthase, which is required for the biosynthesis of purines, thymidylate, methionine, histidine, pantothenic acid and formylmethionyl-tRNA(fMet. The role of this enzyme in translational fidelity had not previously been suspected.

Aminohydrolases↗