Conservative replication of double-stranded RNA in Saccharomyces cerevisiae by displacement of progeny single strands

1984 ◽  
Vol 4 (8) ◽  
pp. 1618-1626
Author(s):  
R A Sclafani ◽  
W L Fangman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
G1 Phase ◽  
Double Stranded Rna ◽  
Virus Like Particles ◽  
Protein Encoding ◽  
In Cells

Saccharomyces cerevisiae contains two double-stranded RNA (dsRNA) molecules, L and M, encapsulated in virus-like particles. After cells are transferred from dense (13C 15N) to light (12C 14N) medium, only two density classes of dsRNA are found, fully light (LL) and fully dense (HH). Cells contain single-stranded copies of both dsRNAs and, at least for L dsRNA, greater than 99% of these single strands are the positive protein-encoding strand. Single-stranded copies of L and M dsRNA accumulate rapidly in cells arrested in the G1 phase. These results parallel previous observations on L dsRNA synthesis and are consistent with a role of the positive single strands as intermediates in dsRNA replication. We propose that new positive strands are displaced from parental molecules and subsequently copied to produce the completely new duplexes.

scholarly journals Conservative replication of double-stranded RNA in Saccharomyces cerevisiae by displacement of progeny single strands.

1984 ◽  
Vol 4 (8) ◽  
pp. 1618-1626 ◽  
Author(s):  
R A Sclafani ◽  
W L Fangman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
G1 Phase ◽  
Double Stranded Rna ◽  
Virus Like Particles ◽  
Protein Encoding ◽  
In Cells

Saccharomyces cerevisiae contains two double-stranded RNA (dsRNA) molecules, L and M, encapsulated in virus-like particles. After cells are transferred from dense (13C 15N) to light (12C 14N) medium, only two density classes of dsRNA are found, fully light (LL) and fully dense (HH). Cells contain single-stranded copies of both dsRNAs and, at least for L dsRNA, greater than 99% of these single strands are the positive protein-encoding strand. Single-stranded copies of L and M dsRNA accumulate rapidly in cells arrested in the G1 phase. These results parallel previous observations on L dsRNA synthesis and are consistent with a role of the positive single strands as intermediates in dsRNA replication. We propose that new positive strands are displaced from parental molecules and subsequently copied to produce the completely new duplexes.

scholarly journals Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (12) ◽  
pp. 6229-6247 ◽  
Author(s):  
S M Miller ◽  
B Magasanik
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glutamine Synthetase ◽  
Nitrogen Source ◽  
Glutamate Dehydrogenase ◽  
Regulatory System ◽  
Sole Source ◽  
Lacz Fusion ◽  
Upstream Region ◽  
In Cells

We analyzed the upstream region of the GDH2 gene, which encodes the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae, for elements important for the regulation of the gene by the nitrogen source. The levels of this enzyme are high in cells grown with glutamate as the sole source of nitrogen and low in cells grown with glutamine or ammonium. We found that this regulation occurs at the level of transcription and that a total of six sites are required to cause a CYC1-lacZ fusion to the GDH2 gene to be regulated in the same manner as the NAD-linked glutamate dehydrogenase. Two sites behaved as upstream activation sites (UASs). The remaining four sites were found to block the effects of the two UASs in such a way that the GDH2-CYC1-lacZ fusion was not expressed unless the cells containing it were grown under conditions favorable for the activity of both UASs. This complex regulatory system appears to account for the fact that GDH2 expression is exquisitely sensitive to glutamine, whereas the expression of GLN1, coding for glutamine synthetase, is not nearly as sensitive.

scholarly journals The Saccharomyces cerevisiae DNA Recombination and Repair Functions of the RAD52 Epistasis Group Inhibit Ty1 Transposition

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 543-556 ◽  
Author(s):  
Alison J Rattray ◽  
Brenda K Shafer ◽  
David J Garfinkel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Dna Recombination ◽  
Recombinational Repair ◽  
Repair Pathway ◽  
Low Frequencies ◽  
Double Stranded Dna ◽  
High Level ◽  
In Cells

Abstract RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or δ) elements. Here we examine the role of the cellular homologous recombination functions on Ty1 transposition. We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes. The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins. However, unincorporated Ty1 cDNA levels are markedly elevated. These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA.

scholarly journals Growth-Independent Regulation of CLN3mRNA Levels by Nutrients in Saccharomyces cerevisiae

1998 ◽  
Vol 180 (2) ◽  
pp. 225-230 ◽  
Author(s):  
Fereshteh Parviz ◽  
Warren Heideman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Control ◽  
Protein A ◽  
Nitrogen Sources ◽  
Mrna Levels ◽  
Rich Medium ◽  
Phase Growth ◽  
Protein Levels ◽  
In Cells

ABSTRACT Saccharomyces cerevisiae cells regulate progress through the G1 phase of the cell cycle in response to nutrients, moving quickly through G1 in rich medium and slowly in poor medium. Recent work has shown that the levels of Cln3 protein, a G1 cyclin, are low in cells growing in poor medium and high in cells growing rapidly in rich medium, consistent with the previously recognized role of Cln3 in promoting passage through Start. Cln3 protein levels appear to be regulated both transcriptionally and posttranscriptionally. We have worked to define the nutrient signals that regulate CLN3 mRNA levels. We find that CLN3 mRNA levels are high during log-phase growth in glucose medium, low in postdiauxic cells growing on ethanol, and slightly lower still in cells in stationary phase. CLN3mRNA levels are induced by glucose in a process that involves transcriptional control, requires metabolism of the glucose, and is independent of the Ras-cyclic AMP pathway. CLN3 mRNA levels are also positively regulated by nitrogen sources, but phosphorus and sulfur limitation do not affect CLN3 message levels.

scholarly journals Role of Nonstructural Protein NS2A in Flavivirus Assembly

2008 ◽  
Vol 82 (10) ◽  
pp. 4731-4741 ◽  
Author(s):  
Jason Y. Leung ◽  
Gorben P. Pijlman ◽  
Natasha Kondratieva ◽  
Jennifer Hyde ◽  
Jason M. Mackenzie ◽  
...  
Keyword(s):  
Virus Assembly ◽  
Nonstructural Protein ◽  
Compensatory Mutation ◽  
Wild Type ◽  
Packaging Cell Line ◽  
Prolonged Incubation ◽  
Hydrophobic Residues ◽  
Virus Like Particles ◽  
In Cells

ABSTRACT Flavivirus nonstructural (NS) proteins are involved in RNA replication and modulation of the host antiviral response; however, evidence is mounting that some NS proteins also have essential roles in virus assembly. Kunjin virus (KUN) NS2A is a small, hydrophobic, transmembrane protein that is part of the replication complex and inhibits interferon induction. Previously, we have shown that an isoleucine (I)-to-asparagine (N) substitution at position 59 of the NS2A protein blocked the production of secreted virus particles in cells electroporated with viral RNA carrying this mutation. We now show that prolonged incubation of mutant KUN NS2A-I59N replicon RNA, in an inducible BHK-derived packaging cell line (expressing KUN structural proteins C, prM, and E), generated escape mutants that rescued the secretion of infectious virus-like particles. Sequencing identified three groups of revertants that included (i) reversions to wild-type, hydrophobic Ile, (ii) pseudorevertants to more hydrophobic residues (Ser, Thr, and Tyr) at codon 59, and (iii) pseudorevertants retaining Asn at NS2A codon 59 but containing a compensatory mutation (Thr-to-Pro) at NS2A codon 149. Engineering hydrophobic residues at NS2A position 59 or the compensatory T149P mutation into NS2A-I59N replicon RNA restored the assembly of secreted virus-like particles in packaging cells. T149P mutation also rescued virus production when introduced into the full-length KUN RNA containing an NS2A-I59N mutation. Immunofluorescence and electron microscopy analyses of NS2A-I59N replicon-expressing cells showed a distinct lack of virus-induced membranes normally present in cells expressing wild-type replicon RNA. The compensatory mutation NS2A-T149P restored the induction of membrane structures to a level similar to those observed during wild-type replication. The results further confirm the role of NS2A in virus assembly, demonstrate the importance of hydrophobic residues at codon 59 in this process, implicate the involvement of NS2A in the biogenesis of virus-induced membranes, and suggest a vital role for the virus-induced membranes in virus assembly.

scholarly journals Replication of double-stranded RNA of the virus-like particles in Saccharomyces cerevisiae.

1981 ◽  
Vol 38 (1) ◽  
pp. 263-271 ◽  
Author(s):  
A M Newman ◽  
S G Elliott ◽  
C S McLaughlin ◽  
P A Sutherland ◽  
R C Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Double Stranded Rna ◽  
Virus Like Particles

Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (12) ◽  
pp. 6229-6247
Author(s):  
S M Miller ◽  
B Magasanik
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glutamine Synthetase ◽  
Nitrogen Source ◽  
Glutamate Dehydrogenase ◽  
Regulatory System ◽  
Sole Source ◽  
Lacz Fusion ◽  
Upstream Region ◽  
In Cells

We analyzed the upstream region of the GDH2 gene, which encodes the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae, for elements important for the regulation of the gene by the nitrogen source. The levels of this enzyme are high in cells grown with glutamate as the sole source of nitrogen and low in cells grown with glutamine or ammonium. We found that this regulation occurs at the level of transcription and that a total of six sites are required to cause a CYC1-lacZ fusion to the GDH2 gene to be regulated in the same manner as the NAD-linked glutamate dehydrogenase. Two sites behaved as upstream activation sites (UASs). The remaining four sites were found to block the effects of the two UASs in such a way that the GDH2-CYC1-lacZ fusion was not expressed unless the cells containing it were grown under conditions favorable for the activity of both UASs. This complex regulatory system appears to account for the fact that GDH2 expression is exquisitely sensitive to glutamine, whereas the expression of GLN1, coding for glutamine synthetase, is not nearly as sensitive.

scholarly journals The role of the lissencephaly protein Pac1 during nuclear migration in budding yeast

2003 ◽  
Vol 160 (3) ◽  
pp. 355-364 ◽  
Author(s):  
Wei-Lih Lee ◽  
Jessica R. Oberle ◽  
John A. Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heavy Chain ◽  
Mitotic Spindle ◽  
Genetic Interactions ◽  
Wild Type ◽  
Dynein Heavy Chain ◽  
Bud Neck ◽  
Cytoplasmic Microtubules ◽  
In Cells

During mitosis in Saccharomyces cerevisiae, the mitotic spindle moves into the mother–bud neck via dynein-dependent sliding of cytoplasmic microtubules along the cortex of the bud. Here we show that Pac1, the yeast homologue of the human lissencephaly protein LIS1, plays a key role in this process. First, genetic interactions placed Pac1 in the dynein/dynactin pathway. Second, cells lacking Pac1 failed to display microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation. Third, Pac1 localized to the plus ends (distal tips) of cytoplasmic microtubules in the bud. This localization did not depend on the dynein heavy chain Dyn1. Moreover, the Pac1 fluorescence intensity at the microtubule end was enhanced in cells lacking dynactin or the cortical attachment molecule Num1. Fourth, dynein heavy chain Dyn1 also localized to the tips of cytoplasmic microtubules in wild-type cells. Dynein localization required Pac1 and, like Pac1, was enhanced in cells lacking the dynactin component Arp1 or the cortical attachment molecule Num1. Our results suggest that Pac1 targets dynein to microtubule tips, which is necessary for sliding of microtubules along the bud cortex. Dynein must remain inactive until microtubule ends interact with the bud cortex, at which time dynein and Pac1 appear to be offloaded from the microtubule to the cortex.

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