scholarly journals Generation of temperature-sensitive cbp1 strains of Saccharomyces cerevisiae by PCR mutagenesis and in vivo recombination: characteristics of the mutant strains imply that CBP1 is involved in stabilization and processing of cytochrome b pre-mRNA.

Genetics ◽  
1993 ◽  
Vol 135 (4) ◽  
pp. 981-991 ◽  
Author(s):  
R R Staples ◽  
C L Dieckmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome B ◽  
Mitochondrial Gene ◽  
Nuclear Gene ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
In Vivo Recombination ◽  
Mutant Strains ◽  
Precursor Rna

Abstract Mitochondrial biogenesis is dependent on both nuclearly and mitochondrially encoded proteins. Study of the nuclearly encoded mitochondrial gene products and their effect on mitochondrial genome expression is essential to understanding mitochondrial function. Mutations in the nuclear gene CBP1 of Saccharomyces cerevisiae result in degradation of mitochondrially encoded cytochrome b (cob) RNA; thus, the cells are unable to respire. Putative roles for the CBP1 protein include processing of precursor RNA to yield the mature 5' end of cob mRNA and/or physical protection of the mRNA from degradation by nucleases. To examine the activity of CBP1, we generated temperature-sensitive cbp1 mutant strains by polymerase chain reaction (PCR) mutagenesis and in vivo recombination. These temperature-sensitive cbp1 strains lack cob mRNA only at the nonpermissive temperature. Quantitative primer extension analyses of RNA from these strains and from a cbp1 deletion strain demonstrated that CBP1 is required for the stability of precursor RNAs in addition to production of the stable mature mRNA. Thus, CBP1 is not involved solely in the protection of mature cob mRNA from nucleases. Moreover, we found that mature mRNAs are undetectable while precursor RNAs are reduced only slightly at the nonpermissive temperature. Collectively, these data lead us to favor a hypothesis whereby CBP1 protects cob precursor RNAs and promotes the processing event that generates the mature 5' end of the mRNA.

scholarly journals Further evidence that the rna2 mutation of Saccharomyces cerevisiae affects mRNA processing.

1982 ◽  
Vol 2 (10) ◽  
pp. 1205-1211 ◽  
Author(s):  
S Bromley ◽  
L Hereford ◽  
M Rosbash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Synthesis ◽  
Relative Rate ◽  
Mrna Processing ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Mature Mrna ◽  
Precursor Rna

The relative rate at which ribosomal protein 51 (rp51) mRNA is synthesized was measured by pulse-labeling cells in vivo with [3H]adenine. Two strains of Saccharomyces cerevisiae were compared: A364A (wild type) and ts368 (rna2), a temperature-sensitive strain in which the level of rp51 mRNA decreases and an intron-containing rp51 precursor RNA increases. When cells were shifted up to the nonpermissive temperature (36 degrees C), the rate of rp51 RNA synthesis was only marginally affected (75% of wild type) by the presence of the rna2 mutation. The precursor RNA was the predominant transcription product at 36 degrees C. This precursor could be converted into RNA equal in size to mature mRNA by further incubation at either 36 or 23 degrees C in the presence of unlabeled adenine. The relative half-life of the rp51 transcripts at 36 degrees C also decreased approximately twofold in ts368 as compared with A364A. All of these data imply that the precursor (intron-containing) RNA is processed inefficiently to mature mRNA and that the rp51 precursor RNA is continuously synthesized and degraded in the mutant strain at 36 degrees C.

Further evidence that the rna2 mutation of Saccharomyces cerevisiae affects mRNA processing

1982 ◽  
Vol 2 (10) ◽  
pp. 1205-1211
Author(s):  
S Bromley ◽  
L Hereford ◽  
M Rosbash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Synthesis ◽  
Relative Rate ◽  
Mrna Processing ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Mature Mrna ◽  
Precursor Rna

The relative rate at which ribosomal protein 51 (rp51) mRNA is synthesized was measured by pulse-labeling cells in vivo with [3H]adenine. Two strains of Saccharomyces cerevisiae were compared: A364A (wild type) and ts368 (rna2), a temperature-sensitive strain in which the level of rp51 mRNA decreases and an intron-containing rp51 precursor RNA increases. When cells were shifted up to the nonpermissive temperature (36 degrees C), the rate of rp51 RNA synthesis was only marginally affected (75% of wild type) by the presence of the rna2 mutation. The precursor RNA was the predominant transcription product at 36 degrees C. This precursor could be converted into RNA equal in size to mature mRNA by further incubation at either 36 or 23 degrees C in the presence of unlabeled adenine. The relative half-life of the rp51 transcripts at 36 degrees C also decreased approximately twofold in ts368 as compared with A364A. All of these data imply that the precursor (intron-containing) RNA is processed inefficiently to mature mRNA and that the rp51 precursor RNA is continuously synthesized and degraded in the mutant strain at 36 degrees C.

In vivo analysis of sequences necessary for CBP1-dependent accumulation of cytochrome b transcripts in yeast mitochondria

1993 ◽  
Vol 13 (7) ◽  
pp. 4203-4213
Author(s):  
T M Mittelmeier ◽  
C L Dieckmann
Keyword(s):  
Cytochrome B ◽  
Mitochondrial Gene ◽  
Microprojectile Bombardment ◽  
Nuclear Gene ◽  
State Level ◽  
Transcription Unit ◽  
Endonucleolytic Cleavage ◽  
In Vivo Analysis ◽  
Nucleotide Region

In Saccharomyces cerevisiae, cytochrome b, an essential component of the respiratory chain, is encoded by the mitochondrial gene cob. The cob transcription unit includes the tRNA(Glu) gene from positions -1170 to -1099 relative to the cob ATG at +1. The initial tRNA(Glu)-cob transcript undergoes several processing events, including removal of tRNA(Glu) and production of the mature 5' end of cob mRNA at nucleotide -954. The nuclear gene product CBP1 is specifically required for the accumulation of cob mRNA. In cbp1 mutant strains, cob transcripts are not detectable by Northern (RNA) blot analysis, but the steady-state level of tRNA(Glu) is similar to that of wild type. The results of a previous study led to the conclusion that a 400-nucleotide region just downstream of tRNA(Glu) is sufficient for CBP1 function. In the present study, the microprojectile bombardment method of mitochondrial transformation was used to introduce deletions within this region of cob. The analysis of cob transcripts in strains carrying the mitochondrial deletion genomes indicates that a 63-nucleotide sequence that encompasses the cleavage site at -954 is sufficient both for CBP1 function and for correct positioning of the cleavage. Furthermore, the data indicate that CBP1 prevents the degradation of unprocessed cob transcripts produced by endonucleolytic cleavage at the 3' end of tRNA(Glu).

In vivo construction of linear vectors based on killer plasmids from Kluyveromyces lactis: selection of a nuclear gene results in attachment of telomeres

1989 ◽  
Vol 9 (9) ◽  
pp. 3931-3937
Author(s):  
J Kämper ◽  
F Meinhardt ◽  
N Gunge ◽  
K Esser
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kluyveromyces Lactis ◽  
Nuclear Gene ◽  
Leu2 Gene ◽  
In Vivo Recombination ◽  
Hybrid Vectors ◽  
Hybrid Molecules ◽  
Specific Sequences ◽  
Selection Of

Linear vectors based on plasmids pGKL1 and pGKL2 from Kluyveromyces lactis were obtained by in vivo recombination in Saccharomyces cerevisiae and selected for integration of the nuclear LEU2 gene. The linear hybrid molecules obtained had no proteins attached to their 5' ends, as is found for native pGKL plasmids. However, telomere-specific sequences were added to the ends of pGKL1. In contrast to the cytoplasmically localized pGKL plasmids, the newly obtained linear hybrid vectors probably replicate within the nucleus and provide evidence that the nuclear LEU2 gene cannot be expressed in the cytoplasm.

scholarly journals In vivo analysis of sequences necessary for CBP1-dependent accumulation of cytochrome b transcripts in yeast mitochondria.

1993 ◽  
Vol 13 (7) ◽  
pp. 4203-4213 ◽  
Author(s):  
T M Mittelmeier ◽  
C L Dieckmann
Keyword(s):  
Cytochrome B ◽  
Mitochondrial Gene ◽  
Microprojectile Bombardment ◽  
Nuclear Gene ◽  
State Level ◽  
Transcription Unit ◽  
Endonucleolytic Cleavage ◽  
In Vivo Analysis ◽  
Nucleotide Region

In Saccharomyces cerevisiae, cytochrome b, an essential component of the respiratory chain, is encoded by the mitochondrial gene cob. The cob transcription unit includes the tRNA(Glu) gene from positions -1170 to -1099 relative to the cob ATG at +1. The initial tRNA(Glu)-cob transcript undergoes several processing events, including removal of tRNA(Glu) and production of the mature 5' end of cob mRNA at nucleotide -954. The nuclear gene product CBP1 is specifically required for the accumulation of cob mRNA. In cbp1 mutant strains, cob transcripts are not detectable by Northern (RNA) blot analysis, but the steady-state level of tRNA(Glu) is similar to that of wild type. The results of a previous study led to the conclusion that a 400-nucleotide region just downstream of tRNA(Glu) is sufficient for CBP1 function. In the present study, the microprojectile bombardment method of mitochondrial transformation was used to introduce deletions within this region of cob. The analysis of cob transcripts in strains carrying the mitochondrial deletion genomes indicates that a 63-nucleotide sequence that encompasses the cleavage site at -954 is sufficient both for CBP1 function and for correct positioning of the cleavage. Furthermore, the data indicate that CBP1 prevents the degradation of unprocessed cob transcripts produced by endonucleolytic cleavage at the 3' end of tRNA(Glu).

scholarly journals Cak1 Is Required for Kin28 Phosphorylation and Activation In Vivo

1998 ◽  
Vol 18 (11) ◽  
pp. 6365-6373 ◽  
Author(s):  
F. Hernán Espinoza ◽  
Alison Farrell ◽  
Jamison L. Nourse ◽  
Holly M. Chamberlin ◽  
Opher Gileadi ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Insect Cells ◽  
Cell Cycle Control ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Loss Of Function ◽  
Regulatory Subunits ◽  
Mutant Strains ◽  
Dependent Protein

ABSTRACT Complete activation of most cyclin-dependent protein kinases (CDKs) requires phosphorylation by the CDK-activating kinase (CAK). In the budding yeast, Saccharomyces cerevisiae, the major CAK is a 44-kDa protein kinase known as Cak1. Cak1 is required for the phosphorylation and activation of Cdc28, a major CDK involved in cell cycle control. We addressed the possibility that Cak1 is also required for the activation of other yeast CDKs, such as Kin28, Pho85, and Srb10. We generated three new temperature-sensitive cak1mutant strains, which arrested at the restrictive temperature with nonuniform budding morphology. All three cak1 mutants displayed significant synthetic interactions with loss-of-function mutations in CDC28 and KIN28. Loss of Cak1 function reduced the phosphorylation and activity of both Cdc28 and Kin28 but did not affect the activity of Pho85 or Srb10. In the presence of the Kin28 regulatory subunits Ccl1 and Tfb3, Kin28 was phosphorylated and activated when coexpressed with Cak1 in insect cells. We conclude that Cak1 is required for the activating phosphorylation of Kin28 as well as that of Cdc28.

scholarly journals ATP25, a New Nuclear Gene ofSaccharomyces cerevisiaeRequired for Expression and Assembly of the Atp9p Subunit of Mitochondrial ATPase

2008 ◽  
Vol 19 (4) ◽  
pp. 1366-1377 ◽  
Author(s):  
Xiaomei Zeng ◽  
Mario H. Barros ◽  
Theodore Shulman ◽  
Alexander Tzagoloff
Keyword(s):  
Mitochondrial Gene ◽  
Nuclear Gene ◽  
Ring Structure ◽  
Northern Analysis ◽  
Temperature Sensitive ◽  
Mitochondrial Atpase ◽  
Translation Product ◽  
Reading Frame ◽  
Inner Membrane Protein

We report a new nuclear gene, designated ATP25 (reading frame YMR098C on chromosome XIII), required for expression of Atp9p (subunit 9) of the Saccharomyces cerevisiae mitochondrial proton translocating ATPase. Mutations in ATP25 elicit a deficit of ATP9 mRNA and of its translation product, thereby preventing assembly of functional F0. Unlike Atp9p, the other mitochondrial gene products, including ATPase subunits Atp6p and Atp8p, are synthesized normally in atp25 mutants. Northern analysis of mitochondrial RNAs in an atp25 temperature-sensitive mutant confirmed that Atp25p is required for stability of the ATP9 mRNA. Atp25p is a mitochondrial inner membrane protein with a predicted mass of 70 kDa. The primary translation product of ATP25 is cleaved in vivo after residue 292 to yield a 35-kDa C-terminal polypeptide. The C-terminal half of Atp25p is sufficient to stabilize the ATP9 mRNA and restore synthesis of Atp9p. Growth on respiratory substrates, however, depends on both halves of Atp25p, indicating that the N-terminal half has another function, which we propose to be oligomerization of Atp9p into a proper size ring structure.

scholarly journals In vivo construction of linear vectors based on killer plasmids from Kluyveromyces lactis: selection of a nuclear gene results in attachment of telomeres.

1989 ◽  
Vol 9 (9) ◽  
pp. 3931-3937 ◽  
Author(s):  
J Kämper ◽  
F Meinhardt ◽  
N Gunge ◽  
K Esser
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kluyveromyces Lactis ◽  
Nuclear Gene ◽  
Leu2 Gene ◽  
In Vivo Recombination ◽  
Hybrid Vectors ◽  
Hybrid Molecules ◽  
Specific Sequences ◽  
Selection Of

Linear vectors based on plasmids pGKL1 and pGKL2 from Kluyveromyces lactis were obtained by in vivo recombination in Saccharomyces cerevisiae and selected for integration of the nuclear LEU2 gene. The linear hybrid molecules obtained had no proteins attached to their 5' ends, as is found for native pGKL plasmids. However, telomere-specific sequences were added to the ends of pGKL1. In contrast to the cytoplasmically localized pGKL plasmids, the newly obtained linear hybrid vectors probably replicate within the nucleus and provide evidence that the nuclear LEU2 gene cannot be expressed in the cytoplasm.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases.

1991 ◽  
Vol 11 (7) ◽  
pp. 3463-3471 ◽  
Author(s):  
S R Schmid ◽  
P Linder
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Clear Correlation ◽  
Temperature Sensitive ◽  
Rna Helicases ◽  
Initiation Of Translation

The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products.

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