Cloning and characterization of LOS1, a Saccharomyces cerevisiae gene that affects tRNA splicing

1987 ◽  
Vol 7 (3) ◽  
pp. 1208-1216 ◽  
Author(s):  
D J Hurt ◽  
S S Wang ◽  
Y H Lin ◽  
A K Hopper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Elevated Temperatures ◽  
Trna Gene ◽  
Single Copy ◽  
Molecular Study ◽  
Trna Splicing ◽  
Intervening Sequences ◽  
Copy Gene

Saccharomyces cerevisiae strains carrying los1-1 mutations are defective in tRNA processing; at 37 degrees C, such strains accumulate tRNA precursors which have mature 5' and 3' ends but contain intervening sequences. Strains bearing los1-1 and an intron-containing ochre-suppressing tRNA gene, SUP4(0), also fail to suppress the ochre mutations ade2-1(0) and can1-100(0) at 34 degrees C. To understand the role of the LOS1 product in tRNA splicing, we initiated a molecular study of the LOS1 gene. Two plasmids, YEpLOS1 and YCpLOS1, that complement the los1-1 phenotype were isolated from the YEp24 and YCp50 libraries, respectively. YEpLOS1 and YCpLOS1 had overlapping restriction maps, indicating that the DNA in the overlapping segment could complement los1-1 when present in multiple or single copy. Integration of plasmid DNA at the LOS1 locus confirmed that these clones contained authentic LOS1 sequences. Southern analyses showed that LOS1 is a single copy gene. The locations of the LOS1 gene within YEpLOS1 and YCpLOS1 were determined by deletion and gamma-delta mapping. Two genomic disruptions of the LOS1 gene were constructed, i.e., an insertion of a 1.2-kilobase fragment carrying the yeast URA3 gene, los1::URA3, and a 2.4-kilobase deletion from the LOS1 gene, los1-delta V. Disruption or deletion of most of the LOS1 gene was not lethal; cells carrying the disrupted los1 alleles were viable and had phenotypes similar to those of cells carrying the los1-1 allele. Thus, it appears that the los1 gene product expedites tRNA splicing at elevated temperatures but is not essential for this process.

scholarly journals Cloning and characterization of LOS1, a Saccharomyces cerevisiae gene that affects tRNA splicing.

1987 ◽  
Vol 7 (3) ◽  
pp. 1208-1216 ◽  
Author(s):  
D J Hurt ◽  
S S Wang ◽  
Y H Lin ◽  
A K Hopper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Elevated Temperatures ◽  
Trna Gene ◽  
Single Copy ◽  
Molecular Study ◽  
Trna Splicing ◽  
Intervening Sequences ◽  
Copy Gene

Saccharomyces cerevisiae strains carrying los1-1 mutations are defective in tRNA processing; at 37 degrees C, such strains accumulate tRNA precursors which have mature 5' and 3' ends but contain intervening sequences. Strains bearing los1-1 and an intron-containing ochre-suppressing tRNA gene, SUP4(0), also fail to suppress the ochre mutations ade2-1(0) and can1-100(0) at 34 degrees C. To understand the role of the LOS1 product in tRNA splicing, we initiated a molecular study of the LOS1 gene. Two plasmids, YEpLOS1 and YCpLOS1, that complement the los1-1 phenotype were isolated from the YEp24 and YCp50 libraries, respectively. YEpLOS1 and YCpLOS1 had overlapping restriction maps, indicating that the DNA in the overlapping segment could complement los1-1 when present in multiple or single copy. Integration of plasmid DNA at the LOS1 locus confirmed that these clones contained authentic LOS1 sequences. Southern analyses showed that LOS1 is a single copy gene. The locations of the LOS1 gene within YEpLOS1 and YCpLOS1 were determined by deletion and gamma-delta mapping. Two genomic disruptions of the LOS1 gene were constructed, i.e., an insertion of a 1.2-kilobase fragment carrying the yeast URA3 gene, los1::URA3, and a 2.4-kilobase deletion from the LOS1 gene, los1-delta V. Disruption or deletion of most of the LOS1 gene was not lethal; cells carrying the disrupted los1 alleles were viable and had phenotypes similar to those of cells carrying the los1-1 allele. Thus, it appears that the los1 gene product expedites tRNA splicing at elevated temperatures but is not essential for this process.

scholarly journals Isolation and characterization of PRT1, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (12) ◽  
pp. 4419-4424 ◽  
Author(s):  
C Keierleber ◽  
M Wittekind ◽  
S L Qin ◽  
C S McLaughlin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Southern Blot Analysis ◽  
Protein Biosynthesis ◽  
Single Copy ◽  
Temperature Sensitive ◽  
Single Copy Gene ◽  
Isolation And Characterization ◽  
Copy Gene ◽  
Dna Fragment

We isolated a cloned DNA fragment containing PRT1, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevisiae, by complementation of the temperature-sensitive prtl-1 mutation. The entire PRT1 gene is contained within a 3.2-kilobase-pair segment of the cloned DNA in YEp13 H1.2. Southern blot analysis demonstrated that PRT1 is a single copy gene which is transcribed into a 2.3-kilobase RNA. We determined the direction of transcription and mapped the 5' and 3' ends of the gene.

Isolation and characterization of PRT1, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (12) ◽  
pp. 4419-4424
Author(s):  
C Keierleber ◽  
M Wittekind ◽  
S L Qin ◽  
C S McLaughlin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Southern Blot Analysis ◽  
Protein Biosynthesis ◽  
Single Copy ◽  
Temperature Sensitive ◽  
Single Copy Gene ◽  
Isolation And Characterization ◽  
Copy Gene ◽  
Dna Fragment

We isolated a cloned DNA fragment containing PRT1, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevisiae, by complementation of the temperature-sensitive prtl-1 mutation. The entire PRT1 gene is contained within a 3.2-kilobase-pair segment of the cloned DNA in YEp13 H1.2. Southern blot analysis demonstrated that PRT1 is a single copy gene which is transcribed into a 2.3-kilobase RNA. We determined the direction of transcription and mapped the 5' and 3' ends of the gene.

scholarly journals Characterization of the Major Antigenic Protein 2 of Ehrlichia canis and Ehrlichia chaffeensis and Its Application for Serodiagnosis of Ehrlichiosis

2003 ◽  
Vol 10 (4) ◽  
pp. 520-524 ◽  
Author(s):  
Tamece T. Knowles ◽  
A. Rick Alleman ◽  
Heather L. Sorenson ◽  
David C. Marciano ◽  
Edward B. Breitschwerdt ◽  
...  
Keyword(s):  
Blot Analysis ◽  
Single Copy ◽  
Ehrlichia Canis ◽  
Specific Method ◽  
Ehrlichia Chaffeensis ◽  
Antigenic Protein ◽  
Canine Monocytic Ehrlichiosis ◽  
Copy Gene ◽  
Species Specific

ABSTRACT Canine monocytic ehrlichiosis, caused by Ehrlichia canis or Ehrlichia chaffeensis, can result in clinical disease in naturally infected animals. Coinfections with these agents may be common in certain areas of endemicity. Currently, a species-specific method for serological diagnosis of monocytic ehrlichiosis is not available. Previously, we developed two indirect enzyme-linked immunosorbent assays (ELISAs) using the major antigenic protein 2 (MAP2) of E. chaffeensis and E. canis. In this study, we further characterized the conservation of MAP2 among various geographic isolates of each organism and determined if the recombinant MAP2 (rMAP2) of E. chaffeensis would cross-react with E. canis-infected dog sera. Genomic Southern blot analysis using digoxigenin-labeled species-specific probes suggested that map2 is a single-copy gene in both Ehrlichia species. Sequences of the single map2 genes of seven geographically different isolates of E. chaffeensis and five isolates of E. canis are highly conserved among the various isolates of each respective ehrlichial species. ELISA and Western blot analysis confirmed that the E. chaffeensis rMAP2 failed to serologically differentiate between E. canis and E. chaffeensis infections.

A synthetic intron in a naturally intronless yeast pre-tRNA is spliced efficiently in vivo

1989 ◽  
Vol 9 (1) ◽  
pp. 329-331
Author(s):  
M Winey ◽  
I Edelman ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Analysis ◽  
Single Copy ◽  
Single Copy Gene ◽  
Copy Gene

Saccharomyces cerevisiae glutamine tRNA(CAG) is encoded by an intronless, single-copy gene, SUP60. We have imposed a requirement for splicing in the biosynthesis of this tRNA by inserting a synthetic intron in the SUP60 gene. Genetic analysis demonstrated that the interrupted gene produces a functional, mature tRNA product in vivo.

Role of nuclear genes in expression of a mitochondrial tRNA gene in Saccharomyces cerevisiae

1983 ◽  
Vol 3 (3) ◽  
pp. 371-379
Author(s):  
M Wesolowski ◽  
C Palleschi ◽  
L Frontali ◽  
H Fukuhara
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trna Gene ◽  
Mitochondrial Gene ◽  
Nuclear Genes ◽  
Yeast Mitochondria ◽  
Mitochondrial Trna ◽  
Genetic Crosses ◽  
Mitochondrial Trna Gene

In yeast mitochondria, most of the isoaccepting species of tyrosyl tRNA are coded by a mitochondrial gene, tyrA. A particular isoaccepting species is coded by a second mitochondrial gene, tyrB. This gene is not expressed in certain strains of yeast which show no deficient phenotype. Genetic crosses between strains expressing or not expressing the tyrB gene demonstrate that expression is controlled by specific nuclear genes and that a mutation of the tyrA gene can be bypassed when the tyrB gene is operative.

scholarly journals Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P.

1991 ◽  
Vol 11 (2) ◽  
pp. 721-730 ◽  
Author(s):  
J Y Lee ◽  
C E Rohlman ◽  
L A Molony ◽  
D R Engelke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Essential Gene ◽  
Rnase P ◽  
Single Copy ◽  
Temperature Sensitive ◽  
Coding Region ◽  
Gene Encoding ◽  
Processing Steps ◽  
Potential Precursor

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.

scholarly journals Systematic multi-level analysis of an organelle proteome reveals new peroxisomal functions

2021 ◽  
Author(s):  
Eden Yifrach ◽  
Duncan Holbrook-Smith ◽  
Jérôme Bürgi ◽  
Alaa Othman ◽  
Miriam Eisenstein ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Targeting ◽  
Cellular Metabolism ◽  
Metabolomic Analysis ◽  
Health And Disease ◽  
Multi Level ◽  
Crucial Part ◽  
Level Analysis

AbstractSeventy years following the discovery of peroxisomes, their proteome remains undefined. Uncovering the complete peroxisomal proteome, the peroxi-ome, is crucial for understanding peroxisomal activities and cellular metabolism. We used high- content microscopy to uncover the peroxi-ome of the model eukaryote – Saccharomyces cerevisiae. This strategy enabled us to expand the known organellar proteome by ∼40% and paved the way for performing systematic, whole-organellar proteome assays. Coupled with targeted experiments this allowed us to discover new peroxisomal functions. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly-identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.

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