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 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 the RNA2, RNA3, and RNA11 genes of Saccharomyces cerevisiae

1984 ◽  
Vol 4 (11) ◽  
pp. 2396-2405
Author(s):  
R L Last ◽  
J B Stavenhagen ◽  
J L Woolford
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Single Copy ◽  
Yeast Genome ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Mrna Accumulation ◽  
Isolation And Characterization ◽  
Polyadenylated Rna

Temperature-sensitive mutations in the genes RNA2 through RNA11 cause accumulation of intervening sequence containing precursor mRNAs in Saccharomyces cerevisiae. Three different plasmids have been isolated which complement both the temperature-sensitive lethality and precursor mRNA accumulation when introduced into rna2, rna3, and rna11 mutant strains. The yeast sequences on these plasmids have been shown by Southern transfer hybridization and genetic mapping to be derived from the RNA2, RNA3, and RNA11 genomic loci. Part of the RNA2 gene is homologous to more than one region of the yeast genome, whereas the RNA3 and RNA11 genes are single copy. RNAs homologous to these loci have been identified by RNA transfer hybridization, and the specific RNAs which are associated with the Rna+ phenotype have been mapped. This was done by a combination of transcript mapping, subcloning, and in vitro mutagenesis. The transcripts are found to be enriched in polyadenylated RNA and are of very low abundance (0.01-0.001% polyadenylated RNA).

scholarly journals Isolation and characterization of the RNA2, RNA3, and RNA11 genes of Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (11) ◽  
pp. 2396-2405 ◽  
Author(s):  
R L Last ◽  
J B Stavenhagen ◽  
J L Woolford
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Single Copy ◽  
Yeast Genome ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Mrna Accumulation ◽  
Isolation And Characterization ◽  
Polyadenylated Rna

Temperature-sensitive mutations in the genes RNA2 through RNA11 cause accumulation of intervening sequence containing precursor mRNAs in Saccharomyces cerevisiae. Three different plasmids have been isolated which complement both the temperature-sensitive lethality and precursor mRNA accumulation when introduced into rna2, rna3, and rna11 mutant strains. The yeast sequences on these plasmids have been shown by Southern transfer hybridization and genetic mapping to be derived from the RNA2, RNA3, and RNA11 genomic loci. Part of the RNA2 gene is homologous to more than one region of the yeast genome, whereas the RNA3 and RNA11 genes are single copy. RNAs homologous to these loci have been identified by RNA transfer hybridization, and the specific RNAs which are associated with the Rna+ phenotype have been mapped. This was done by a combination of transcript mapping, subcloning, and in vitro mutagenesis. The transcripts are found to be enriched in polyadenylated RNA and are of very low abundance (0.01-0.001% polyadenylated RNA).

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.

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 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.

Specific transcripts are elevated in Saccharomyces cerevisiae in response to DNA damage

1984 ◽  
Vol 4 (11) ◽  
pp. 2356-2363
Author(s):  
T McClanahan ◽  
K McEntee
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Southern Hybridization ◽  
Single Copy ◽  
Strand Break ◽  
Yeast Genome ◽  
Dna Strand Break ◽  
Isolation And Characterization ◽  
Dna Strand

Differential hybridization has been used to identify genes in Saccharomyces cerevisiae displaying increased transcript levels after treatment of cells with UV irradiation or with the mutagen/carcinogen 4-nitroquinoline-1-oxide (NQO). We describe the isolation and characterization of four DNA damage responsive genes obtained from screening ca. 9,000 yeast genomic clones. Two of these clones, lambda 78A and pBR178C, contain repetitive elements in the yeast genome as shown by Southern hybridization analysis. Although the genomic hybridization pattern is distinct for each of these two clones, both of these sequences hybridize to large polyadenylated transcripts ca. 5 kilobases in length. Two other DNA damage responsive sequences, pBRA2 and pBR3016B, are single-copy genes and hybridize to 0.5- and 3.2-kilobase transcripts, respectively. Kinetic analysis of the 0.5-kilobase transcript homologous to pBRA2 indicates that the level of this RNA increases more than 15-fold within 20 min after exposure to 4-nitroquinoline-1-oxide. Moreover, the level of this transcript is significantly elevated in cells containing the rad52-1 mutation which are deficient in DNA strand break repair and gene conversion. These results provide some of the first evidence that DNA damage stimulates transcription of specific genes in eucaryotic cells.

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.

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