scholarly journals Disruption of single-copy genes encoding acidic ribosomal proteins in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (5) ◽  
pp. 2182-2190 ◽  
Author(s):  
M Remacha ◽  
C Santos ◽  
J P Ballesta
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Minimal Medium ◽  
Single Copy ◽  
Diploid Strain ◽  
Tetrad Analysis ◽  
Genes Encoding ◽  
Acidic Proteins ◽  
Dna Fragment ◽  
His3 Gene

Using the cloned genes coding for the ribosomal acidic proteins L44 and L45, constructions were made which deleted part of the coding sequence and inserted a DNA fragment at that site carrying either the URA3 or HIS3 gene. By gene disruption techniques with linearized DNA from these constructions, strains of Saccharomyces cerevisiae were obtained which lacked a functional gene for either protein L44 or protein L45. The disrupted genes in the transformants were characterized by Southern blots. The absence of the proteins was verified by electrofocusing and immunological techniques, but a compensating increase of the other acidic ribosomal proteins was not detected. The mutant lacking L44 grew at a rate identical to the parental strain in complex as well as in minimal medium. The L45-disrupted strain also grew well in both media but at a slower rate than the parental culture. A diploid strain was obtained by crossing both transformants, and by tetrad analysis it was shown that the double transformant lacking both genes is not viable. These results indicated that proteins L44 and L45 are independently dispensable for cell growth and that the ribosome is functional in the absence of either of them.

Disruption of single-copy genes encoding acidic ribosomal proteins in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (5) ◽  
pp. 2182-2190
Author(s):  
M Remacha ◽  
C Santos ◽  
J P Ballesta
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Minimal Medium ◽  
Single Copy ◽  
Diploid Strain ◽  
Tetrad Analysis ◽  
Genes Encoding ◽  
Acidic Proteins ◽  
Dna Fragment ◽  
His3 Gene

Using the cloned genes coding for the ribosomal acidic proteins L44 and L45, constructions were made which deleted part of the coding sequence and inserted a DNA fragment at that site carrying either the URA3 or HIS3 gene. By gene disruption techniques with linearized DNA from these constructions, strains of Saccharomyces cerevisiae were obtained which lacked a functional gene for either protein L44 or protein L45. The disrupted genes in the transformants were characterized by Southern blots. The absence of the proteins was verified by electrofocusing and immunological techniques, but a compensating increase of the other acidic ribosomal proteins was not detected. The mutant lacking L44 grew at a rate identical to the parental strain in complex as well as in minimal medium. The L45-disrupted strain also grew well in both media but at a slower rate than the parental culture. A diploid strain was obtained by crossing both transformants, and by tetrad analysis it was shown that the double transformant lacking both genes is not viable. These results indicated that proteins L44 and L45 are independently dispensable for cell growth and that the ribosome is functional in the absence of either of them.

scholarly journals Transcriptional regulation of ribosomal proteins during a nutritional upshift in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (7) ◽  
pp. 2429-2435 ◽  
Author(s):  
D M Donovan ◽  
N J Pearson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Protein Gene ◽  
Relative Rate ◽  
Ribosomal Protein Gene ◽  
Single Copy ◽  
Protein Product ◽  
Yeast Genome ◽  
Base Pairs ◽  
Beta Galactosidase

The relative rates of synthesis of Saccharomyces cerevisiae ribosomal proteins increase coordinately during a nutritional upshift. We constructed a gene fusion which contained 528 base pairs of sequence upstream from and including the TATA box of ribosomal protein gene rp55-1 (S16A-1) fused to a CYC1-lacZ fusion. This fusion was integrated in single copy at the rp55-1 locus in the yeast genome. During a nutritional upshift, in which glucose was added to cells growing in an ethanol-based medium, we found that the increase in the relative rate of synthesis of the beta-galactosidase protein product followed the same kinetics as the change in relative rates of synthesis of several ribosomal proteins measured in the same experiment. This demonstrates that the nontranscribed sequences upstream from the rp55-1 gene, which are present in the fusion, are sufficient to mediate the change in rates of synthesis characteristic of ribosomal proteins under these conditions. The results also suggest that a change in transcription rates is mainly responsible for the increase in relative rates of synthesis of ribosomal proteins during a nutritional upshift in S. cerevisiae.

scholarly journals Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (10) ◽  
pp. 5235-5243 ◽  
Author(s):  
D M Baronas-Lowell ◽  
J R Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Wild Type ◽  
Diploid Strain ◽  
Chain Reaction ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ribosomal Subunits ◽  
Strong Homology ◽  
Polymerase Chain

In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes. We have cloned and sequenced RPL30B, which shows strong homology to RPL30A. Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region. This intron has an unusual 5' splice site, C/GUAUGU. The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination. Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores. Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.

Genetic analysis of small nuclear RNAs in Saccharomyces cerevisiae: viable sextuple mutant

1988 ◽  
Vol 8 (8) ◽  
pp. 3150-3159
Author(s):  
R Parker ◽  
T Simmons ◽  
E O Shuster ◽  
P G Siliciano ◽  
C Guthrie
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Start Site ◽  
Single Copy ◽  
Gene Replacement ◽  
Null Alleles ◽  
Wild Type ◽  
Small Nuclear Rnas ◽  
Apparent Correlation ◽  
Genes Encoding ◽  
Sm Antigen

Saccharomyces cerevisiae contains at least 24 distinct small nuclear RNAs (snRNAs), several of which are known to be essential for viability and to participate in the splicing of pre-mRNAs; the RNAs in this subset contain binding sites for the Sm antigen, a hallmark of metazoan snRNAs involved in mRNA processing. In contrast, we showed previously that the single-copy genes for three other snRNAs (snR3, snR4, and snR10) are not required for viability, although cells lacking snR10 are growth impaired at low temperature. None of these RNAs associates with the Sm antigen. To assess this apparent correlation, we cloned and sequenced the genes encoding three additional non-Sm snRNAs. Comparison of these genes with nine additional yeast snRNA genes revealed a highly conserved TATA box located 92 +/- 8 nucleotides 5' of the transcriptional start site. By using the technique of gene replacement with null alleles, each of these three single copy genes was shown to be completely dispensable. We constructed multiple mutants to test the hypothesis that, individually, each of these snRNAs is nonessential because the snRNAs play functionally overlapping roles. A mutant lacking five snRNAs (snR3, snR4, snR5, snR8, snR9) was indistinguishable from the wild type, and growth of the sextuple mutant was no more impaired than that in strains lacking only snR10. This widespread dispensability of snRNAs was completely unexpected and forces us to reconsider the possible roles of these ubiquitous RNAs.

Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae

1990 ◽  
Vol 10 (10) ◽  
pp. 5235-5243
Author(s):  
D M Baronas-Lowell ◽  
J R Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Wild Type ◽  
Diploid Strain ◽  
Chain Reaction ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ribosomal Subunits ◽  
Strong Homology ◽  
Polymerase Chain

In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes. We have cloned and sequenced RPL30B, which shows strong homology to RPL30A. Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region. This intron has an unusual 5' splice site, C/GUAUGU. The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination. Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores. Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.

Transcriptional regulation of ribosomal proteins during a nutritional upshift in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (7) ◽  
pp. 2429-2435
Author(s):  
D M Donovan ◽  
N J Pearson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Protein Gene ◽  
Relative Rate ◽  
Ribosomal Protein Gene ◽  
Single Copy ◽  
Protein Product ◽  
Yeast Genome ◽  
Base Pairs ◽  
Beta Galactosidase

The relative rates of synthesis of Saccharomyces cerevisiae ribosomal proteins increase coordinately during a nutritional upshift. We constructed a gene fusion which contained 528 base pairs of sequence upstream from and including the TATA box of ribosomal protein gene rp55-1 (S16A-1) fused to a CYC1-lacZ fusion. This fusion was integrated in single copy at the rp55-1 locus in the yeast genome. During a nutritional upshift, in which glucose was added to cells growing in an ethanol-based medium, we found that the increase in the relative rate of synthesis of the beta-galactosidase protein product followed the same kinetics as the change in relative rates of synthesis of several ribosomal proteins measured in the same experiment. This demonstrates that the nontranscribed sequences upstream from the rp55-1 gene, which are present in the fusion, are sufficient to mediate the change in rates of synthesis characteristic of ribosomal proteins under these conditions. The results also suggest that a change in transcription rates is mainly responsible for the increase in relative rates of synthesis of ribosomal proteins during a nutritional upshift in S. cerevisiae.

Organization of genes encoding the L11, L1, L10, and L12 equivalent ribosomal proteins in eubacteria, archaebacteria, and eucaryotes

1989 ◽  
Vol 35 (1) ◽  
pp. 164-170 ◽  
Author(s):  
Lawrence C. Shimmin ◽  
C. Hunter Newton ◽  
Celia Ramirez ◽  
Janet Yee ◽  
Willa Lee Downing ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Sulfolobus Solfataricus ◽  
Mrna Translation ◽  
Restriction Fragments ◽  
E Coli ◽  
Transcription Pattern ◽  
Genes Encoding ◽  
Cytoplasmic Ribosomes

Archaebacterial and eucaryotic cytoplasmic ribosomes contain proteins equivalent to the L11, L1, L10, and L12 proteins of the eubacterium Escherichia coli. In E. coli the genes encoding these ribosomal proteins are clustered, cotranscribed, and autogenously regulated at the level of mRNA translation. Genomic restriction fragments encoding the L11e, L1e, L10e, and L12e (equivalent) proteins from two divergent archaebacteria, Halobacterium cutirubrum and Sulfolobus solfataricus, and the L10e and L12e proteins from the eucaryote Saccharomyces cerevisiae have been cloned, sequenced, and analyzed. In the archaebacteria, as in eubacteria, the four genes are clustered and the L11e, L1e, L 10e, and L12e order is maintained. The transcription pattern of the H. cutirubrum cluster is different from the E. coli pattern and the flanking genes on either side of the tetragenic clusters in E. coli, H. cutirubrum, and Sulfolobus solfataricus are all unrelated to each other. In the eucaryote Saccharomyces cerevisiae there is a single L10e gene and four separate L12e genes that are designated L12eIA, L12eIB, L12eIIA, and L12eIIB. These five genes are not closely linked and each is transcribed as a monocistronic mRNA; the L10e, L12eIA, L12eIB, and the L12eIIA genes are contiguous and uninterrupted, whereas the L12eIIB gene is interrupted by a 301 nucleotide long intron located between codons 38 and 39.Key words: archaebacteria, ribosome, Halobacterium, Sulfolobus.

Chromosome location of a family of genes encoding different acidic ribosomal proteins in Saccharomyces cerevisiae

1990 ◽  
Vol 17 (6) ◽  
pp. 535-536 ◽  
Author(s):  
M. Remacha ◽  
L. Ramirez ◽  
I. Marin ◽  
J. P. G. Ballesta
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Chromosome Location ◽  
Genes Encoding

mRNA transcription in nuclei isolated from Saccharomyces cerevisiae

1986 ◽  
Vol 6 (5) ◽  
pp. 1633-1639
Author(s):  
J F Jerome ◽  
J A Jaehning
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Blot Hybridization ◽  
Single Copy ◽  
Rna Polymerases ◽  
Slot Blot Hybridization ◽  
Genes Encoding ◽  
Diploid Cells ◽  
Alpha Amanitin

We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II.

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