scholarly journals The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases.

1986 ◽  
Vol 6 (7) ◽  
pp. 2500-2510 ◽  
Author(s):  
C A Woolford ◽  
L B Daniels ◽  
F J Park ◽  
E W Jones ◽  
J N Van Arsdell ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genomic Dna ◽  
Predicted Amino Acid Sequence ◽  
Posttranslational Regulation ◽  
Aspartyl Protease ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Pep4 Gene ◽  
Precursor Molecules ◽  
Recombination Experiments

pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.

The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases

1986 ◽  
Vol 6 (7) ◽  
pp. 2500-2510
Author(s):  
C A Woolford ◽  
L B Daniels ◽  
F J Park ◽  
E W Jones ◽  
J N Van Arsdell ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genomic Dna ◽  
Predicted Amino Acid Sequence ◽  
Posttranslational Regulation ◽  
Aspartyl Protease ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Pep4 Gene ◽  
Precursor Molecules ◽  
Recombination Experiments

pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome

1986 ◽  
Vol 6 (12) ◽  
pp. 4516-4525
Author(s):  
K G Coleman ◽  
H Y Steensma ◽  
D B Kaback ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Cloning ◽  
Genomic Dna ◽  
Shuttle Vector ◽  
Blot Hybridization ◽  
Yeast Genome ◽  
Dna Library ◽  
Isolation And Characterization ◽  
Genomic Dna Library ◽  
Chromosome I

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.

scholarly journals Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome.

1986 ◽  
Vol 6 (12) ◽  
pp. 4516-4525 ◽  
Author(s):  
K G Coleman ◽  
H Y Steensma ◽  
D B Kaback ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Cloning ◽  
Genomic Dna ◽  
Shuttle Vector ◽  
Blot Hybridization ◽  
Yeast Genome ◽  
Dna Library ◽  
Isolation And Characterization ◽  
Genomic Dna Library ◽  
Chromosome I

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.

Molecular characterization of cell cycle gene CDC7 from Saccharomyces cerevisiae

1986 ◽  
Vol 6 (5) ◽  
pp. 1590-1598
Author(s):  
M Patterson ◽  
R A Sclafani ◽  
W L Fangman ◽  
J Rosamond
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genomic Dna ◽  
Genetic Recombination ◽  
Multiple Roles ◽  
Predicted Amino Acid Sequence ◽  
Cell Cycle Gene ◽  
Temperature Sensitive ◽  
Multicopy Plasmid ◽  
Reading Frame ◽  
Genomic Insert

The product of the CDC7 gene of Saccharomyces cerevisiae appears to have multiple roles in cellular physiology. It is required for the initiation of mitotic DNA synthesis. While it is not required for the initiation of meiotic DNA replication, it is necessary for genetic recombination during meiosis and for the formation of ascospores. It has also been implicated in an error-prone DNA repair pathway. Plasmids capable of complementing temperature-sensitive cdc7 mutations were isolated from libraries of yeast genomic DNA in the multicopy plasmid vectors YRp7 and YEp24. The complementing activity was localized within a 3.0-kilobase genomic DNA fragment. Genetic studies that included integration of the genomic insert at or near the CDC7 locus and marker rescue of four cdc7 alleles proved that the cloned fragment contains the yeast chromosomal CDC7 gene. The RNA transcript of CDC7 is about 1,700 nucleotides. Analysis of the nucleotide sequence of a 2.1-kilobase region of the cloned fragment revealed the presence of an open reading frame of 1,521 nucleotides that is presumed to encode the CDC7 protein. Depending on which of two possible ATG codons initiates translation, the calculated size of the CDC7 protein is 58.2 or 56 kilodaltons. Comparison of the predicted amino acid sequence of the CDC7 gene product with other known protein sequences suggests that CDC7 encodes a protein kinase.

PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors

1986 ◽  
Vol 6 (7) ◽  
pp. 2490-2499
Author(s):  
G Ammerer ◽  
C P Hunter ◽  
J H Rothman ◽  
G C Saari ◽  
L A Valls ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Structural Gene ◽  
Yeast Cells ◽  
Linkage Data ◽  
Predicted Amino Acid Sequence ◽  
Multicopy Plasmid ◽  
Proteinase A ◽  
Pep4 Gene ◽  
Cloned Gene

The proteinase A structural gene of Saccharomyces cerevisiae was cloned by using an immunological screening procedure that allows detection of yeast cells which are aberrantly secreting vacuolar proteins (J. H. Rothman, C. P. Hunter, L. A. Valls, and T. H. Stevens, Proc. Natl. Acad. Sci. USA, 83:3248-3252, 1986). A second cloned gene was obtained on a multicopy plasmid by complementation of a pep4-3 mutation. The nucleotide sequences of these two genes were determined independently and were found to be identical. The predicted amino acid sequence of the cloned gene suggests that proteinase A is synthesized as a 405-amino-acid precursor which is proteolytically converted to the 329-amino-acid mature enzyme. Proteinase A shows substantial homology to mammalian aspartyl proteases, such as pepsin, renin, and cathepsin D. The similarities may reflect not only analogous functions but also similar processing and intracellular targeting mechanisms for the two proteins. The cloned proteinase A structural gene, even when it is carried on a single-copy plasmid, complements the deficiency in several vacuolar hydrolase activities that is observed in a pep4 mutant. A strain carrying a deletion in the genomic copy of the gene fails to complement a pep4 mutant of the opposite mating type. Genetic linkage data demonstrate that integrated copies of the cloned proteinase A structural gene map to the PEP4 locus. Thus, the PEP4 gene encodes a vacuolar aspartyl protease, proteinase A, that is required for the in vivo processing of a number of vacuolar zymogens.

scholarly journals Isolation and characterization of PEP5, a gene essential for vacuolar biogenesis in Saccharomyces cerevisiae.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 739-752 ◽  
Author(s):  
C A Woolford ◽  
C K Dixon ◽  
M F Manolson ◽  
R Wright ◽  
E W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mass ◽  
Open Reading Frame ◽  
Relative Molecular Mass ◽  
Cell Fractionation ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Dna Library ◽  
Isolation And Characterization ◽  
Genomic Dna Library

Abstract pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. The PEP5 gene was isolated from a genomic DNA library by complementation of the pep5-8 mutation. Deletion analysis localized the complementing activity to a 3.3-kb DNA fragment. DNA sequence analysis of the PEP5 gene revealed an open reading frame of 1029 codons with a calculated molecular mass for the encoded protein of 117,403 D. Deletion/disruption of the PEP5 gene did not kill the cells. The resulting strains grow very slowly at 37 degrees. The disruption mutant showed greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY, and the repressible alkaline phosphatase. Apparently normal precursors forms of the proteases accumulated in pep5 mutants, as did novel forms of PrB antigen. Antibodies raised to a fusion protein that contained almost half of the PEP5 open reading frame allowed detection by immunoblot of a protein of relative molecular mass 107 kD in extracts prepared from wild-type cells. Cell fractionation showed the PEP5 gene product is enriched in the vacuolar fraction and appears to be a peripheral vacuolar membrane protein.

Eimeria species: studies using rRNA and rDNA probes

Parasitology ◽  
1990 ◽  
Vol 101 (1) ◽  
pp. 1-6 ◽  
Author(s):  
J. Ellis ◽  
J. Bumstead
Keyword(s):  
Genomic Dna ◽  
Southern Hybridization ◽  
Eimeria Species ◽  
Small Subunit ◽  
Rrna Genes ◽  
Rdna Probe ◽  
Dna Library ◽  
Rdna Sequences ◽  
Genomic Dna Library

SUMMARYrRNA and a heterologous cloned rDNA probe have been used to detect the rRNA genes of Eimeria species which infe the chicken, and has allowed the isolation and preliminary characterization of cloned rDNA sequences from a genomic DNA library of Eimeria tenella. It is demonstrated that rRNA and rDNA probes can be used to identify individual Eimeria species by the restriction fragment patterns detected after Southern hybridization. In addition, studies have shown that the large and small subunit rRNAs are expressed throughout sporulation.

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