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 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 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.

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.

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.

Production of a Genomic DNA Library

1991 ◽  
Vol 13 (1) ◽  
Author(s):  
Thomas Quertermous
Keyword(s):  
Genomic Dna ◽  
Dna Library ◽  
Genomic Dna Library

Genomic DNA Library Preparation for Resistance Gene Enrichment and Sequencing (RenSeq) in Plants

2014 ◽  
pp. 291-303 ◽  
Author(s):  
Florian Jupe ◽  
Xinwei Chen ◽  
Walter Verweij ◽  
Kamel Witek ◽  
Jonathan D. G. Jones ◽  
...  
Keyword(s):  
Resistance Gene ◽  
Genomic Dna ◽  
Library Preparation ◽  
Dna Library ◽  
Gene Enrichment ◽  
Genomic Dna Library
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