Gel Electrophoresis Analysis of rDNA Instability in Saccharomyces cerevisiae

2020 ◽  
pp. 403-425
Author(s):  
Mariko Sasaki ◽  
Takehiko Kobayashi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Electrophoresis

scholarly journals Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28.

1982 ◽  
Vol 2 (4) ◽  
pp. 412-425 ◽  
Author(s):  
S I Reed ◽  
J Ferguson ◽  
J C Groppe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
In Vitro Mutagenesis ◽  
Coding Region ◽  
Loop Analysis ◽  
Partial Digestion ◽  
Polyadenylic Acid

The CDC28 gene was subcloned from a plasmid containing a 6.5-kilobase-pair segment of Saccharomyces cerevisiae DNA YRp7(CDC28-3) by partial digestion with Sau3A and insertion of the resulting fragments into the BamHI sites of YRp7 and pRC1. Recombinant plasmids were obtained containing inserts of 4.4 and 3.1 kilobase pairs which were capable of complementing a cdc28(ts) mutation. R-loop analysis indicated that each yeast insert contained two RNA coding regions of about 0.8 and 1.0 kilobase pairs, respectively. In vitro mutagenesis experiments suggested that the smaller coding region corresponded to the CDC28 gene. When cellular polyadenylic acid-containing RNA, separated by agarose gel electrophoresis after denaturation with glyoxal and transferred to nitrocellulose membrane, was reacted with labeled DNA from the smaller coding region, and RNA species of about 1 kilobase in length was detected. Presumably, the discrepancy in size between the R-loop and electrophoretic determinations is due to a segment of polyadenylic acid which is excluded from the R-loops. By using hybridization of the histone H2B mRNAs to an appropriate probe as a previously determined standards, it was possible to estimate the number of CDC28 mRNA copies per haploid cell as between 6 and 12 molecules. Hybrid release translation performed on the CDC29 mRNA directed the synthesis of a polypeptide of 27,000 daltons, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This polypeptide was not synthesized when mRNA prepared from a cdc28 nonsense mutant was translated in a parallel fashion. However, if the RNA from a cell containing the CDC28 gene on a plasmid maintained at a high copy number was translated, the amount of in vitro product was amplified fivefold.

scholarly journals Chromosomal transformation in Saccharomyces cerevisiae with DNA isolated by pulse field gel electrophoresis.

1990 ◽  
Vol 54 (6) ◽  
pp. 1499-1504 ◽  
Author(s):  
Kuniyasu GOTO ◽  
Tohru MOTOYOSHI ◽  
Gakuzo TAMURA ◽  
Takaji OBATA ◽  
Shodo HARA
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Electrophoresis ◽  
Pulse Field ◽  
Pulse Field Gel Electrophoresis

scholarly journals Structure and molecular analysis of RGR1, a gene required for glucose repression of Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (8) ◽  
pp. 4130-4138 ◽  
Author(s):  
A Sakai ◽  
Y Shimizu ◽  
S Kondou ◽  
T Chibazakura ◽  
F Hishinuma
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Electrophoresis ◽  
Daughter Cell ◽  
Glucose Repression ◽  
Pulsed Field Gel Electrophoresis ◽  
Null Mutation ◽  
Electron Microscopic ◽  
Transmission Electron ◽  
Transmission Electron Microscopic ◽  
Carboxy Terminal

An RGR1 gene product is required to repress expression of glucose-regulated genes in Saccharomyces cerevisiae. The abnormal morphology of rgr1 cells was studied. Scanning and transmission electron microscopic observations revealed that the cell wall of the daughter cell remained attached to that of mother cell. We cloned the RGR1 gene by complementation and showed that the cloned DNA was tightly linked to the chromosomal RGR1 locus. The cloned RGR1 gene suppressed all of the phenotypes caused by the mutation and encoded a 3.6-kilobase poly(A)+ RNA. The RGR1 gene is located on chromosome XII, as determined by pulsed-field gel electrophoresis, and we mapped rgr1 between gal2 and pep3 by genetic analysis. rgr1 was shown to be a new locus. We also determined the nucleotide sequence of RGR1, which was predicted to encode a 123-kilodalton protein. The null mutation resulted in lethality, indicating that the RGR1 gene is essential for growth. On the other hand, a carboxy-terminal deletion of the gene caused phenotypes similar to but more severe than those caused by the original mutation. The amount of reserve carbohydrates was reduced in rgr1 cells. Possible functions of the RGR1 product are discussed.

Assignment of cloned genes to the seven electrophoretically separated Candida albicans chromosomes

1988 ◽  
Vol 8 (11) ◽  
pp. 4721-4726
Author(s):  
B B Magee ◽  
Y Koltin ◽  
J A Gorman ◽  
P T Magee
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Unknown Function ◽  
Gel Electrophoresis ◽  
Homogeneous Field ◽  
Electrophoretic Karyotype ◽  
Chromosome Size ◽  
Homologous Chromosomes ◽  
Orthogonal Field ◽  
Chromosomal Bands

By using orthogonal-field alternating gel electrophoresis (OFAGE), field-inversion gel electrophoresis (FIGE), and contour-clamped homogeneous field gel electrophoresis (CHEF), we have clearly resolved 11 chromosomal bands from various Candida albicans strains. OFAGE resolves the smaller chromosomes better, while FIGE, which under our conditions causes the chromosomes to run in the reverse order of OFAGE, is more effective in separating the larger chromosomes. CHEF separates all chromosomes under some conditions, but these conditions do not often resolve homologs. The strains examined are highly polymorphic for chromosome size. Fourteen cloned Candida genes, isolated on the basis of conferral of new properties to or complementation of auxotrophic deficiencies in Saccharomyces cerevisiae, and three sequences of unknown function have been hybridized to Southern transfers of CHEF, FIGE, and OFAGE gels. Four sets of resolvable bands have been shown to be homologous chromosomes. On the basis of these data, we suggest that C. albicans has seven chromosomes. Genes have been assigned to the seven chromosomes. Two chromosomes identified genetically have been located on the electrophoretic karyotype.

Many yeast chromosomes lack the telomere-specific Y' sequence

1989 ◽  
Vol 9 (12) ◽  
pp. 5754-5757
Author(s):  
D Jäger ◽  
P Philippsen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Hybridization ◽  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Pulsed Field ◽  
Different Strains

Chromosomal DNAs of 26 different strains representing Saccharomyces species were analyzed by pulsed-field gel electrophoresis and subsequent hybridization to Y' telomere DNA. Hybridization to Y' was found exclusively in Saccharomyces cerevisiae strains, and among these strains, Y' sequences were found to be lacking in small, middle-sized, and large chromosomes.

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