Genetic modification of cytochrome b deficient mutants in Saccharomyces cerevisiae

1974 ◽  
Vol 131 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Stanislaw Ułaszewski ◽  
Tadeusz M. Lachowicz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome B ◽  
Genetic Modification

scholarly journals In vitro splicing of the terminal intervening sequence of Saccharomyces cerevisiae cytochrome b pre-mRNA.

1987 ◽  
Vol 7 (7) ◽  
pp. 2545-2551 ◽  
Author(s):  
A Gampel ◽  
A Tzagoloff
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome B ◽  
Cytochrome B Gene ◽  
Group I Introns ◽  
Vitro Assay ◽  
Group I ◽  
Mitochondrial Cytochrome B ◽  
Nuclease Mapping

A region of the Saccharomyces cerevisiae mitochondrial cytochrome b gene encompassing the entire terminal intron plus flanking exonic sequences has been cloned in an SP6 vector. A runoff transcript prepared from this construct as well as the native cytochrome b pre-mRNA containing the terminal intervening sequence were found to act as substrates for the autocatalytic excision of the intervening sequence in vitro. This reaction proceeds under conditions previously shown by Cech and co-workers to promote protein-independent excision of the Tetrahymena rRNA intervening sequence. The 5' and 3' termini of the excised intervening sequence, determined by S1 nuclease mapping and sequence analysis, are consistent with the known sequence of the cytochrome b mRNA. The same region of the cytochrome b gene from a yeast mutant, defective in splicing due to a mutation in a critical sequence inside the terminal intron, has also been cloned in an SP6 vector. The mutant transcript fails to self-splice in the in vitro assay. These observations provide strong presumptive evidence that in vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA occurs by an autocatalytic mechanism analogous to that shown for other group I introns. In vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA, however, exhibits complete dependence on a protein factor previously shown to be encoded by the nuclear gene CBP2.

Construction of integrative plasmids suitable for genetic modification of industrial strains of Saccharomyces cerevisiae

Plasmid ◽  
2013 ◽  
Vol 69 (1) ◽  
pp. 114-117 ◽  
Author(s):  
Fernanda Cristina Bezerra Leite ◽  
Rute Salgues Gueiros dos Anjos ◽  
Anna Carla Moreira Basilio ◽  
Guilherme Felipe Carvalho Leal ◽  
Diogo Ardaillon Simões ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Modification ◽  
Industrial Strains

scholarly journals Generation of temperature-sensitive cbp1 strains of Saccharomyces cerevisiae by PCR mutagenesis and in vivo recombination: characteristics of the mutant strains imply that CBP1 is involved in stabilization and processing of cytochrome b pre-mRNA.

Genetics ◽  
1993 ◽  
Vol 135 (4) ◽  
pp. 981-991 ◽  
Author(s):  
R R Staples ◽  
C L Dieckmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome B ◽  
Mitochondrial Gene ◽  
Nuclear Gene ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
In Vivo Recombination ◽  
Mutant Strains ◽  
Precursor Rna

Abstract Mitochondrial biogenesis is dependent on both nuclearly and mitochondrially encoded proteins. Study of the nuclearly encoded mitochondrial gene products and their effect on mitochondrial genome expression is essential to understanding mitochondrial function. Mutations in the nuclear gene CBP1 of Saccharomyces cerevisiae result in degradation of mitochondrially encoded cytochrome b (cob) RNA; thus, the cells are unable to respire. Putative roles for the CBP1 protein include processing of precursor RNA to yield the mature 5' end of cob mRNA and/or physical protection of the mRNA from degradation by nucleases. To examine the activity of CBP1, we generated temperature-sensitive cbp1 mutant strains by polymerase chain reaction (PCR) mutagenesis and in vivo recombination. These temperature-sensitive cbp1 strains lack cob mRNA only at the nonpermissive temperature. Quantitative primer extension analyses of RNA from these strains and from a cbp1 deletion strain demonstrated that CBP1 is required for the stability of precursor RNAs in addition to production of the stable mature mRNA. Thus, CBP1 is not involved solely in the protection of mature cob mRNA from nucleases. Moreover, we found that mature mRNAs are undetectable while precursor RNAs are reduced only slightly at the nonpermissive temperature. Collectively, these data lead us to favor a hypothesis whereby CBP1 protects cob precursor RNAs and promotes the processing event that generates the mature 5' end of the mRNA.

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