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  • 1
    ISSN: 1432-0983
    Keywords: Key words Mitochondrial transcription apparatus ; Mitochondrial RNA polymerases ; Nuclear pet mutations ; Saccharomyces cerevisisae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The core enzyme of mitochondrial RNA polymerase in yeast is homologous to those of bacteriophages T3, T7 and SP6. In previous studies the identification of the first conditional yeast mutant for this enzyme helped to identify the corresponding specificity factor and to elucidate their interaction inside mitochondria. In the present study we report the identification of a second nuclear mutation located in the gene for mitochondrial RNA polymerase. A comparison of the two temperature-sensitive mutants demonstrates that the new mutant has a phenotype distinct from the first one and characterizes a new important domain of the enzyme. Two different suppressor genes which both rescue the first mutant do not abolish the defect of the second one and, in addition, an extremely high instability of mitochondrial genomes is observed in the new mutant. The enzymatic defect is caused by a single nucleotide exchange which results in the replacement of the serine938 residue by phenylalanine. This amino acid is located in the middle part of the protein in an as yet poorly characterized region that is not highly conserved between mitochondrial core enzymes and bacteriophage-type RNA polymerases. However, the affected amino acid and the respective protein domain are specific for mitochondrial RNA polymerase core enzymes and may help to define enzymatic functions specific for the mitochondrial transcription apparatus.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Current genetics 38 (2000), S. 1-7 
    ISSN: 1432-0983
    Keywords: Key words F1-ATPase ; ɛ-Subunit ; ρo-lethality ; Kluyveromyces lactis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The mitochondrial F1-ATPase is a multimeric enzyme, comprised of 3α, 3β, γ, δ and ɛ subunits, that is primarily responsible for the synthesis of ATP in eukaryotic cells. Recent work has shown that the F1 complex of the petite-negative yeast Kluyveromyces lactis, with specific mutations in the α, β or γ subunits, has a novel function that suppresses lethality caused by loss of mtDNA. Previously, genes for the four largest subunits of K. lactis F1 have been identified and characterised. In this study the gene coding for the ɛ-subunit of F1, KlATPɛ, has been isolated and found to encode a polypeptide of 61 amino acids with only 32 residues identical to those in the protein from Saccharomyces cerevisiae. Strains carrying a null mutation of KlATPɛ are respiratory deficient while the introduction of ATPɛ from S. cerevisiae restores growth on non-fermentable carbon sources. In contrast to S. cerevisiae, K. lactis disrupted in ATPɛ does not have a detectable F1-related mitochondrial ATP hydrolysis activity, suggesting that the ɛ-subunit plays a critical role in the formation of the catalytic sector of F1. With a disrupted KlATPɛ, the ρo-lethality suppressor function of F1 carrying the atp2-1 and atp1-6 alleles is abolished. However, inactivation of the ɛ subunit does not eliminate the ρo-viable phenotype of the atp1-1, atp2-9, atp3-2 mutants. It is suggested that the absence of ɛ may effect the assembly or stability of F1 in the wild-type, atp 2-1 and atp1-6 strains, whereas the defect can be suppressed by the atp1-1, atp2-9 and atp3-2 mutations in the α, β and γ subunits respectively.
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  • 3
    ISSN: 1432-0983
    Keywords: Kluyveromyces ; Transformation ; 2μ-like plasmids ; G418 resistance ; pKDI
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary pKDI is a 2μ-like circular plasmid found in the yeast Kluyveromyces drosophilarum that can also stably replicate in Kluyveromyces lactis. We have found a short intergenic region in this genome that appears to be functionally neutral; that is, the introduction of foreign sequences into the single EcoRI restriction site located near one of the inverted repeats did not affect the high stability of the natural plasmid. By introducing a G418 resistance gene at this site, we constructed an autonomous recombinant plasmid. Since this vector did not require cir + hosts for its stable maintenance, it could be used to examine the transformation host range of pKD1 among all the species belonging to the genus Kluyveromyces. Both species closely related to K. drosophilarum as well as a few other species that are very different in chromosomal GC % could be transformed to yield highly stable transformant clones.
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  • 4
    ISSN: 1432-0983
    Keywords: Key wordsKluyveromyces lactis ; Mitochondrial genome integrity ; F1-ATPase δ-subunit gene ; ATPδ -disruption
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Kluyveromyces lactis is a petite-negative yeast that does not form viable mitochondrial genome-deletion mutants (petites) when treated with DNA-targeting drugs. Loss of mtDNA is lethal for this yeast but mutations at three loci termed MGI, for mitochondrial genome integrity, can suppress this lethality. The three loci encode the α-, β- and γ-subunits of mitochondrial F1-ATPase. In this study we report the isolation and characterization of the KlATPδ gene encoding the δ-subunit of F1-ATPase. The deduced protein contains 158 amino acids showing 72% identity to the protein from Saccharomyces cerevisiae and a putative mitochondrial targeting sequence of 23 amino acids. Disruption of the gene causes cells to become respiratory deficient while the introduction of ATPδ from S. cerevisiae restores growth on glycerol. Cells with a disrupted ATPδ gene, like strains with disruptions of α-, β- and γ-F1-subunits, do not produce petite mutants when treated with ethidium bromide. However, unlike strains with disruptions in the three largest F1-subunits, disruption of ATPδ in the presence of some mgi alleles does not abolish the Mgi– phenotype. By contrast, elimination of ATPδ in other mgi strains removes resistance to ethidium bromide and ρ 0 mutants are not formed. Hence the ATPδ subunit of F1-ATPase, while not mandatory for a Mgi– phenotype, aids some mgi alleles in suppressing ρ 0 lethality.
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  • 5
    ISSN: 1432-0983
    Keywords: Key wordsKluyveromyces lactis ; Mitochondrial ribosomal protein ; ρo-lethality ; L23
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Kluyveromyces lactis nuclear gene, MRP-L23, encodes a polypeptide of 155 amino acids that shares 70% and 43% identity to the ribosomal proteins L23 and L13 of Saccharomyces cerevisiae and Escherichia coli. The deduced protein, designated KlL23, is a likely component of the large subunit of mitochondrial ribosomes as it can complement the respiratory deficient phenotype of a S. cerevisiae mrp-L23 mutant. As in S. cerevisiae, KlMRP-L23 is essential for respiratory growth of K. lactis because disruption of the gene in a “petite-positive” strain carrying a ρo-lethality suppressor atp mutation rendered cells unable to grow on a non-fermentable carbon source. However, in contrast to S. cerevisiae, disruption of MRP-L23 in wild type K. lactis is lethal. Meiotic segregants of K. lactis with a disrupted MRP-L23 allele form microcolonies with cell numbers varying from 32 to 300. These data clearly indicate an essential role of mitochondrial protein synthesis for viability of the petite-negative yeast K. lactis.
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  • 6
    ISSN: 1617-4623
    Keywords: Key words Mitochondrial DNA ; Kluyveromyceslactis ; MGM101/MGI genes ; Petite-negative yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Petite-negative yeasts do not form viable respiratory-deficient mutants on treatment with DNA-targeting drugs that readily eliminate the mitochondrial DNA (mtDNA) from petite-positive yeasts. However, in the petite-negative yeast Kluyveromyces lactis, specific mutations in the nuclear genes MGI2 and MGI5 encoding the α- and γ-subunits of the mitochondrial F1-ATPase, allow mtDNA to be lost. In this study we show that wild-type K. lactis does not survive in the absence of its mitochondrial genome and that the function of mgi mutations is to suppress lethality caused by loss of mtDNA. Firstly, we find that loss of a multicopy plasmid bearing a mgi allele readily occurs from a wild-type strain with functional mtDNA but is not tolerated in the absence of mtDNA. Secondly, we cloned the K. lactis homologue of the Saccharomyces cerevisiae mitochondrial genome maintenance gene MGM101, and disrupted one of the two copies in a diploid. Following sporulation, we find that segregants containing the disrupted gene form minicolonies containing 6-8000 inviable cells. By contrast, disruption of MGM101 is not lethal in a haploid mgi strain with a specific mutation in a subunit of the mitochondrial F1-ATPase. These observations suggest that mtDNA in K. lactis encodes a vital function which may reside in one of the three mitochondrially encoded subunits of F0.
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  • 7
    ISSN: 1617-4623
    Keywords: Kluyveromyces lactis ; Transcriptional regulation ; Promoter ; Sugar transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary TheRAG1 gene encodes a membrane protein involved in the low-affinity glucose/fructose transport system of the yeastKluyveromyces lactis. Analysis of steady-state mRNA levels analysis and quantitation of expression by β-galactosidase fromRAG1-lacZ fusions assays revealed that theRAG1 gene was poorly expressed in cells grown under gluconeogenesis conditions, but was induced more than ten-fold when they were grown on various sugars. These sugars included glucose, fructose, mannose, sucrose, raffinose, as well as galactose. Nucleotide sequence and deletion analysis of the 5′ fianking region of theRAG1 gene showed that an essentialcis-acting element required for induced transcription of theRAG1 gene resided between − 615 and − 750 from the coding sequence. This region contained a 22 by purine stretch, and a pair of 11 by direct repeat sequences. The 11 by repeats harbor a CCAAT motif, a consensus sequence for binding of the yeast and mammalian HAP2/3/4-type protein complex. The transcription of theRAG1 gene was dramatically affected by three unlinked mutations,rag4, rag5 andrag8. We discuss the possible roles ofRAG4,RAG5 andRAG8 gene products in the expression of theRAG1 gene, as well as the importance of the inducibleRAG1 gene in the fermentative growth ofK. lactis.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 8 (1992), S. 801-804 
    ISSN: 0749-503X
    Keywords: Kluyveromyces lactis ; LEU2 gene ; rat ribosomal protein L7 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A DNA fragment that can complement the leu2 mutation of Saccharomyces cerevisiae was cloned from the genomic library of Kluyveromyces lactis.The nucleotide sequence revealed an open reading frame of 362 codons, 75% homologous to S. cerevisiae LEU2 gene. The upstream region contained a CCGGAACCGG sequence identical to the site of leucine-specific control of LEU2. Further upstream, there is a partial open reading frame homologous to rat ribosmal protien L7.
    Additional Material: 1 Ill.
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  • 9
    ISSN: 0749-503X
    Keywords: recombinant DNA ; K. lactis genomic library ; pCXJ22 ; arginine biosynthesis ; KlARG8 ; mitochondrial transformation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A recombinant plasmid was isolated from a Kluyveromyces lactis genomic DNA library which complements a Saccharomyces cerevisiae arg8 mutant defective in the gene encoding acetylornithine aminotransferase. The complementation activity was found to reside within a 2.0 kb DNA fragment. Nucleotide sequence analysis revealed an open reading frame able to encode a 423-residue protein sharing 68·1% and 35·0% sequence identities with the products of the ARG8 and argD genes of S. cerevisiae and Escherichia coli. That the cloned gene, KlARG8, is the functional equivalent of S. cerevisiae ARG8 was supported by a gene disruption experiment which showed that K. lactis strains carrying a deleted chromosomal copy of KlARG8 are auxotrophic for arginine. The nucleotide sequence of KlARG8 has been submitted to GenBank under Accession Number U93209.
    Additional Material: 3 Ill.
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