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  • 1
    ISSN: 1432-0983
    Keywords: Intervening sequence mutations ; RNA blotting ; Transcript hybridization ; RNA processing
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Electrophoretic separation of mitochondrial RNA followed by hybridization with restriction fragments of mtDNA has been used to identify transcripts of the split gen COB which codes for apocytochrome b. In wild type a major transcript of 18S is detected besides a 10S RNA and a series of transcripts with electrophoretic mobilities higher than 18S. Mutations in coding sequences do not significantly alter this transcript pattern. In contrast, mutations in intervening sequences give rise to different patterns: The 18S RNA, the putative messenger for apocytochrome b, is lacking; depending on the intervening sequence affected by mutation, one or the other of the larger transcripts (23S, 24S, 32S, 34S) is accumulated instead. In most mutants a 10S RNA species is present; it has not been detected, however, in case of a small cluster of mutations which lead to the accumulation of the largest transcript (34S) observed, which most likely contains all coding and intervening sequences of the split gene COB. These results suggest a pathway of splicing.
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  • 2
    ISSN: 1432-0983
    Keywords: MitDNA restriction analysis ; Nucleotide sequences ; Possible initiation site of DNA replication
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A class of suppressive “petite” mutants of S. cerevisiae, called here supersuppressive, is characterized by a) the fact that their unmodified mitochondrial genomes are the only ones found in the progeny of crosses with wild-type cells; b) very short repeat units (400–900 base pairs) in their mitochondrial genomes. The repeat units of the three supersuppressive “petites” investigated here share a common 83 nucleotide sequence, which seems to correspond to an initiation site of DNA replication; the multiple copies of this site in the mitochondrial genomes of supersuppressive “petites” might explain why these genomes can compete out those of wild-type cells.
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  • 3
    ISSN: 1432-0983
    Keywords: Yeast ; Ribosomal protein alteration ; Cycloheximide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A spontaneous high-level cycloheximide-resistant mutant of the yeast Saccharomyces cerevisiae (strain cy32) is found to have an altered protein of the large subunit (60S) of cytoplasmic ribosomes, namely protein L29. The resistance character segregates together with this biochemical defect and is semidominant in heterozygous diploids. Judged from in vitro susceptibility to inhibition by cycloheximide there are at least 50% resistant ribosomes present in such diploid strains. From these results it is concluded that cycloheximide resistance of mutant cy32 is caused by mutation of a single gene and that it is the structural gene for L29 which is affected. Preliminary genetic mapping data are also reported. They indicate a location of cyhx-32 marker on chromosome 7 near met13.
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  • 4
    ISSN: 1432-0983
    Keywords: Ascobolus ; Fungi ; Gene conversion ; Octospored asci
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The cross b2-17-A4 x b2+ involving 2 mutant sites located one at each end of the b2 locus in Ascobolus immersus was analysed in order to look for aberrant segregations at one site among asci with aberrant 4:4 segregation at the other site. Most double site conversion asci with an aberrant 4:4 at one site showed a 5:3 segregation at the other site. Almost always, the aberrant 4:4 involved the low conversion end site and the 5:3 involved the high conversion end site. It is concluded that, in each individual meiosis, symmetrical hybrid DNA is often, if not always, physically associated with asymmetrical hybrid DNA. The asymmetrical hybrid DNA portion lies near the high conversion end and the symmetrical hybrid DNA portion near the low one.
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  • 5
    ISSN: 1432-0983
    Keywords: rate of synthesis of proteins ; ts block of initiation ; decay of polysomes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The synthesis of thirty five ribosomal proteins has been studied in Saccharomyces cerevisiae using a temperature sensitive mutant blocked in the initiation of polypeptides. The synthesis of all the ribosomal proteins was nearly equally affected by the block. This absence of polarity, together with previous genetic and biochemical data, indicates that eukaryotic ribosomal proteins are coded by monocistronic mRNAs.
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  • 6
    ISSN: 1432-0983
    Keywords: Mitochondrial DNA ; Restriction mapping
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A restriction endonuclease cleavage map of rat liver mitochondrial DNA is presented for the following enzymes: Xba I, Bgl II, Hae II, Bam HI, Hpa I, Hha I, Bcl I, Hind II, Hind III, Eco RI, Hpa II, Hae III, and Sau 3A. It was derived from complete and partial digestions with these enzymes, double digestions, and redigestions of defined fragments obtained with one enzyme with other restriction enzymes. By the use of these and further enzymes (Taq YI, Alu I) the mitochondrial DNA (ca. 15.6 Kb) can be dissected into a large number of defined fragments.
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  • 7
    ISSN: 1432-0983
    Keywords: Mitochondrial rRNA ; Nuclear/mitochondrial interactions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have isolated two non-allelic nuclear mutants of Neurospora crassa that are temperature-sensitive for the production of cytochromes aa 3 and b. When grown at 23 °C the mutants are virtually indistinguishable from the parent wild-type strains. When grown at 41 °C the mutants have large amounts of KCN-insensitive respiration and lack cytochromes aa 3 and b. Further examination of the mutants revealed that they were extremely deficient in their capacity for mitochondrial protein synthesis when grown at 41 °C. This protein synthesis deficiency appears to be related to a virtual absence of both small and large mitochondrial ribosomal subunits following growth at 41 °C. Examination of the mitochondrial RNAs of the mutants suggests that mitochondrial rRNAs are synthesized in greatly reduced amounts or that they are misprocessed when these mutants are grown at the non-permissive temperature.
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  • 8
    ISSN: 1432-0983
    Keywords: Yeast ; Mitochondrial DNA ; Recombination
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Mitochondrial genes recombine extensively in yeast zygotes. In heteropolar crosses (ω+ × ω−) in which the ω− “allele” consists of an insertion, there is preferential recovery of ω+ and markers closely linked to it. This polarity has been postulated to be a consequence of one-way gene conversion beginning at the ω locus (ω- to ω+). We have shown that most or all mitochondrial recombination in homopolar and heteropolar crosses, and the phenomenon of polarity itself, does not require products of protein synthesis on mitochondrial ribosomes. (i) Yeast strains were grown and mated, and the zygotes plated and grown, on glucose medium with erythromycin to inhibit and dilute out the products of mitochondrial protein synthesis. Recombination frequencies and polarity at the cap1 and oli1 loci were normal compared to controls in some homopolar (ω+ × ω−) and heteropolar crosses. Apparent changes in recombination frequencies and polarity were seen in other crosses but are attributable to locus-specific petite induction by erythromycin. (ii) Homopolar (ω+ × ω+) and heteropolar crosses between pairs of petite mutants retaining the cap1, ery1, and oli1 loci also showed nearly normal recombination at the cap1 and oli1 loci, as determined by test-crossing the petite progeny. The petite mutants and zygotes cannot do mitochondria) protein synthesis. These results support the recombinational model of polarity.
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  • 9
    ISSN: 1432-0983
    Keywords: Ribosomal RNA ; mitochondrial DNA ; physical map ; direction of transcription
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary 1. We have constructed a physical map of the yeast mtDNA region coding for the small RNA component of the mitochondrial ribosome. The map includes restriction sites for HindII, BamHI, MboI, MboII, HapII, HaeIII and Sau-961. 2. We have cloned Mbol mtDNA fragments containing parts of the 15S ribosomal gene in the BamHI site of the plasmid pBR325, using standard recombinant DNA techniques. With the exception of one border fragment, more than 5,800 base pairs long, all four MboI DNA fragments were cloned. 3. We have digested DNA fragments, containing part of the 15S ribosomal gene at one end only, with the 3′-specific exonuclease III of Escherichia coli and the 5′-specific exonuclease of bacteriophage lambda in opposite directions with respect to the physical map. From the hybridization of 32P-labelled 15S RNA to the single-stranded ends of these DNA fragments we conclude that transcription of this gene runs clock-wise on the map (i.e. from oxi-2 towards oxi-3). 4. We have studied the position of the 15S ribosomal gene on the physical map with the S1 nuclease mapping procedure of Berk and Sharp (Cell 12 (1977) 721–732). It is asymmetrically localized on a 2,000 base pair HapII fragment and the length of the protected hybrids adds to approximately 1,600 base pairs. 5. We have found no evidence for an intervening sequence in the 15S ribosomal gene, since S1 nucleasetreated hybrids, analysed by electrophoresis in alkaline gels, showed no indication for interruptions in the DNA strand of the hybrid.
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  • 10
    ISSN: 1432-0983
    Keywords: meiotic and mitotic recombination ; general and specific marker effects
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Meiotic and mitotic fine-structure maps of two efficient UGA suppressors of Schizosaccharomyces pombe which are known (sup3-e) or inferred (sup9-e) to code for two serine tRNAs carrying the mutant anticodon U*CA (Kohli et al. 1979a, b, Rafalski et al. 1979) are presented. Maps based on spontaneous meiotic, spontaneous mitotic and MMS induced mitotic recombination between the primary site of the anticodon mutation and a number of inactivating second-site mutations are similar. Specific marker effects, which drastically increase the frequency of spontaneous meiotic and mitotic recombination in crosses involving one or the other of four exceptional sites (including the anticodon sites of both sup3-e and sup9-e), disappear when mapping is based on MMS induced mitotic recombination. The meiotic marker effect characterizing the anticodon site of one of the two efficient UGA suppressors (sup3-e) also disappears upon further mutation to an inefficient UAA suppressor allele (sup3-i), as shown by its absence in a fine-structure map based on meiotic recombination between the anticodon mutation of this ochre suppressor allele and a new set of inactivating second-site mutations derived from it.
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