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F Kaudewitz

Publications and source records attributed to F Kaudewitz.

At least 19 recordsLinked to original sources
PubMed1987

A mitochondrial frameshift-suppressor (+1) [corrected] of the yeast S. cerevisiae maps in the mitochondrial 15S rRNA locus.

The first case of a +1 "extrageneic" frameshift suppressor (MF1), mapping in the yeast mitochondrial 15S rRNA gene is reported. The suppressor was identified by genetic analyses in a leaky mitochondrial oxil frameshift mutant and the respective wild-type strain 777-3A of the yeast S. cerevisiae. This is in accordance with the finding that all mitochondrial frameshift mutants isolated from this strain tend to be leaky to a variable degree. MF1 does not suppress known nonsense mutations created by a direct basepair exchange in strain 777-3A. These mutants exhibit a non-leaky phenotype (Weiss-Brummer et al. 1984).

Chromosome Deletion↗
PubMed1986

Molecular cloning of the yeast nuclear genes CBS1 and CBS2.

The yeast nuclear genes CBS1 and CBS2 are both required for translation of the mitochondrial COB transcripts. Here we report on the identification of two unique chromosomal DNA-sequences of 2 kb and 2.3 kb from yeast wild type gene banks which functionally complement cbs1 and cbs2 mutants, respectively. Disruption of the homologous DNA-fragments by insertion of the URA3 gene generates respiratory deficient cells which fail to complement the original mutants. Cells with these gene disruptions are phenotypically identical to the original cbs1 and cbs2 mutants with respect to cytochrome spectra and mitochondrial translation products. The results exclude the possibility that suppressor genes have been cloned and confirm the conclusion that both genes, CBS1 and CBS2, specifically are involved in translation of mitochondrial COB RNA.

Cell Nucleus↗
PubMed1985

Mitochondrial suppression of a yeast nuclear mutation which affects the translation of the mitochondrial apocytochrome b transcript.

We describe a mitochondrial suppressor mutation, which restores respiratory competence to the nuclear pet- -mutant MK2. This mutant lacks the message of the mitochondrial cob-gene and instead accumulates a partially spliced pre-mRNA which is not translated. Complete processing and translation of the cob-RNA is restored by a rearrangement of the mitochondrial DNA, leading to a fusion of the cob-coding sequences with the leader of oli1, the mitochondrial gene coding for subunit IX of the ATPase. We conclude that the nuclear gene affected in MK2 is essential to allow translation of transcripts which contain the cob-leader sequence.

Apoproteins↗
PubMed1984

Sequencing of the nuclear gene for the yeast cytochrome c1 precursor reveals an unusually complex amino-terminal presequence.

Cytochrome c1 is a component of the mitochondrial respiratory chain in most eukaryotes. The protein is coded by nuclear DNA, synthesized as a larger precursor outside the mitochondria and then cleaved to the mature form in two successive steps during its import into the mitochondria. We have cloned the structural gene for yeast cytochrome c1 by functional complementation of a cytochrome c1-deficient yeast mutant with a yeast genomic library in the yeast-Escherichia coli 'shuttle' vector YEp 13. The complete nucleotide sequence of the gene and of its 5'- and 3'-flanking regions was determined. The deduced amino acid sequence of the yeast cytochrome c1 precursor reveals an unusually long transient amino-terminal presequence of 61 amino acids. This presequence consists of a strongly basic amino-terminal region of 35 amino acids, a central region of 19 uncharged amino acids and an acidic carboxy-terminal region of seven amino acids. This tripartite structure of the presequence resembles that of the precursor of cytochrome c peroxidase and supports a previous suggestion on the import pathways of these two precursors.

Amino Acid Sequence↗
PubMed1984

Leakiness of termination codons in mitochondrial mutants of the yeast Saccharomyces cerevisiae.

Seven mutants in exon 1 of the mitochondrial cob gene in yeast are described with respect to their translation products, RNA pattern, and deoxyribonucleotide sequence alteration(s). Sequence analysis of the mutations, which previously were shown to cause premature termination of apocytochrome b, revealed that two of them directly transform sense codons to chain-termination codons, whereas the other four are frame-shift mutations (+1/-1, insertions/deletions). Only the latter mutants are found to be leaky in that (a) RNA splicing occurs, and (b) in three of them, to a minor degree an apocytochrome b homologue is synthesized, which, however, does not lead to respiratory competence. Both require translation through exon 1 into downstream introns to produce 'RNA maturases' necessary for splicing the primary transcript (Lazowska et al. 1980; Weiss-Brummer et al. 1982). These and other previously published data show that mitochondrial frame-shift mutants tend to be leaky to a variable degree. Several possible mechanisms of 'frame-shift suppression' are discussed.

Base Sequence↗
PubMed1983

Expression of the "split gene" cob in yeast mtDNA. Nuclear mutations specifically block the excision of different introns from its primary transcript.

Five nuclear mutants falling into five different complementation groups are shown to block the maturation of long form mitochondrial cob RNA at five different processing steps. At the same time they prevent complete processing of the oxi 3 RNA, thus exhibiting the same phenotype as mitochondrial box mutants (cyt b- and oxi 3-). The different nuclear factors in question have varying ranges of specificity for the removal of introns from cob RNA, from only one to at the most three introns. Two mutated nuclear elements are shown to be specific for the processing of introns present only in the long form cob gene. One such mutation shows, as expected, no deleterious effect on the processing of the short form cob RNA exchanged into the mutant via cytoduction. The role of nuclear coded factors in the possible translation or activity of introncoded products ("maturases") is discussed for two mutants. Striking parallels are found between diverse polypeptide products, presumably translated from accumulated cob RNA intermediates, in pet- and mit- mutants blocked in the excision of the same intron.

Base Sequence↗
PubMed1983

Processing of yeast mitochondrial RNA: involvement of intramolecular hybrids in splicing of cob intron 4 RNA by mutation and reversion.

Revertants have been obtained from six mutants of the box9 cluster, which are supposed to be defective in RNA splicing as a result of alterations in a splice signal sequence. This sequence is in the 5' part of intron 4 of the cob gene, 330 to 340 bp downstream from the 5' splice site. Sequencing reveals that reversion to splicing competence is achieved by restoration of the wild-type box9 sequence; by creation of novel box9 sequences; and by introduction of a second site or suppressor mutation (sup-) compensating for the effect of the primary box9- mutation. The sup- mutation alters a sequence in intron 4,293 bp upstream from the box9- primary mutation. The box9 sequence and this upstream sequence can base pair to form an intramolecular hybrid in intron RNA in which box9- and sup- are compensatory base pair exchanges (G----A and C----U, respectively). Thus intramolecular hybrid structures of intron RNA are essential for RNA splicing.

Base Sequence↗
PubMed1982

Expression of the split gene cob in yeast: evidence for a precursor of a "maturase" protein translated from intron 4 and preceding exons.

Intron 4 (14) of the split gene cob in mitochondrial DNA contains a long open reading frame in phase with the preceding exon. Mutations in this intron block the excision of the 14 sequence from the cob precursor RNA and, at the same time, generate a series of new polypeptides, parts of which apparently result from translation of 14 sequences. We sequenced six mutations clustered in the upstream part of the open reading frame, about 340 bp from the exon-intron boundary (box9 cluster). Four are base pair exchanges in the same triplet of this region; these form the polypeptides typical for 14 plus a trans-acting product encoded by 14, as shown by complementation studies. The other two mutations--a -2 bp deletion at the same site, causing frameshift with a chain-terminating codon within a few triplets, and a base pair exchange at a nearby site--affect both the formation of 14 typical translation products and the trans-acting function. These results on box9 mutants combined with results on box7 mutants suggest that an 14-encoded "maturase" protein (apparent molecular weight, 27,000) is cleaved off a precursor protein (apparent molecular weight, 55,000) encoded by exon sequences B1 to B4 and the intron open reading frame. We further discuss the role of the box9 nucleotide sequence in the maturation of cob-specific RNA.

Base Sequence↗
PubMed1981

Pathways of transcript splicing in yeast mitochondria. Mutations in intervening sequences of the split gene COB reveal a requirement for intervening sequence-encoded products.

We have studied the transcript processing of the split gene COB (or BOX) in yeast mtDNA, in both wild type and cob- mutants. Using various DNA fragments specific for coding or intervening sequences of this gene, we have determined the composition of splicing intermediates by DNA/RNA hybridization. The pattern of splicing intermediates detected in wild type reveals differing rates of the five splicings resulting in an apparent pathway of processing rather than an absolute order among the five cut and splice events. Effects of mutations in four of the five sequences have been studied. All of them interfere with transcript processing. Some block the excision of the sequence mutated only, but allow other splicing events to occur essentially as in the wild type. They suggest that in these mutants any order of splicings is possible, but that some are preferred. In contrast, other mutations located in four different sequences block several splicings simultaneously and thus suggest the existence of an obligatory order of events. In order to reconcile these findings we discuss the following hypotheses. (i) Some intervening sequences in COB specify products which are involved in transcript splicing; (ii) the biosynthesis of trace amounts of these products occurs on splicing intermediates. Their formation requires a certain order of splicing events to occur on a small number of COB transcripts. (iii) If expressed and functional, the intervening sequence-encoded products, together with other components, act on the bulk of COB transcripts, resulting in the steady state pattern of splicing intermediates observed in wild type.

Base Sequence↗
PubMed1981

Expression of the "split gene" COB in yeast mtDNA. Translation of intervening sequences in mutant strains.

This study deals with the effects that mutations in the COB region of yeast mtDNA have on the expression of mitochondrially made polypeptides. Based on the detection of two series of polypeptide chain-terminating mutations, we conclude that two proteins are specified by this region, apocytochrome b (Mr = 30,000) and a polypeptide of Mr = 42,000. One series of mutations generates new polypeptides ranging in size from 8,000 to 29,000 daltons; all of them are precipitated by serum direct against apocytochrome b. These mutations are located in five distinct segments of the COB region, the sequences alpha to epsilon coding for apocytochrome b. The second series of mutations, generating new polypeptides ranging in size from 17,000 to 41,000 daltons, is located within the first intervening sequence (alpha/beta) of the split gene for apocytochrome b. These mutations cause premature chain termination in the COOH-terminal part of a 42,000-dalton polypeptide. Its NH2-terminal part is likely to be specified by sequence alpha and thus to be homologous to that of apocytochrome b. We conclude that the 42,000-dalton polypeptide is translated on a processing intermediate of th COB transcript by reading through sequence alpha into sequence alpha/beta. We discuss the hypothesis that this polypeptide has a function in the expression of the COB region, possibly at the level of transcript processing.

DNA, Mitochondrial↗
PubMed1979

The mitochondrial COB region in yeast codes for apocytochrome b and is mosaic.

Mitochondrial mutants of Saccharomyces cerevisiae defective in cytochrome b were analyzed genetically and biochemically in order to elucidate the role of the mitochondrial genetic system in the biosynthesis of this cytochrome. The mutants mapped between OLI1 and OLI2 on mitochondrial DNA in a region called COB. A fine structure map of the COB region was constructed by rho- deletion mapping and recombination analysis. The combined genetic and biochemical data indicate that the COB region is mosaic and contains at least five distinct clusters of mutants, A-E, with A being closest to OLI2 and E being closest to OLI1. Clusters A, C and E are probably coding regions for apocytochrome b, whereas clusters B and D seem to be involved in as yet unknown functions. These conclusions rest on the following evidence. 1. Most mutants in clusters A, C and E have specifically lost cytochrome b. Many of them accumulate smaller mitochondrial translation products; some of these were identified as fragments of apocytochrome b by proteolytic fingerprinting. The molecular weight of these fragments depends on the map position of the mutant, increasing in the direction OLI2 leads to OLI1. The mutant closest to OLI1 accumulates an apocytochrome b which is slightly larger than that of wild type. 2. A mutant in cluster C exhibits a spectral absorption band of cytochrome b that is shifted 1.5 nm to the red. 3. Mutants in clusters B and D are pleiotropic. A majority of them are conditional and lack the absorption bands of both cytochrome b and cytochrome aa3; these mutants also fail to accumulate apocytochrome b and subunit I of cytochrome c oxidase and instead form a large number of abnormal translation products whose nature is unknown. 4. Zygotic complementation tests reveal at least two complementation groups: The first group includes all mutants in cluster B and the second group includes mutants in clusters (A + C + D + E).

Apoproteins↗
PubMed1979

The identification of apocytochrome b as a mitochondrial gene product and immunological evidence for altered apocytochrome b in yeast strains having mutations in the COB region of mitochondrial DNA.

The yeast mitochondrial translation product of Mr 30 000 is identical with apocytochrome b. After labelling in vivo with [35S]sulphate in the presence of cycloheximide, the radioactivity in this product present in solubilized submitochondrial particles, was completely recovered in pure cytochrome bc1 complex as a single polypeptide. We show that this translation product is identical with apocytochrome b using peptide mapping by limited proteolysis according to Cleveland et al. [J. Biol. Chem. 250 (1977) 8236-8242] and by immunoprecipitation with a specific antiserum against apocytochrome b. New mitochondrial translation products in 36 strains of Saccharomyces cerevisiae having mutations in the COB region of the mitochondrial DNA, are precipitated by this antiserum. This is consistent with the assumption that many of the cob mutations are localized in the structural gene for apolcytochrome b on mitochondrial DNA. Mutations in two intervening sequences can give rise to products related to apocytochrome b that are considerably longer than normal apocytochrome b. We discuss the hypothesis that in these mutants splicing of the messenger RNA does not occur correctly and that, as a consequence of this, ribosomes read through in an intervening sequence.

Apoproteins↗
PubMed1978

Extrachromosomal inheritance in Schizosaccharomyces pombe. VII. Studies by zygote clone analysis on transmission, segregation, recombination, and uniparental inheritance of mitochondrial markers conferring resistance to antimycin, chloramphenicol, and erythromycin.

Crosses involving mitochondrial markers conferring resistance to antimycin (anar, AR), chloramphenicol (capr, CR), and erythromycin (eryr, ER) in cis- and trans-configuration were studied by zygote clone analysis. Mutant anar-8, from which all other drug--resistant isolates were derived, exhibits a highly biased transmission (6.8% anar) in an analysis of 100 individual zygote clones. Important results of zygote clone analyses were:--Zygote clones may contain one, two, three, or four mitochondrial genotypes.--The proportion of the two parental and the two recombinant genotypes in individual zygote clones can vary almost over the entire range of percentages.--Proportions of the two corresponding recombinant types in individual clones are usually unequal.--Transmission rates of markers are higher in trans- than in cis-crosses, indicating additivity of bias by two mutated alleles in coupling.--Transmission rates are different for the three markers both in cis- and trans-crosses, being lowest for CR and highest for ER.--Up to more than 80% uniform clones, expressing only one genotype, can be produced in cis- and trans-crosses. In cis-crosses always the double-sensitive parental type becomes uniform, in trans-crosses this may be the case for parental and/or recombinant genotypes. A tentative map is presented using data from cis- and trans-crosses, including a correction by omission of uniform clones. Phenomena of transmission, segregation, and formation of uniform clones are discussed with special regard to the difference brought about by fission versus budding. A comparison with relevant data from Saccharomyces cerevisiae and other organisms is presented.

Anti-Bacterial Agents↗
PubMed1978

On the formation of rho - petites in yeast. III. Effects of temperature on transmission and recombination of mitochondrial markers and on rho - cell formation in temperature sensitive mutants of Saccharomyces cerevisiae.

The rho-factor stability is shown to be affected by four conditional mutations, tsm-8 (mitochondrial), tsp-20, tsp-25 and tsp-30 (nuclear). Growth of mutant cells at high temperature (35 degrees C) results in the rapid production of rho - cells and concomittantly in the decrease of the ability to transmit mitochondrial genetic information to the rho + progeny of crosses. Kinetics of rho - cell formation during growth at 35 degrees C have been compared with variations in transmission and recombination of mitochondrial markers in crosses. In all cases the transmission of mitochondrial markers of the ts-parent decreases as the number of cell generations increases. The frequencies of recombinants between mitochondrial markers either increase or decrease depending on the markers considered and the alleles of the omega-locus involved in the crosses. The results of all crosses performed have been compared with the predictions of the model for recombination and segregation of mitochondrial genes proposed by Dujon et al. (1974). This comparison indicates that the main result of high temperature treatment is a diminution of the input of mitochondrial information from the ts-parent into zygotes. Consequences of the induced variations of input follow the predictions of the model. The correlation found in ts-strains between the reduction of input in crosses and the formation of rho - cells is discussed in terms of molecular events occurring in mitDNA molecules during high temperature induction of rho + to rho - mutation.

Crosses, Genetic↗
PubMed1977

Extrachromosomal inheritance in Schizosaccharomyces pombe. IV. Isolation and genetic characterization of mutants resistant to chloramphenicol and erythromycin using the mutator properties of mutant anar-8.

Spontaneous chloramphenicol (capr)- and erythromycin (eryr)-resistant mutants were isolated from strain ade7-50 h- and the antimycin-resistant mutant anar-8 ade 7-50 h- of Schizosaccharomyces pombe (Sch. p.). By mitotic segregation analysis all 154 capr- and 120 eryr-mutants derived from ade 7-50 h- proved to be recessive chromosomal, whereas all 108 capr- and 200 eryr-mutants originating from anar-8 were extrachromosomally inherited. The rate of spontaneous capr- and eryr-mutants was about hundredfold in anar-8 compared to ade 7-50 h-. Growth of capr- and eryr-mutants was not inhibited by chloramphenicol or erythromycin, respectively, in glucose-medium and only slightly in glycerol-medium at concentrations which completely inhibited anar-8. By mitotic segregation-, tetrad-, and mitotic haploidization-analysis the extrachromosomal inheritance of mutants derived from anar-8 was established. Segregational patterns of capr- and eryr-determinants during mitosis, meiosis, and mitotic haploidization of diploids are discussed.

Ascomycota↗