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G Michaelis

Publications and source records attributed to G Michaelis.

At least 55 records · Page 3Linked to original sources

Cytochrome b, the var 1 protein, and subunits I and III of cytochrome c oxidase are synthesized without transient presequences in Saccharomyces cerevisiae.

The N-termini of four mitochondrial translation products, the var 1 protein, cytochrome b, and subunits I and III of cytochrome c oxidase have been characterized in Saccharomyces cerevisiae and compared with the known DNA sequences of the respective structural genes. The four mature proteins correspond to the predicted primary translation products and retain the formylated methionine residue. Thus, subunit II of cytochrome c oxidase studied previously [Pratje et al. (1983) EMBO J.2, 1049-1054] is so far the only mitochondrial translation product carrying a N-terminal-extended transient presequence in S. cerevisiae.

Amino Acid Sequence↗

A nuclear mutation prevents processing of a mitochondrially encoded membrane protein in Saccharomyces cerevisiae.

Subunit II of cytochrome oxidase is encoded by the mitochondrial OXI1 gene in Saccharomyces cerevisiae. The temperature-sensitive nuclear pet mutant ts2858 has an apparent higher mol. wt. subunit II when analyzed on lithium dodecylsulfate (LiDS) polyacrylamide gels. However, on LiDS-6M urea gels the apparent mol. wt. of the wild-type protein exceeds that of the mutant. Partial revertants of mutant ts2858 that produce both the wild-type and mutant form of subunit II were isolated. The two forms of subunit II differ at the N-terminal part of the molecule as shown by constructing and analyzing nuclear ts2858 and mitochondrial chain termination double mutants. The presence of the primary translation product in the mutant and of the processed form in the wild-type lacking 15 amino-terminal residues was demonstrated by radiolabel protein sequencing. Comparison of the known DNA sequence with the partial protein sequence obtained reveals that six of the 15 residues are hydrophilic and, unlike most signal sequences, this transient sequence does not contain extended hydrophobic parts. The nuclear mutation ts2858 preventing post-translational processing of cytochrome oxidase subunit II lies either in the gene for a protease or an enzyme regulating a protease.

Cell Nucleus↗

Mitochondrial and nuclear myxothiazol resistance in Saccharomyces cerevisiae.

Mitochondrial and nuclear mutants resistant to myxothiazol were isolated and characterized. The mitochondrial mutants could be assigned to two loci, myx1 and myx2, by allelism tests. The two loci map in the box region, the split gene coding for apocytochrome b. Locus myx1 maps in the first exon (box4/5) whereas myx2 maps in the last exon (box6). The nuclear mutants could be divided into three groups: two groups of recessive mutations and one of dominant mutations. Respiration of isolated mitochondria from mitochondrial mutants is resistant to myxothiazol. These studies support the conclusion that myxothiazol is an inhibitor of the respiratory chain of yeast mitochondria. The site of action of myxothiazol is mitochondrial cytochrome b.

Antifungal Agents↗

Sporulation of mitochondrial respiratory deficient mit- mutants of Saccharomyces cerevisiae.

The role of mitochondrial protein synthesis, electron transport, and four specific mitochondrial gene products on sporulation were studied in respiratory deficient mit- mutants. These mutants were isolated in an op 1 strain and localized on the mitochondrial genome by petite deletion mapping. All 153 mutations studied could be assigned to the four mitochondrial regions OXI1, OXI2, OXI3 and COB, known to affect cytochrome c oxidase and cytochrome b. The specific loss of one mitochondrially translated polypeptide was found in some mutants of each locus: OXI1--cytochrome c oxidase subunit 2, OXI2--subunit 3, OXI3--subunit 1, and COB--cytochrome b. The ability of diploid mit- mutants to sporulate was systematically investigated. About one third of the mutants, representing three loci, were incapable of forming spores. All other cultures produced either respiratory competent mit+ tetrads, both mit+ and mit- tetrads, or only mit- tetrads. Mutants forming mit- tetrads mapped in all four loci. These results demonstrate that in contrast to petite mutants some mit- mutants have retained the ability to perform meiosis and sporulation.

DNA, Mitochondrial↗

Mapping of the two mitochondrial antimycin A resistance loci in Saccharomyces cerevisiae.

Retention or loss of the two new mitochondrial antimycin A resistance loci AI and AII has been analyzed in a large number of stable cytoplasmic petite mutants. Using these deletion mutants it was possible to localize the two antimycin A resistance loci in the OI--OII region of mitochondrial DNA. The genetic loci are mapped in the following order: OII--AI--AII--cobl--OI. The mapping relationship of mutants resistant to antimycin A or funiculosin to various cob mutants is described.

Antimycin A↗

Recombined molecules of mitochondrial DNA obtained from crosses between cytoplasmic petite mutants of Saccharomyces cerevisiae: the stoichiometry of parental DNA repeats within the recombined molecule.

We have studied recombination between repetitive mitochondrial DNAs from cytoplasmic petite mutants of Saccharomyces cerevisiae. Mitochondrial DNA was isolated from two parental p- mutants, carrying respectively the CR and the ER mitochondrial genetic markers, and from two p- CRER diploid genetic recombinants. These two recombinants, obtained from the same parental petites, differ in their degrees of suppressiveness. The p- mitochondrial DNAs were analyzed by DNA-DNA hybridization, high resolution melting and reassociation kinetics. It was found that the repeating unit of the CR parental p- DNA is 3 to 4 times longer than that of the ER parent. There is very little sequence homology between these two p- mitochondrial DNAs and almost all parental sequences are integrated into the recombined molecules. Mitochondrial DNA from both types of recombinants seems to contain the two parental repeating units in the ratio 1:1.

Base Sequence↗

Cytoplasmic inheritance of antimycin A resistance in Saccharomyces cerevisiae.

Three antimycin resistant mutants of Saccharomyces cerevisiae are characterized genetically. The mutations have been shown to be cytoplasmically inherited by four criteria. The phenotype persists in diploids formed by a cross with a pO strain of yeast of the opposite mating type. Diploids heterozygous for the antimycin marker, however, show segregation of the resistance and sensitivity during mitosis. Tetrad analysis indicates a non-Mendelian segregation (4:0 and 0:4) of the mutations. The antimycin marker can be eliminated by ethidium bromide treatment under conditions that should have deleted all of the mitochondrial DNA.

Antimycin A↗

Genetic analysis of mitochondrial biogenesis and function in Saccharomyces cerevisiae.

Different mitochondrial mutants have been isolated that affect mitochondrial ribosome function. These mutants were used to establish most of the known methods and principles of mitochondrial genetics in yeast. Another class of mitochondrial mutants have been shown to affect mitochondrial ATPase and, more specifically, the "membrane factor" of mitochondrial ATPase. These mutants might be very useful in studying the energy-conserving function, and the interaction between the hydrophobic and hydrophylic parts, of the ATPase complex. New types of mitochondrial point mutations, concerning cytochrome a-a3 or b, will soon open up new fields of investigation. The biochemical and genetic analysis of numerous mutants belonging to that category and recently obtained [31] is being currently pursued in Tzagoloff's and Slonimski's laboratories.

Adenosine Triphosphatases↗

Lipid-lipid and lipid-protein interactions as studied with a novel type of fluorescent fatty acid and phospholipid probes.

A novel fluorescent-labelled group of fatty acids and phospholipids has been applied to determine phase transitions in liposomes by fluorescence intensity and polarisation measurements. The chromophore of these amphiphilic lipids proved to be very suitable to demonstrate temperature-dependent lipid-lipid interactions. Liposomes from 1,2-dipalmitoyl-3-sn-glycero-phosphoethanolamine and from lipids isolated from membranes of E. coli K 1062 mutant grown on elaidic acid were used in these studies. These probes also made it possible to observe conformational changes in membrane proteins in isolated plasma membranes from this mutant. The changes in protein conformation were dependent on structural changes in the lipid phase.

Binding Sites↗

Chemical syntheses of novel fluorescent-labelled fatty acids, phosphatidylcholines and cholesterol esters.

The synthesis of a novel class of fluorescent-labelled fatty acids of different chain lengths and unsaturation, phospholipids and cholesterol esters has been developed. The following omega-anthracene-labelled cis-unsaturated fatty acids have been synthesized: omega-(9-anthryl)-6c-octenoic, -7c-nonenoic, -10c-dodecenoic, -6c,9c-undecadienoic, -10c,13c-pentadecadienoic acid. They have been introduced into the 2-position of 1-stearoyl- and 1-linoleoyl-3-sn-glycerophosphocholine and cholesterol. Mass spectroscopy, 1H-NMR, IR and fluorescence spectroscopy and different chromatographic procedures have been applied to confirm and characterize their structures. The properties of the different fluorescent-labelled phosphatidylcholines in monomolecular films have been determined by the Langmuir technique.

Cholesterol↗

Biosynthetic labelling of membrane lipids of eukaryotic cells in tissue culture by a novel type of fluorescent fatty acids.

W-Anthryl labelled fatty acids with hydrocarbon chains of different lengths (C8, C11, C15) and different degrees of unsaturation have been incorporated into the membrane lipids of three different cell lines in tissue culture by addition of these 3H-labelled precursor fatty acids to the growth medium. The cell lines were baby hamster kidney cells (BHK 21), Chang liver cells and the RN6 cell line derived from a chemically induced Schwannoma tumor cell clone. Cell growth was normal. The quantitative analysis on the basis of radioactivity determinations demonstrated that the fluorescent-labelled fatty acids were introduced into the neutral lipid fraction (triglycerides, diglycerides, and cholesterol esters, all present in small amounts), but mainly into the phospholipid classes phosphatidylcholine, -ethanolamine and -serine, and to a lesser extent, as N-acyl component of sphingolipids (sphingomyelins, ceramides, mono- and diglycosylceramides). Cell fractionation studies indicated that the membranes of all subcellular particles were labelled with the fluorescent probes in their lipid moieties. These w-anthryl fatty acids are the first type of fluorescent lipid precursors which can be incorporated biosynthetically in vivo into membrane lipids of eukaryotic cells. The effective incorporation of the bulky fluorescent anthryl group in the terminal position of fatty acids of different chain lengths into the complex membrane lipids of the cell gives proff of 1) their uninhibited membrane transport, 2) their activation by the acyl-CoA synthetase and 3) their substrate properties for the O- acyl and N-acyl transferases in phospho- and sphingolipid biosynthesis.

Animals↗

DNA-dependent RNA polymerase from yeast mitochondria.

The DNA-dependent RNA polymerase present in the mitochondria of Saccharomyces cerevisiae has been solubilized using a 0.5 M KCI solution, and the soluble enzyme has been purified. Two forms of mitochondrial enzyme were obtained; they differ in their template specificity and metal-ion dependency. The mitochondrial RNA polymerase also differs from enzyme obtained from the nucleus with respect to antibiotic sensitivity and template specificity. Only the nuclear enzyme is sensitive to alpha-amanitin inhibition. The relative activities of the isolated nuclear and mitochondrial polymerases toward their homologous DNAs are consistent with their in vivo functions. The possibilities of bacterial- and nuclear-enzyme contamination of the mitochondrial enzyme preparation have been ruled out. RNA polymerase activity in two petite mutants has been studied. Isolated mitochondria from a petite mutant with no detectable mitochondrial DNA has greatly diminished mitochondrial DNA-dependent RNA polymerase activity, while a petite mutant with only a small change in mitochondrial DNA base composition has normal amounts of enzyme.

Anti-Bacterial Agents↗