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M Rabinowitz

Publications and source records attributed to M Rabinowitz.

At least 109 records · Page 6Linked to original sources
PubMed1978

Physical mapping of genes on yeast mitochondrial DNA: localization of antibiotic resistance loci, and rRNA and tRNA genes.

We have physically mapped the loci conferring resistance to antibiotics that inhibit mitochondrial protein synthesis (erythromycin, chloramphenicol and paromomycin) or respiration (oligomycin I and II), as well as the 21s and 14s rRNA and tRNA genes on the restriction map of the mitochondrial genome of the yeast Saccharomyces cerevisiae. The mitochondrial genes were localized by hybridization of labeled RNA probes to restriction fragments of grande (strain MH41-7B) mitochondrial DNA (mtDNA) generated by endonucleases EcoRI, HpaI, BamHI, HindIII, SalI, PstI and HhaI. We have derived the HhaI restriction fragment map of MH41-7B mit DNA, to be added to our previously reported maps for the six other endonucleases. The antibiotic resistance loci (antR) were mapped by hybridization of 3H-cRNA transcribed from single marker petite mtDNA's of low kinetic complexity to grande restriction fragments. We have chosen the single Sal I site as the origin of the circular physical map and have positioned the antibiotic loci as follows: C (99.5-1.Ou)--P (27-36.Ou)--OII (58.3-62u--OI (80-84u)--E (94.4-98.4u). The 21s rRNA is localized at 94.4-99.2u, and the 14s rRNA is positioned between 36.2-39.8u. The two rRNA species are separated by 36% of the genome. Total mitochondrial tRNA labeled with 125I hybridized primarily to two regions of the genome, at 99.5-11.5u and 34-44u. A third region of hybridization was occasionally detected at 70--76u, which probably corresponds to seryl and glutamyl tRNA genes, previously located to this region by petite deletion mapping.

Chloramphenicol↗
PubMed1978

Restriction enzyme analysis of mitochondrial DNAs of petite mutants of yeast: classification of petites, and deletion mapping of mitochondrial genes.

We have analyzed the restriction digest patterns of the mitochondrial DNA from 41 cytoplasmic petite strains of Saccharomyces cerevisiae, that have been extensively characterized with respect to genetic markers. Each mitochondrial DNA was digested with seven restriction endonucleases (EcoRI, HPaI, HindIII, BamHI, HhaI, SalI, and PstI) which together make 41 cuts in grande mitochondrial DNA and for which we have derived fragment maps. The petite mitochondrial DNAs were also analyzed with HpaII, HaeIII, and AluI, each of which makes more than 80 cleavages in grande mitochondrial DNA. On the basis of the restriction patterns observed (i.e., only one fragment migrating differently from grande for a single deletion, and more than one for multiple deletions) and by comparing petite and grande mitochondrial DNA restriction maps, the petite clones could be classified into two main groups: (1) petites representing a single deletion of grande mitochondrial DNA and (2) petites containing multiple deletions of the grande mitochondrial DNA resulting in rearranged sequences. Single deletion petites may retain a large portion of the grande mitochondrial genome or may be of low kinetic cimplexity. Many petites which are scored as single continuous deletions by genetic criteria were later demonstrated to be internally deleted by restriction endonuclease analysis. Heterogeneous sequences, manifested by the presence of sub-stoichiometric amounts of some restriction fragments, may accompany the single or multiple deletions. Single deletions with heterogeneous sequences remain useful for mapping if the low concentration sequences represent a subset of the stoichiometric bands. Using a group of petites which retain single continuous regions of the grande mitochondrial DNA, we have physically mapped antibiotic resistance and mit- markers to regions of the grande restriction map as follows: C (99.3--1.4 map units)--OXI-1 (2.5--15.7)--OXI-2 (18.5--25)--P (28.1--34.2)--OXI-3 (32.2--61.2--OII (60--62)--COB (64.6--80.8--0I (80.4--85.7)--E (95--98.9).

DNA Restriction Enzymes↗
PubMed1978

Biochemical and stereological analysis of rat liver mitochondria in different thyroid states.

The concentrations of the inner mitochondrial membrane markers cardiolipin and cytochrome alpha have been measured in liver homogenates and in purified mitochondria after thyroxine administration to thyroidectomized and normal rats. The biochemical results have been correlated with stereological electron micrographic analyses of hepatocytes in liver sections, and of isolated mitochondrial pellets. There were progressive and parallel increases in homogenate and mitochondrial cardiolipin concentration, and in mitochondrial cytochrome alpha concentration, after administration of 20 microgram of thyroxine on alternate days to thyroidectomized rats, and of 300 microgram on alternate days to normal rats. Electron microscope measurements showed marked differences in the shape of the mitochondria and in the number of cristae in different thyroid states. Hypothyroid mitochondria were shorter and wider than controls, and hyperthyroid mitochondria longer but of similar width. Mitochondrial volume per unit cell volume was virtually unchanged in hypo- and hyperthyroid animals. The most striking changes were a decrease in the area of the inner membrane plus cristae in thyroidectomized rats, and a substantial increase in membrane area after thyroxine administration. The biochemical and electron micrographic results indicate that, in rat liver, thyroid hormone administration leads to a selective increase in the relative amount of mitochondrial inner membranes, with little or no change in the mitochondrial volume per unit cell volume, or in total mitochondrial protein per unit total cell protein.

Animals↗
PubMed1977

Yeast mitochondrial DNA specifies tRNA for 19 amino acids. Deletion mapping of the tRNA genes.

We have previously identified 14 aminoacyl tRNAs that are specified by yeast mitochondrial DNA (mtDNA). We now report four more amino acids (Arg, Cys, Trp, Thr) that acylate tRNAs which hybridize with mtDNA. Furthermore one of the two mitochondrial tRNAs that we had earlier demonstrated to be directly charged with glutamic acid responds to glutamine but not to glutamic acid codons. Thus Gln-tRNAGln appears to be formed by transamidation of a missense intermediate Glu-tRNAGln. This brings to 19 the number of amino acids which have corresponding tRNAs specified by mtDNA. Only tRNAAsn has not yet been shown to be a mtDNA transcript. We have also mapped the genes for the newly identified mitochondrial tRNAs, as well as several others that were previously identified but unmapped, by hybridization to the mtDNA of a series of petite deletion mutants. We now have ordered 20 mitochondrial tRNA genes (including two methionyl-tRNAs) wtih respect to the antibiotic resistance markers chloramphenicol (CR), erythromycin (ER), paromomycin (PR), and oligomycin I and II (ORI, ORII). Eighteen tRNA genes map between the C and E resistance markers. Only the serinyl-tRNA and glutamyl-tRNA genes are localized near the OI and OII resistance markers.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Measurements of half-life of rat cardiac myosin heavy chain with leucyl-tRNA used as precursor pool.

The kinetics of labeling of myosin heavy chain, following a single intravenous injection of L-[4,5-3H]leucine, were analyzed with the help of a computer, in conjunction with the labeling kinetics of the specific radioactivities of the precursor amino acid pool. As precursor we used leucyl-tRNA which, as we show here, differs significantly from the intracellular free leucine pool. The half-life of myosin heavy chain was determined from the initial period (0 to 60 min) of incorporation of label into protein after a single injection of tritiated leucine, and also from the period (7 to 14 days) when there is exponential decay of the labeled protein. Myosin heavy chain was separated from other myofibrillar proteins by polyacrylamide gel electrophoresis before measurement of leucine specific radioactivity. The specific radioactivity was measured in both protein and precursor pools by a sensitive isotope dilution procedure (range, 100 to 1500 pmol). The values for the half-life of myosin heavy chain determined at both intervals were similar (5.4 and 5.9 days). Substitution of the specific radioactivity of the intracellular free leucine pool decreased the half-life to 2.7 dyas. Similar values were obtained when the half-life was calculated by simple graphical integration of the experimental curves.

Amino Acids↗
PubMed1977

Restriction cleavage map of mitochonrial DNA from the yeast Saccharomyces cerevisiae.

Mitochondrial DNA (mtDNA) from the yeast Saccharomyces cerevisiae was cleaved by restriction endonucleases Eco RI, Hpa I, Bam HI, Hind III, Pst I, and Sal I, yielding 10, 7, 5, 6, 1, and 1 fragments, respectively. A physical ordering of the restriction sites on yeast mtDNA has been derived. Yeast mtDNA cannot be isolated as intact molecules, and it contains nicks and gaps which complicate the use of conventional fragment mapping procedures. Nevertheless, the position of each of the restriction sites was obtained primarily by reciprocal redigestion of isolated restriction fragments. This procedure was supplemented by co-digestion of mtDNA with a multisite enzyme and a single-site enzyme (i.e., Sal I or Pst I) which provided a unique orientation for overlapping fragments cleaved by Sal I or Pst I. The data obtained from these approaches were confirmed by analysis of double and triple enzyme digests. Analysis of partial digest fragments was used for positioning of the smallest Eco RI fragment. A comparison of mtDNA from four grande strains (MH41-7B, 19d, TR3-15A, and MH32-12D) revealed similar, but slightly varying restriction patterns, with an identical genome size for each of approximately 5 X 10(-7) d or 75 kb. A fifth grande strain, D273-10B from S. cerevisiae, revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb.

DNA Restriction Enzymes↗
PubMed1977

New hue '77.

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Dental Technicians↗
PubMed1976

Control of protein balance in hypertrophied cardiac muscle.

The levels of intracellular proteins are determined by a balance between their rates of synthesis and degradation. During the development of regression of cardiac hypertrophy, both of these rates can be expected to change. Possible control sites of synthetic and degradation processes are discussed in this article. The following experimental results are presented: (1) Cardiac mitochondrial cytochromes accumulate early after imposition of pressure overload, as a result of an increased rate of synthesis and decreased rate of degradation. (2) The half-life of myosin heavy chains (HC) in the steady state was determined from incorporation kinetics, using leucyl-tRNA as precursor, to be 5-6 days. (3) The existence of a pool of newly synthesized myofilaments which are not fully incorporated into the core of myofibrils is indicated by the incorporation data.

Animals↗
PubMed1975

Cytochrome synthesis in synchronous cultures of the yeast, Saccharomyces cerevisiae.

The synthesis of cytochromes aa3, b, and c has been investigated during synchronous growth in the yeast, Saccharomyces cerevisiae. These cytochromes increase in concentration continuously throughout each cell cycle, with an approximate doubling in rate during successive cycles. The rates of cytochrome formation are considerably higher in galactose-grown cultures than in cells grown in glucose. Although cytochrome aa3 increases at a continuous rate, its functional counterpart, cytochrome c oxidase, increases in stepwise fashion, with the increments occurring at the beginning of each new cell cycle. Chloramphenicol, a specific inhibitor of intramitochondrial protein synthesis, inhibits the formation of cytochrome aa3 at all stages of the cell cycle, but does not inhibit cytochrome c. Chloramphenicol exhibits a somewhat intermediate effect on cytochrome b synthesis, with transient inhibition occurring only when the drug is added prior to or during the initial part of the first cell cycle. After this time, chloramphenicol had no effect on the rate of cytochrome b synthesis. The data indicate that under our conditions of cell synchrony mitochondrial membrane formation as reflected by increments in mitochondrial cytochromes occurs by continuous accretion of new material throughout the cell cycle. Intramitochondrially synthesized polypeptide products, responsible for the formation of new cytochrome aa3, appear to be synthesized throughout the cell cycle.

Cell Division↗
PubMed1975

Removal of Z-lines and alpha-actinin from isolated myofibrils by a calcium-activated neutral protease.

A calcium-activated factor (CaAF) has been isolated and partially purified from the post-myofibrillar supernatant fraction of rabbit skeletal muscle. The 200-fold purified CaAF hydrolyzed denatured casein, [3-H]acetyl hemoglobin, and N-ethyl[3-H]maleimide-labeled alpha-actinin. The proteolytic activity has a pH optimum at 6.9 and is dependent on the presence of Ca2+ (optimum concentration, 10 mM). Digestion of isolated myofibrils with CaAF results in removal of Z-lines and in a parallel loss of a 90, 000-dalton protein that has a mobility identical with that of alpha-actinin as determined by polyacrylamide gel electrophoresis. A protein with the properties of alpha-actinin (identical electrophoretic mobility, and ability to accelerate the Mg2+-activated ATPase of reconstituted actomyosin) was isolated from the supernatant of CaAF-treated myofibrils. The release of alpha-actinin from myofibrils by the calcium-activated neutral protease occurs in the absence of detectable change in the electrophoretic profiles of the other myofibrillar proteins, or in the ethylene glycol bis(beta-aminoethyl ether)-N, N' tetraacetic acid (EGTA) sensitivity of Mg2+-activated ATPase. In contrast to the specific removal of Z-lines and of alpha-actinin by CaAF, trypsin treatment of myofibrils results in extensive degradation of myosin heavy chains and of the inhibitory component of troponin (TN-I), and in loss of EGTA sensitivity of myofibrillar ATPase. The degradation of TN-I and loss of EGTA sensitivity occur before the Z-line disappearance.

Animals↗