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G S Getz

Publications and source records attributed to G S Getz.

At least 91 records · Page 5Linked to original sources
PubMed1986

Transcriptional regulation of the mitochondrial genome of yeast Saccharomyces cerevisiae.

The relative rates of transcription of several classes of the mitochondrial genes of the yeast Saccharomyces cerevisiae have been determined. The rates were measured by pulse labeling whole yeast with [32P]O4, isolating the total RNA, hybridization to single-stranded M13 DNA probes containing segments of the gene of interest, digestion with RNase A or T1, and separation of the protected fragment by gel electrophoresis. This analysis indicated that, among the genes analyzed, transcriptional promoters varied in strength by 20-fold while the rates of transcription varied by more than 50-fold. The strengths of the promoters of the genes were ordered: tRNAMetf greater than tRNAPhe greater than 14 S rRNA greater than 21 S rRNA greater than tRNAGlu greater than Oli-1 much greater than tRNACys. In addition, transcription rates were measured within polygenic transcription units. This analysis indicated that there was transcriptional attenuation within all the polygenic transcription units with the greatest attenuation factor being as much as 17-fold, occurring after the tRNAGlu and tRNAMetf genes. This analysis indicated that regulation of the rates of transcription in the yeast mitochondrial genome occurs by two distinct mechanisms, modulation of the rate of transcriptional initiation and attenuation of transcriptional elongation.

Base Sequence↗
PubMed1986

The charge polymorphism of rat apoprotein E.

Rat apolipoprotein E (apo-E) exists in plasma as four unique isoelectric forms (designated E-1, E-2, E-3, or E-4 from acidic to basic, respectively). We have examined the processes accounting for this polymorphism using intact rats or cultured rat hepatocytes. Intrahepatic precursors of rat apo-E were isolated and analyzed on isoelectric focusing gels. The primary translation product of rat liver apo-E mRNA focused as two isoproteins with more basic pI values than the isoproteins of plasma apo-E. The microsome-processed translation product also focused as two isoproteins having pI values corresponding to apo-E-4 and apo-E-3 isoproteins of plasma apo-E. Following a bolus injection of [3H]leucine into the portal vein, intrahepatic isoproteins corresponding to plasma apo-E-2 and apo-E-1 isoproteins were first detected in the rough endoplasmic reticulum (RER) and Golgi fractions, respectively. The apparent molecular weight of intrahepatic apo-E increased as it passed from the RER to the Golgi. Only the most acidic isoform, apo-E-1, of plasma apo-E was sensitive to neuraminidase treatment indicating that sialic acid residues are responsible, in part, for the polymorphism of rat apo-E. Using cultured hepatocytes, tunicamycin (1 microgram/ml) inhibited the incorporation of [3H]glucosamine into both molecular weight forms of apolipoprotein B but did not influence the synthesis, glycosylation (as measured by [3H]glucosamine incorporation), or secretion of apo-E. Tunicamycin-inhibited hepatocytes secreted the normal complement of apo-E isoforms including apo-E-1, thus confirming that apo-E-1 is not an N-linked glycoprotein. These results suggest that post-translational modifications involving both RER and Golgi-specific reactions contribute to the polymorphism of rat apo-E.

Animals↗
PubMed1986

A critical base in the yeast mitochondrial nonanucleotide promoter. Abolition of promoter activity by mutation at the -2 position.

The yeast mitochondrial promoter comprises a highly conserved nonanucleotide sequence, 5'-ATATAAGTA-3'. Transcription is initiated both in vivo and in vitro at the 3'-terminal A (+1). The G at the -2 position is strictly conserved. The requirement for this nucleotide is established by comparing the in vitro promoter activity of nonanucleotides containing any of the four bases at the -2 position, obtained as synthetic oligonucleotides. Only the synthetic promoter having G at the -2 position exhibits any promoter activity.

Base Sequence↗
PubMed1986

In vitro transcription and promoter strength analysis of five mitochondrial tRNA promoters in yeast.

The in vitro transcriptional initiation sites of four yeast mitochondrial tRNA genes have been investigated in a run-off transcription assay. Precise initiation originating within the 9-nucleotide mitochondrial promoter sequence was detected for the phenylalanine, initiator formyl methionine, cysteine, and one of the two threonine tRNA genes. The relative promoter strength of each of these tRNA promoters as well as that for the previously described glutamate tRNA promoter was determined in a competition assay. This assay measured the utilization of a particular tRNA promoter relative to the amount of transcription arising from a control 14 S rRNA promoter present in the same reaction. The competition strength of the tRNAPhe, tRNAMetf, and tRNAGlu promoters is 20-fold greater than that for the tRNAThrACN and tRNACys promoters. Comparison of the nucleotide sequences at the +2 and +3 positions in the transcripts reveals a homology among the strong promoters not duplicated in the weak promoters.

Base Sequence↗
PubMed1986

Nucleotides flanking the promoter sequence influence the transcription of the yeast mitochondrial gene coding for ATPase subunit 9.

The conserved nonanucleotide sequence functions as a promoter in the yeast (Saccharomyces cerevisiae) mitochondrial genome. A mitochondrial gene, Oli 1, which codes for ATPase subunit 9, has two identical nonanucleotide promoter sequences separated by 78 nucleotides, but they initiate transcription with very different efficiencies in vivo and in vitro. Deletion analysis has revealed that the nucleotide at position +2 of the weak downstream promoter accounts for its poor in vitro transcriptional activity. This finding was confirmed with site-specific mutations at +2 and +3 positions of a consensus synthetic promoter. The nonanucleotide mitochondrial promoter with a pyrimidine at position +2 acts as a weak promoter, whereas the same sequence with a purine at the +2 position functions as a strong promoter. The nucleotide at the +3 position further contributes to the relative promoter strength. These results suggest not only that the conserved nine-nucleotide sequence is required for the correct transcriptional initiation but also that other neighboring nucleotides influence the efficiency of promoter function.

Base Sequence↗
PubMed1985

Regulation of apolipoprotein E synthesis in rat ovarian granulosa cells.

Apoprotein E (apo-E) is a surface component of several classes of plasma lipoproteins. It functions as a ligand for receptor-mediated uptake of lipoproteins. Granulosa cells from ovaries of diethylstilbestrol-stimulated hypophysectomized immature rats cultured in serum-free medium with [35S]methionine secretes a 34-kDa protein which reacts with a monospecific anti-rat apo-E antibody and represents 0.2% of total secreted protein. Protease mapping confirms that this protein is apoprotein E. The secreted apoprotein E may be complexed with lipid since it floats in the ultracentrifuge at density less than 1.21 micrograms/ml. Freshly isolated granulosa cells contain receptors for follicle stimulating hormone (FSH) but not for human chorionic gonadotropin (hCG) or prolactin. Apoprotein E secretion is stimulated 2-fold by FSH, but hCG and prolactin have no effect. When granulosa cells develop hCG and prolactin receptors after 48 h of culture with FSH, apoprotein E secretion is not stimulated by addition of FSH, hCG, or prolactin although steroidogenesis is induced. The addition of 10(-7) M androgen plus FSH stimulates a marked increase in progestin synthesis over FSH alone, but androgen has little added effect on apoprotein E secretion. Cholera toxin (1.25 micrograms/ml) and dibutyryl cAMP (5 mg/ml), both of which increase intracellular cAMP, stimulate apo-E secretion 9-fold and 12-fold, respectively. The dibutyryl cAMP effect is dependent on both dose (greater than or equal to 0.5 mg/ml required) and time (onset at 24 h, maximum at 48 h, and back to near baseline at 96 h). Isobutylmethylxanthine, a phosphodiesterase inhibitor, augments FSH-stimulated apoprotein E synthesis 2.5-fold, supporting a role for cAMP in mediating the FSH effect. This is the first demonstration of the hormonal regulation of apoprotein E synthesis in an extrahepatic tissue.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Characterization of a yeast mitochondrial promoter by deletion mutagenesis.

We have generated collections of mutants of the promoter for the small rRNA gene from the mitochondria of yeast deleted from either the 3' or 5' end. Plasmids containing the partially deleted promoter were assayed for their ability to direct correct transcriptional initiation in a homologous in vitro system. We find that the region required for high-efficiency promoter function lies between positions -10 and +2. Our methods detected no effect of flanking sequences on the strength of this promoter.

Base Sequence↗
PubMed1985

Effect of Reye's syndrome serum on isolated chinchilla liver mitochondria.

A general impairment of liver mitochondrial enzymes is central to Reye's syndrome (RS). The respiration of isolated liver mitochondria was measured after the addition of concentrated normal serum or RS serum derived from 12 patients. RS serum stimulates oxygen consumption in isolated rat liver mitochondria. This effect is due to the oxidation of uric acid by peroxisomes contaminating the preparation and a stimulation of mitochondrial respiration (1.05 +/- 0.14 nmol of O2/min X mg of protein; control 0.30 +/- 0.08 nmol O2/min X mg). The stimulation of respiration occurs in the presence of all respiratory substrates, is dependent on the amount of serum added, and represents an uncoupling of oxidative phosphorylation. RS serum reduces ATP formation by 15-76%. The uncoupling effect correlates with the amount of free fatty acid in the serum sample and resembles the effect induced by the addition of a dicarboxylic fatty acid. Dicarboxylic fatty acids, especially long-chain dicarboxylic acids, impair ATP formation. Dicarboxylic acids were found in the serum of all RS patients and comprised as much as 54% of the total serum free fatty acids. 90% of the serum dicarboxylic acids were of 16-18 carbon lengths. The amount of dicarboxylic acids in the RS serum corresponded directly with the reduction in ATP formation by the RS serum. This demonstrates that dicarboxylic acids occur in RS and may be important in the general impairment of mitochondrial function in RS and other disorders where they are present.

Adolescent↗
PubMed1985

Suppression of plasma testosterone leads to an increase in serum total and high density lipoprotein cholesterol and apoproteins A-I and B.

Men have lower high density lipoprotein (HDL) and higher low density lipoprotein (LDL) levels than women. To dynamically evaluate the role of endogenous testosterone on the lipoprotein profile, eight normal men received a long-acting gonadotropin releasing hormone analog (LHRHA) for 10 weeks by SC injection. Plasma testosterone levels were acutely lowered below 1 ng/ml after 4 weeks of LHRHA treatment and remained depressed at this level for the duration of administration of the analog. There were prompt increases in total cholesterol [baseline vs. peak (milligrams per dl) mean +/- SEM, 177 +/- 18 vs. 208 +/- 22; P less than 0.005], apoprotein B (apo B; 69 +/- 12 vs. 97 +/- 13; P less than 0.05), HDL-cholesterol (23 +/- 2 vs. 33 +/- 2; P less than 0.005), and apo A-I (80 +/- 7 vs. 112 +/- 5; P less than 0.005), but not in apo A-II (40 +/- 3 vs. 40 +/- 4; P = NS) levels. The peaks occurred after 10 weeks of treatment and were followed by a fall in these values after discontinuing LHRHA. These changes were largely prevented in a second study (six men) in which LHRHA was administered together with im testosterone enanthate, which was given every 2 weeks. These results show that suppression of endogenous testosterone leads to increases in HDL and LDL, demonstrating that testosterone has an important effect on lipoprotein metabolism and plays a key role in defining the lipoprotein profile in men.

Adult↗
PubMed1984

Extrahepatic synthesis of apolipoprotein E.

Apolipoprotein E (apoE) synthesis has been examined in rat and guinea pig tissues using in vitro translation and [35S]methionine labeling of tissue slices. A number of tissues not involved in lipoprotein synthesis synthesize a protein very similar to apoE, including the spleen, adrenal, kidney, testis, ovary, heart, and lung. Although the intestine is involved in lipoprotein synthesis, apoE synthesis could not be detected in intestinal mucosa. The protein synthesized by the extrahepatic tissues was identified as apoE by its electrophoretic mobility, its immunologic reactivity with a monospecific antibody and by limited proteolysis mapping with Staphylococcus aureus V8 protease. ApoE represented between 0.02 and 0.7% of the total protein synthesized in the extrahepatic tissues, indicating that apoE mRNA is a fairly abundant mRNA in these tissues. ApoE mRNA was also detected by hybridization with a rat apoE cDNA clone, which hybridized to a single mRNA 1250 nucleotides in length in rat liver and in extrahepatic tissues. Hybridization of the apoE clone to rat genomic DNA demonstrated that the apoE gene was more heavily methylated in intestinal mucosa, which did not synthesize apoE, than in liver, testis, or kidney. 35S labeling of peritoneal macrophages revealed that both rat and guinea pig macrophages synthesized and secreted apoE in vitro. Rhesus aortic smooth muscle cells also synthesized and secreted apoE. The possible functions of apoE synthesized in the peripheral tissues are considered.

Animals↗
PubMed1984

Physiology of a temperature-sensitive mutant of Saccharomyces cerevisiae defective in phosphofructokinase activity.

In this paper, we describe a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae (P5-9) which at a restrictive temperature (36 degrees C) shows a pleiotropic defect for transport of many different metabolites. The temperature sensitivity of the mutant is closely related to a reduction in phosphofructokinase activity. This conclusion is based on the following criteria. (i) Both the primary isolate, designated P5-9 (ts [rho-] Ino-), which is an inositol auxotroph and respiration deficient, and a purified derivative, SB4 (ts [rho+] Ino+ ), which is respiration competent and capable of growing in the absence of inositol, are temperature sensitive for growth and ethanol production in media containing glucose or fructose as the sole carbon source. (ii) The respiration-competent derivative SB4 is not temperature sensitive in media containing glycerol or glycerol-pyruvate; glucose inhibits its growth at 36 degrees C in these media. (iii) Assays of glycolytic enzymes in P5-9 and SB4 extracts, prepared from cells incubated for 1 to 2 h at 36 degrees C before harvesting, show selective reduction in phosphofructokinase activity. Analysis of tetrads derived from the cross of mutant and nonmutant haploids indicates that temperature sensitivity for growth is due to a single gene or to two closely linked genes. The biochemical analysis of spores from seven such tetrads revealed a uniform cosegregation of temperature sensitivity for growth and phosphofructokinase activity. Transport and ATP levels were drastically reduced in SB4 cells incubated at 36 degrees C for 1 to 2 h with glucose as the carbon source, but not when glycerol-pyruvate or lactate was the energy source. Therefore, depletion of energy as a result of phosphofructokinase inactivation appears to be the cause of the pleiotropic transport defect observed in the mutant.

Adenosine Triphosphatases↗
PubMed1984

Characterization of lipoprotein produced by the perfused rhesus monkey liver.

Isolated livers from rhesus monkeys (Macaca mulatta) were perfused in order to asses the nature of newly synthesized hepatic lipoprotein. Perfusate containing [3H]leucine was recirculated for 1.5 hr, followed by an additional 2.5-hr perfusion with fresh perfusate. Equilibrium density gradient ultracentrifugation clearly separated VLDL from LDL. The apoprotein composition of VLDL secreted by the liver was similar to that of serum VLDL. The perfusate LDL contained some poorly radiolabeled, apoB-rich material, which appeared to be contaminating serum LDL. There was also some material of an LDL-like density, which was rich in radiolabeled apoE. Rate zonal density gradient ultracentrifugation fractionated HDL. All perfusate HDL fractions had a decreased cholesteryl ester/unesterified cholesterol ratio, compared to serum HDL. Serum HDL distributed in one symmetric peak near the middle of the gradient, with coincident peaks of apoA-I and apoA-II. The least dense fractions of the perfusate gradient were rich in radiolabeled apoE. The middle of the perfusate gradient contained particles rich in radiolabeled apoA-I and apoA-II. The peak of apoA-I was offset from the apoA-II peak towards the denser end of the gradient. The dense end of the HDL gradient contained lipoprotein-free apoA-I, apoE, and small amounts of apoA-II, probably resulting from the relative instability of nascent lipoprotein compared to serum lipoprotein. Perfusate HDL apoA-I isoforms were more basic than serum apoA-I isoforms. Preliminary experiments, using noncentrifugal methods, suggest that some hepatic apoA-I is secreted in a lipoprotein-free form. In conclusion, the isolated rhesus monkey liver produces VLDL similar to serum VLDL, but produces LDL and HDL which differ in several important aspects from serum LDL and HDL.

Animals↗
PubMed1984

Processing of rat liver apoprotein E primary translation product.

The primary translation product of rat liver apoE mRNA was isolated from wheat germ cell-free translation systems. Plasma apoE and the primary translation product migrated similarly on SDS-polyacrylamide gels, had similar partial proteolytic peptide maps, and bound to and coeluted from heparin-Sepharose columns. Comparison of the partial amino acid sequence of the primary translation product with the amino-terminal sequence of plasma apoE indicated that rat apoE is initially synthesized with an 18 amino acid amino-terminal extension. This entire segment was removed cotranslationally by canine microsomes possessing signal peptidase activity. The microsome-processed translation product did not contain an endoglycosidase H-sensitive oligosaccharide, suggesting that rat apoE is O-glycosylated.

Amino Acid Sequence↗
PubMed1982

The apoprotein B-independent hepatic uptake of chylomicron remnants.

Rat lymph chylomicrons were treated with Pronase resulting in particles completely devoid of surface apoproteins. On re-incubation with serum, the Pronase-treated chylomicrons re-acquired, by transfer from other lipoproteins, all apoproteins except apoprotein B, which is water-insoluble and non-transferable. When two groups of rats were injected with [3H]cholesterol-labelled control or Pronase-treated chylomicrons, radioactivity was incorporated into the liver of both groups at similar rates. It is concluded that the remnants of the control and Pronase-treated chylomicrons formed in the vascular space were recognized and taken up by liver cells by a process that does not require apoprotein B.

Animals↗
PubMed1981

Phospholipid accumulation during the cell cycle in synchronous cultures of the yeast, Saccharomyces cerevisiae.

Phospholipid concentrations have been examined throughout successive cell cycles in synchronously growing cultures of the yeast, Saccharomyces cerevisiae. Total phospholipid phosphorus, as well as lecithin and phosphatidylethanolamine levels, exhibited stepwise increases during the cell cycle with step increments beginning just prior to new rounds of bud formation. Phosphatidylinositol and phosphatidylserine levels, on the other hand, showed what have been interpreted to be peak concentrations near the time of bud formation. Cardiolipin content varied considerably and was dependent upon the carbon source of the growth medium. Glucose-grown cells exhibited peak concentrations of cardiolipin near the time of bud formation, with marked decreases after this time. In contrast, galactose-grown synchronous cells exhibited stepwise increments in cardiolipin content, with step increases occurring near the time of new rounds of bud formation. Step or peak increases in cardiolipin, as well as all other phospholipids, were found to coincide with the time of stepwise increases in cytochrome c oxidase activity in these cells. No correlations were observed between the elaboration of mitochondrial membranes during the synchronous cell cycle and the observed patterns of phospholipid increase.

Cardiolipins↗
PubMed1981

New form of acid phosphatase during lysosome biogenesis.

Lysosome formation was induced in cells of the renal medulla by feeding rats on a K+-deficient diet. The role of the endoplasmic reticulum in the production of acid phosphatase, a typical lysosomal enzyme, was examined. Lysosomal and microsomal fractions were prepared for study by differential centrifugation of homogenates of renal papilla and inner stripe of red medulla. Acid phosphatase activity in the microsomal fraction was distinguished from the activity in the lysosomal fraction in normal tissue by differences in pH optima, tartrate inhibition, distribution of multiple forms after polyacrylamide-gel electrophoresis and detergent-sensitivity. During progressive K+ depletion, acid phosphatase activity in both microsomal and lysosomal fractions of the tissue increased 3-fold. In the lysosomes, K+ depletion was associated with the appearance of a new band of acid phosphatase. The neuraminidase-sensitivity of this band on polyacrylamide-gel electrophoresis indicated that the enzyme protein had been modified by the addition of sialic acid residues. K+ depletion also altered the lysosomal enzyme so that thiol compounds were able to stimulate its activity.

Acid Phosphatase↗