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J C Rabinowitz

Publications and source records attributed to J C Rabinowitz.

At least 37 records · Page 2Linked to original sources
PubMed1989

The effect of Escherichia coli ribosomal protein S1 on the translational specificity of bacterial ribosomes.

Ribosomes from Gram-negative bacteria such as Escherichia coli exhibit non-specific translation of bacterial mRNAs. That is, they are able to translate mRNAs from a variety of sources in a manner independent of the "strength" of the Shine-Dalgarno region, in contrast to ribosomes from many Gram-positive bacteria, such as Bacillus subtilis, which show specific translation in only being able to translate other Gram-positive mRNA, or mRNAs that have "strong" Shine-Dalgarno regions. There is an evolutionary correlation between the translational specificity and the absence of a protein analogous to E. coli ribosomal protein S1. The specificity observed with B. subtilis ribosomes is a function of their 30 S subunit which lacks S1; translation of Gram-negative mRNA can occur with heterologous ribosomes containing the 30 S subunit of E. coli ribosomes and the 50 S subunit of B. subtilis ribosomes. However, the addition of E. coli S1 alone to B. subtilis ribosome does not overcome their characteristic inability to translate mRNA from Gram-negative organisms. By contrast, the removal of S1 from E. coli ribosomes results in translational behavior similar to that shown by B. subtilis ribosomes in that the S1-depleted E. coli ribosomes can translate mRNA from Gram-positive sources in the absence of added S1, although addition of S1 stimulates further translation of such mRNAs by the E. coli ribosomes.

Bacteria↗
PubMed1989

Judgments of origin and generation effects: comparisons between young and elderly adults.

In 2 experiments, young and elderly adults were required to both read words, and generate words by completing word fragments. Subjects were then required to recognize those words that had been presented earlier; for those words that they recognized they judged whether the items had initially been presented in read or generate form. Generation effects (better memory for words that were generated as compared with words that were read) of similar magnitude were observed for both young and older adults. The older adults were consistently less accurate than the younger adults in their judgments of origin. In addition, the young adults exhibited a bias to respond "read" for these judgments. In contrast, the older adults either exhibited a neutral response bias or were biased to respond "generate." Age-related differences in the encoding or retrieval of information about cognitive operations do not provide a good account of the results. Alternative accounts are described.

Adult↗
PubMed1989

Age deficits in recall under optimal study conditions.

Young and older adults studied lists of words under both standard and optimal study conditions for subsequent free recall. Under optimal conditions, the participants studied each word for as long as they wished, were allowed to take notes, and were encouraged to actively use whatever strategies they thought would maximize recall. Both age groups recalled more words under optimal study conditions than under standard conditions, but the improvement was greater for the young adults. This increase in the age-related recall deficit was not due to differences in study time. The results suggest that standard laboratory memory tasks do not overestimate the memory deficits of older adults because of a failure to provide either optimal learning conditions or sufficient study time.

Adolescent↗
PubMed1988

Isolation and characterization of the Saccharomyces cerevisiae MIS1 gene encoding mitochondrial C1-tetrahydrofolate synthase.

C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is found in both the cytoplasm and the mitochondria. The gene encoding yeast mitochondrial C1-tetrahydrofolate synthase was isolated using synthetic oligonucleotide probes based on the amino-terminal sequence of the purified protein. Hybridization analysis shows that the gene (designated MIS1) has a single copy in the yeast genome. The predicted amino acid sequence of mitochondrial C1-tetrahydrofolate synthase shares 71% identity with yeast C1-tetrahydrofolate synthase and shares 39% identity with clostridial 10-formyltetrahydrofolate synthetase. Chromosomal deletions of the mitochondrial C1-tetrahydrofolate synthase gene were generated using the cloned MIS1 gene. Mutant strains which lack a functional MIS1 gene are viable and can grow in medium containing a nonfermentable carbon source. In fact, deletion of the MIS1 locus has no detectable effect on cell growth.

Amino Acid Sequence↗
PubMed1988

Distribution of 10-formyltetrahydrofolate synthetase in eubacteria.

The distribution of 10-formyltetrahydrofolate synthetase, which activates formate for use as a one-carbon donor in a variety of biosynthetic reactions, was determined for a variety of eubacteria. Organisms from several genera were found to lack detectable synthetase activity; however, all organisms tested were found to contain 5,10-methylenetetrahydrofolate dehydrogenase activity.

Bacteria↗
PubMed1988

Nucleotide sequence of the Clostridium acidiurici ("Clostridium acidi-urici") gene for 10-formyltetrahydrofolate synthetase shows extensive amino acid homology with the trifunctional enzyme C1-tetrahydrofolate synthase from Saccharomyces cerevisiae.

The nucleotide sequence of the gene for 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) from Clostridium acidiurici ("Clostridium acidi-urici") was determined. The synthetase mRNA initiation and termination regions were determined by primer extension and S1 nuclease mapping. Two potential -10 and -35 promoter regions were identified upstream of mRNA initiation. The terminator region was found to be in a large region of dyad symmetry. A comparison of the amino acid sequences of the monofunctional synthetase and the eucaryotic trifunctional enzyme, C1-tetrahydrofolate synthase, from Saccharomyces cerevisiae demonstrated a region of strong homology.

Amino Acid Sequence↗
PubMed1987

Heparin-agarose chromatography for the purification of tetrahydrofolate utilizing enzymes: C1-tetrahydrofolate synthase and 10-formyltetrahydrofolate synthetase.

Rapid and convenient purification procedures based upon heparin-agarose chromatography for C1-tetrahydrofolate synthase from Saccharomyces cerevisiae and 10-formyltetrahydrofolate synthetase from Clostridium acidi-urici have been developed. The purification of the yeast enzyme involves three chromatographic steps that can be done rapidly, with no intervening dialyses, and results in high yield. The first step alone, heparin-agarose chromatography, is sufficient to purify the enzyme from yeast bearing a cloned copy of the ADE3 gene that overexpresses the protein. The other steps in the purification from wild-type yeast are matrex gel red A and phenyl-Sepharose chromatography. The purification of the clostridial enzyme involves protamine sulfate fractionation and heparin-agarose chromatography. Heparin-agarose also binds two other enzymes that use tetrahydrofolate, 5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylenetetrahydrofolate dehydrogenase. Thus, heparin-agarose should prove useful in purification of a variety of enzymes that utilize tetrahydrofolate or its derivatives as a cofactor.

Aminohydrolases↗
PubMed1986

Purification and characterization of a mitochondrial isozyme of C1-tetrahydrofolate synthase from Saccharomyces cerevisiae.

C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is encoded by the ADE3 locus, yet ade3 mutants have low but detectable levels of these enzyme activities. Synthetase, cyclohydrolase, and dehydrogenase activities in an ade3 deletion strain co-purify 4,000-fold to yield a single protein species as seen on sodium dodecyl sulfate-polyacrylamide gels. The native molecular weight of the isozyme (Mr = 200,000 by gel exclusion chromatography) and the size of its subunits (Mr = 100,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are similar to those of C1-tetrahydrofolate synthase. Cell fractionation experiments show that the isozyme, but not C1-tetrahydrofolate synthase, is localized in the mitochondria. Genetic studies indicate that the isozyme is encoded in the nuclear genome. Peptide mapping experiments show that C1-tetrahydrofolate synthase and the isozyme are not structurally identical. However, immunotitration experiments and amino acid sequence analysis suggest that C1-tetrahydrofolate synthase and the isozyme are structurally related. We propose to call the isozyme "mitochondrial C1-tetrahydrofolate synthase."

Amino Acid Sequence↗
PubMed1986

In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. Evidence for "extended" promoter elements in gram-positive organisms.

Analysis of Clostridium pasteurianum genomic DNA indicates that the ferredoxin (Fd) gene is present in a single copy. The cloned Fd gene previously described (Graves, M.C., Mullenbach, G. T., and Rabinowitz, J. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1653-1657) was used to map in vivo and in vitro synthesized Fd transcripts. The in vivo mRNA was sized in two ways: by Northern hybridization analysis, and more directly from the known DNA sequence after the 5'- and 3'-termini were identified. The 5'-end was determined by primer extension-dideoxy sequencing and the 3'-end by S1 nuclease mapping. The monocistronic Fd mRNA contains about 255 nucleotides and, thus, is one of the shortest bacterial mRNAs yet described. We also examined the Fd transcripts produced by Escherichia coli transformed with the plasmid containing the Fd gene. E. coli RNA polymerase most likely recognizes the same promoter (P1) as the clostridial polymerase, and furthermore, efficiently uses an additional promoter (P2) that is poorly recognized by the normal host enzyme. For comparison, in vitro transcripts were generated by E. coli and Bacillus subtilis RNA polymerases. In vitro, only promoter P1 is used by either E. coli or B. subtilis RNA polymerase. The 3'-end of each of the four types of transcripts occurs essentially at the same location and maps to within a large dyad symmetry element. Comparison of the Fd promoter with other Gram-positive promoters reveals that some sequences outside of the traditional Pribnow and -35 regions are conserved. This analysis indicates that an "extended" promoter recognition site may be required in these organisms.

Base Sequence↗
PubMed1986

Nucleotide sequence of the Saccharomyces cerevisiae ADE3 gene encoding C1-tetrahydrofolate synthase.

The sequence of a cloned copy of the yeast ADE3 gene, which encodes the trifunctional enzyme C1-5,6,7,8-tetrahydrofolate (THF) synthase, was determined. Yeast cells transformed with a multicopy yeast plasmid containing this ADE3 gene overexpress C1-THF synthase 20-60-fold relative to wild-type yeast cells. C1-THF synthase from transformed cells is identical with that isolated from wild-type cells by all the criteria tested. The translated DNA sequence and amino-terminal protein sequences are identical and the amino acid composition predicted from the DNA sequence agrees closely with that determined by hydrolysis of C1-THF synthase protein. Correlation of the genetic map of the ADE3 region and of proteolysis experiments with the protein sequence suggests locations for two functional domains within yeast C1-THF synthase. The sequence of C1-THF synthase does not appear to be homologous to any other sequenced protein, including other proteins that use folate substrates. The 5' and 3' untranslated regions of the ADE3 gene suggest initiation and termination signals similar to transcription signals associated with other yeast genes. No special regulatory features have been associated with the ADE3 sequence. An unusual open reading frame that is encoded by a very unbiased set of codons follows the ADE3 gene.

Amino Acid Sequence↗
PubMed1986

Substrate and inhibitor activities of the screw sense isomers of metal-nucleotide complexes in the formyltetrahydrofolate synthetase reaction.

Phosphorothioate analogues of ATP and isomers of CrATP and CrADP were used to examine the nucleotide stereoselectivity of formyltetrahydrofolate synthetase from procaryotic and eucaryotic sources. Substrate activity of the thio-ATP analogues increased as the site of sulfur substitution was changed from the gamma to the alpha position. Thus, adenine nucleotide analogues substituted with sulfur at an alpha nonbridging position (ATP alpha S isomers) were the most active, and ATP gamma S was inactive. When Mg2+ was used as the divalent cation, both enzymes showed a clear preference (higher V/Km value) for the Sp isomer of ATP beta S although the magnitude of the preference was greater with the bacterial enzyme. With Cd2+ as the divalent cation the Rp isomer was preferred, but the difference was greater with the yeast enzyme. Both (Sp)-MgATP beta S and (Rp)-CdATP beta S have the delta or right-hand screw sense configuration of the metal chelate ring. The reversal of stereoselectivity when the cation was changed indicates that the metal ion is coordinated to the beta-phosphate group. No stereoselectivity was observed when ATP alpha S isomers were used in the presence of Mg2+ or Cd2+, suggesting that the metals are not coordinated to the alpha-phosphate. ATP beta S was also found to be a competitive inhibitor of MgATP and CdATP, and the lowest Ki values were obtained with the lambda screw sense isomers. The screw sense isomers of bidentate CrATP exhibited no detectable substrate activity but were competitive inhibitors of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1986

Priming in episodic memory.

Young and old adults studied related and unrelated word pairs and were given both cued recall and recognition tests. The recognition test required speeded responses to single words. The test order was constructed so that half of the B items from each A-B pair were preceded by its paired A item whereas the other half of the B items were preceded by some other old item. Priming was measured as the difference in reaction time between these two types of items. Significant age differences were found in both recall and recognition accuracy, but young and old adults showed equal amounts of priming. There were significant main effects of relatedness on all three dependent measures, but only in cued recall was there a larger age deficit for unrelated items. The results are inconsistent with an age-related deficit for integrating pairs of words at encoding and suggest, instead, an impairment of effortful retrieval processes.

Adult↗
PubMed1986

Prior retrieval effects in young and old adults.

Previous work has shown that successful recall boosts subsequent retrieval of the recalled items. Two experiments tested the hypothesis that this benefit from prior retrieval would be less strong in older adults. Contrary to expectation, the older adults showed as great a benefit from prior retrieval as did the young adults in both experiments. It is suggested that successful cued recall may act like an orienting task at encoding, to guide retrieval processes effectively, and that the benefit obtained from a successful retrieval is a function of the information used to guide the retrieval process.

Adolescent↗
PubMed1986

Cloning and expression in Escherichia coli of the gene for 10-formyltetrahydrofolate synthetase from Clostridium acidiurici ("Clostridium acidi-urici").

The gene for 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) from the purinolytic anaerobic bacterium Clostridium acidiurici ("Clostridium acidi-urici") was cloned into Escherichia coli JM83 with plasmid pUC8. A C. acidiurici genomic library was prepared in E. coli from a partial Sau3A digest and screened with antibody against the synthetase. Of 10 antibody-positive clones, 1 expressed a high level of synthetase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis demonstrated that the protein synthesized in E. coli had the same subunit molecular weight as the C. acidiurici enzyme. The gene was located on an 8.3-kilobase genomic insert and appeared to be transcribed from its own promoter. Analysis of genomic digests with a fragment of the synthetase gene indicated that one copy of the gene was present in the C. acidiurici chromosome.

Cloning, Molecular↗
PubMed1985

Inefficient translation of T7 late mRNA by Bacillus subtilis ribosomes. Implications for species-specific translation.

Bacillus subtilis 30 S subunits inefficiently recognize initiation sites in mRNAs from Gram-negative bacteria, but they are able to efficiently recognize initiation sites in mRNA derived from Gram-positive bacteria. McLaughlin et al. (McLaughlin, J. R., Murray, C. L., and Rabinowitz, J. C. (1981) J. Biol. Chem. 256, 11283-11291) have suggested that B. subtilis ribosomes require a strong Shine-Dalgarno sequence for translation initiation. To test whether this criterion is sufficient to explain the translational specificity of B. subtilis ribosomes, T7 late mRNA, which contains strong Shine-Dalgarno sequences before many of the late genes (Dunn, J. J., and Studier, F. W. (1983) J. Mol. Biol. 166, 477-535), was translated in vitro with both Escherichia coli and B. subtilis ribosomes. The identification of several of the in vitro products upon gel electrophoresis indicated that B. subtilis ribosomes recognize correct translation initiation sites in late T7 mRNA, but they do not translate these products efficiently. Competition experiments demonstrated that late T7 mRNA does not inhibit B. subtilis ribosomal translation of B. subtilis derived mRNA (from the bacteriophage phi 29). It is concluded that strong Shine-Dalgarno sequences may be necessary in B. subtilis translation initiation sites; however, additional determinants of initiation which differ from those found in the translation initiation sites of E. coli mRNAs must exist.

Bacillus subtilis↗
PubMed1985

Evidence for overlapping active sites in a multifunctional enzyme: immunochemical and chemical modification studies on C1-tetrahydrofolate synthase from Saccharomyces cerevisiae.

The relationship of the active sites which catalyze the three reactions in the trifunctional enzyme C1-tetrahydrofolate synthase (C1-THF synthase) from Saccharomyces cerevisiae has been examined with immunochemical and chemical modification techniques. Immunotitration of the enzyme with a polyclonal antiserum resulted in identical inhibition curves for the dehydrogenase and cyclohydrolase activities which were distinctly different from the inhibition curve for the synthetase activity. During chemical modification with diethyl pyrocarbonate (DEPC), the three activities were inactivated at significantly different rates, indicating that at least three distinct essential residues are involved in the reaction with DEPC. The pH dependence of the reaction with DEPC was consistent with the modification of histidyl residues. Treatment of C1-THF synthase with N-ethylmaleimide (NEM) resulted in significant inactivation of only the dehydrogenase and cyclohydrolase activities, with the cyclohydrolase at least an order of magnitude more sensitive than the dehydrogenase. Inactivation of cyclohydrolase was biphasic at NEM concentrations above 0.1 mM, suggesting two essential cysteinyl residues were being modified. NADP+, a dehydrogenase substrate, protected both dehydrogenase and cyclohydrolase activities, but not synthetase activity, against inactivation by either reagent. Synthetase substrates had no protective ability. Pteroylpolyglutamates and p-aminobenzoic acid polyglutamates exhibited some protection of all three activities. The p-aminobenzoic acid polyglutamate series showed progressive protection with increasing chain length. These results are consistent with an overlapping site for the dehydrogenase and cyclohydrolase reactions, independent from the synthetase active site. Possible active-site configurations and the role of the polyglutamate tail in substrate binding are discussed.

Aminohydrolases↗
PubMed1985

Regulation of expression of the ADE3 gene for yeast C1-tetrahydrofolate synthase, a trifunctional enzyme involved in one-carbon metabolism.

C1-THF (5,6,7,8-tetrahydrofolate) synthase is a trifunctional protein catalyzing the sequential reactions specified by the enzymes 10-formyl-THF synthetase (EC 6.3.4.3), 5,10-methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5). These three activities supply the activated one-carbon units required for the biosynthesis of purines, thymidylate, the amino acids histidine and methionine, the vitamin pantothenic acid, and the formyl group of mitochondrial fMet-tRNAfMet. Extracts of Saccharomyces cerevisiae whose growth is dependent on the three activities of C1-THF synthase contain 2-3 times the level of enzyme activity of extracts from cells grown under conditions where they are independent of this enzyme. Repression of C1-THF synthase activity requires the simultaneous presence of adenine, histidine, methionine, and pantothenic acid. Starvation of the cells for any one of these nutrients leads to derepression of the enzyme. Drug-induced folate starvation also leads to derepression of enzyme activity. The response to changing nutritional conditions occurs within 1 h and is due to changes in the steady-state concentration of C1-THF synthase enzyme, rather than to activation or deactivation of a pre-existing pool of enzyme. Determination of the amount of C1-THF synthase mRNA under the various growth conditions by an in vitro translation/immunoprecipitation assay indicates that regulation of the enzyme occurs predominantly at a pretranslational level since steady-state levels of C1-THF synthase mRNA are 2-3-fold higher in derepressed cells than in repressed cells.

Acetates↗