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J C Rabinowitz

Publications and source records attributed to J C Rabinowitz.

At least 55 records · Page 3Linked to original sources
PubMed1985

Cloning and nucleotide sequence determination of the Clostridium pasteurianum ferredoxin gene.

We have constructed a library of Clostridium pasteurianum DNA cloned in the plasmid pBR322. Based on the known amino acid sequence for C. pasteurianum ferredoxin, a 64-fold degenerate heptadecanucleotide pool was synthesized. This mixed probe hybridized to two clones which were shown to contain greater than 6 kilobase pairs of the same genomic DNA. Sequence analysis of a common Sau3A1 0.6-kilobase-pair fragment revealed that it contains the information for the apoferredoxin structural gene. According to the DNA sequence, the only post-translational processing of this small apoprotein is the hydrolysis of the initiator methionine. Putative transcription and translation start and stop signals are present within the sequence.

Base Sequence↗
PubMed1985

The effects of presentation rate and encoding task on age-related memory deficits.

Young and old adults studied several lists of words at each of three presentation durations (1.5, 3, and 6 s/word). Under standard, intentional learning instructions the magnitude of the age-related recall deficit increased with longer presentation durations. When a semantic orienting question was used to guide the encoding of the items on each list, however, an equivalent benefit of longer study times was observed for the two age groups. In delayed recognition testing, the age groups also showed equivalent benefits from longer study times. These results were interpreted within the context of a processing deficit framework of age differences in memory, in which age differences are due to differences in the spontaneous use of organizational strategies. The results are not well described by a cognitive slowing account of age differences in memory.

Adolescent↗
PubMed1984

Aging and recognition failure.

Young and old adults studied lists of A-B paired associates that were subsequently tested for both cued recall and recognition of the B items. Significant age deficits were found for both recall and recognition. The age deficit in recognition was attributed to differences in the effectiveness of the retrieval process in recognition, with no age deficit in the process of evaluating an item's familiarity. Recognition failure, the failure to recognize items that are subsequently recalled, was also observed; older adults showed a higher recognition failure rate than younger adults. These results are discussed within the context of current theories of the recognition failure phenomenon.

Adolescent↗
PubMed1983

Chemical synthesis of folylpolyglutamates, their reduction to tetrahydro derivatives, and their activity with yeast C1-THF synthase.

Pteroyldi- through -pentaglutamic acids were synthesized through the use of the carbodiimide method, rather than the anhydride method or solid phase synthesis. The compounds were converted to the tetrahydro derivatives by enzymatic reduction. The Km values of the folate coenzymes for yeast C1-THF synthase were determined. The determination of the values for the formyltetrahydrofolate synthetase activity of the multifunctional enzyme was possible through the use of newly devised assay based on the fluorescence properties of the pteridine derivatives. The value for the tetraglutamyl coenzyme derivative was approximately 1000-fold lower than that of the monoglutamyl coenzyme, tetrahydrofolate.

Aminohydrolases↗
PubMed1983

Immunological crossreactivity of eukaryotic C1-tetrahydrofolate synthase and prokaryotic 10-formyltetrahydrofolate synthetase.

Antiserum to yeast C1-tetrahydrofolate (C1-H4folate) synthase reacts with other eukaryotic C1-H4folate synthases and prokaryotic 10-formyltetrahydrofolate (10-CHO-H4folate) synthetases [formate:tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3] even though these enzymes vary in subunit size and function and probably vary widely in sequence. The comigration of the purified enzymes with the immunoreactive material establishes the specificity of the reaction for C1-H4folate synthase proteins. Reciprocal crossreaction of the antibody to Clostridium acidiurici 10-CHO-H4folate synthetase with the eukaryotic proteins indicates that such broad cross-species reactions are not specific to the antisera elicited in response to the yeast C1-H4folate synthase. These specific crossreactions among divergent species have been observed only on an electrophoretic transfer blot of a denaturing polyacrylamide gel. These observations may have been possible because of the sensitivity and specificity of the technique, which differ from more conventional immunochemical methods.

Aminohydrolases↗
PubMed1982

Nucleotide sequences of transcription and translation initiation regions in Bacillus phage phi 29 early genes.

phi 29 DNA directs the synthesis of three major proteins of Mr = 22,400, 13,900, and 10,500 in a cell-free transcription-translation system derived from Bacillus subtilis. We have determined the locations of the coding regions for these early proteins on the phi 29 genome, and our results are in agreement with genetic evidence that the 22.4-kilodalton protein is the product of cistron 17 (p17) and the 13.9-kilodalton protein is the product of cistron 6 (p6). The 13.9-kilodalton and 10.5-kilodalton proteins are encoded on a polycistronic mRNA previously designated G3b RNA. We have determined the nucleotide sequence of a HindIII restriction fragment of phi 29 DNA, the H fragment, that encodes the 13.9-kilodalton protein and contains two early promoters, G3a and G3b. The nucleotide sequence of the ribosome binding site contains a polypurine region capable of forming a very stable complex with nine bases on the 3' end of B. subtilis 16 S rRNA. This strong Shine-Dalgarno complementarity supports the hypothesis that an extensive mRNA . rRNA interaction is a requirement for efficient translation by B. subtilis ribosomes (McLaughlin, J. R., Murray, C. L., and Rabinowitz, J. C. (1981) J. Biol. Chem. 256, 11283-11291). The nucleotide sequences of both promoters are very similar to the "consensus" sequence for Escherichia coli promoters. While E. coli RNA polymerase initiates transcription from both of these promoters in vitro, only the G3b promoter is utilized by B. subtilis RNA polymerase under the same conditions.

Bacillus subtilis↗
PubMed1982

Aging and metamemory: the roles of relatedness and imagery.

The hypothesis that age deficits in long-term episodic memory tasks are due to imparied metamemorial skills was investigated in a paired-associate task that required participants to predict the likelihood of recalling individual items. Both young and old adults were able to predict their ability to recall, and both age groups were equally sensitive to the effects of differences in the degree of relatedness between word-pair members on subsequent recall. However, both old and young adults were insensitive to the beneficial effects of using interactive imagery at encoding. It was concluded that differences in metamemorial skills are not responsible for age differences in memory.

Adolescent↗
PubMed1981

Plasmid-directed expression of Staphylococcus aureus beta-lactamase by Bacillus subtilis in vitro.

A plasmid carrying the Gram-positive Staphylococcus aureus PC1 beta-lactamase gene is active in directing a cell-free transcription and translation system from Bacillus subtilis. The major protein synthesized has been identified as the S. aureus beta-lactamase on the basis of peptide mapping. The protein is larger than the extracellular enzyme by about Mr = 3100. Significant in vitro translation of the beta-lactamase mRNA occurs in the absence of the initiation factor fraction as is characteristic of translation of mRNAs of Gram-positive origin. The 1250 base transcript that encodes the beta-lactamase and leader sequence has been mapped on the plasmid molecule.

Bacillus subtilis↗
PubMed1981

Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus beta-lactamase gene.

The base sequence of the ribosome binding site region of the Gram-positive Staphylococcus aureus beta-lactamase gene has been determined. The leader peptide sequence of 24 amino acids which precedes the NH2 terminus of extracellular S. aureus beta-lactamase has also been established. This initiation site possesses two unique features not observed for most initiation sites recognized by Escherichia coli ribosomes. A novel initiation codon, UUG, initiates protein synthesis with methionine; and a very strong Shine-Dalgarno complementarity containing five G-C base pairs precedes the UUG initiation codon. The strong Shine-Dalgarno complementarity may explain the reduced translational dependence on initiation factor IF-3 function that has been observed for the beta-lactamase mRNA and other mRNAs from Gram-positive bacteria. We suggest that this extent of complementarity between the mRNA and this extent of complementarity between the mRNA and the ribosome may be a requirement for efficient initiation by Bacillus subtilis and other Gram-positive ribosomes, and may provide the basis for the observed inability of the Gram-positive systems to translate most of the mRNAs from Gram-negative bacteria.

Amino Acid Sequence↗
PubMed1981

RNA polymerase from Clostridium acidi-urici. Characterization of a naturally occurring rifampicin-resistant bacterial enzyme.

We report here the isolation of a prokaryotic RNA polymerase that shows pronounced template specificity. The enzyme from Clostridium acidi-urici is highly active on DNA templates isolated from phage that infect Gram-positive organisms and is essentially inactive at either high or low ionic strength on DNA from phage associated with Gram-negative bacteria. The enzyme is also unique among RNA polymerases isolated from wild type bacteria in being highly resistant to inhibition by rifampicin. These properties are characteristic of the enzyme present in several independently isolated strains of C. acidi-urici. However, RNA polymerase present in other clostridial species resembles the enzyme present in Bacillus subtilis in sensitivity to rifampicin and template specificity.

Clostridium↗
PubMed1981

Initiation factor-independent translation of mRNAs from Gram-positive bacteria.

Initiation factor-independent translation of mRNA derived from bacillus phage phi29 DNA occurs with translation systems derived from Bacillus subtilis or Escherichia coli. This is in sharp contrast to the strict dependence on ribosome salt wash fraction of E. coli ribosomes for the translation of T7 and other mRNAs derived from Gram-negative organisms.

Bacillus subtilis↗
PubMed1980

Specificity of promoter site utilization in vitro by bacterial RNA polymerases on Bacillus phage phi 29 DNA. Transcription mapping with exonuclease III.

Bacillus subtilis RNA polymerase holoenzyme transcribes phi 29 DNA in vitro producing five major RNA species defined by characteristic electrophoretic mobilities. In addition to these products, Escherichia coli RNA polymerase transcribes phi 29 DNA to yield three RNA species not detected when transcribing with the B. subtilis enzyme under the same optimal reaction conditions for RNA synthesis. Transcriptional analysis of purified restriction fragments and exonuclease III-digested DNA established locations of six promoter and three termination sites defining the eight transcripts. The transcription map shows that E. coli RNA polymerase initiates transcription at three sites not efficiently utilized by the B. subtilis enzyme. However, initiation by the B. subtilis polymerase from at least two of these sites could be detected at E:DNA ratios greater than 10 in the absence of competing promoters. These results indicate that differences between the two polymerases in promoter utilization are not explained by specificity of promoter binding, but represent differences in responding to promoter strength. Transcription of phi 29 DNA and T7 DNA by E. coli core polymerase with either B. subtilis or E. coli sigma subunits results in formation of transcripts identical with those produced by E. coli holoenzyme, suggesting that core polymerase contains elements important in determining relative promoter strength. The efficiency of rifampicin-resistant complex formation on phi 29 and T7 promoters is also dependent upon the source of core polymerase.

Bacillus subtilis↗
PubMed1980

Clostridial apoferredoxin messenger ribonucleic acid. Assay and partial purification.

An assay for Clostridium pasteurianum apoferredoxin messenger ribonucleic acid (mRNA) was developed, based on the synthesis of the protein in vitro. Quantitation of apoferredoxin synthesis was accomplished by trypsinization of the cell-free incubation labeled with 3H- or 14C-labeled amino acids, separation of the products by SDS-urea polyacrylamide gel electrophoresis, and excising and counting the NCS-solubilized gel band corresponding to the unique 52-amino acid tryptic peptide derived from apoferredoxin. Its synthesis was shown to be RNA dependent, and was optimized with respect to several parameters of the in vitro protein-synthesizing system. The specificity of the assay was examined with RNA from Clostridium acidi urici, a related species the ferredoxin of which does not yield the 52-amino acid tryptic peptide, and by the use of [3H]leucine, which is not present in C. pasteurianum apoferredoxin. By these methods, the overestimation of apoferredoxin synthesis due to the comigration of fragments from other in vitro products with the legitimate apoferredoxin-derived peptide could be accounted for. The apoferredoxin mRNA was partially purified by the sequential zonal sucrose gradient centrifugation of total RNA followed by Sephadex G-200 chromatography of the enriched RNA, after which a fraction was obtained in which apoferredoxin mRNA was 20-fold enriched. The enriched RNA fraction can now be used for further purification of the apoferredoxin-coding sequences by cloning procedures.

Apoproteins↗
PubMed1980

Methylenetetrahydrofolate-dependent biosynthesis of ribothymidine in transfer RNA of Streptococcus faecalis. Evidence for reduction of the 1-carbon unit by FADH2.

The methyl carbon of ribothymidine in Loop IV of the tRNA of Streptococcus faecalis, Bacillus subtilis, and some other microorganisms is derived directly from 5,10-methylenetetrahydrofolate, not S-adenosylmethionine. The pure enzyme from S. faecalis also requires FADH2. We have obtained evidence that tetrahydrofolate is a product of the reaction and demonstrated that label from [5-3H]5-deazaFMNH2 is incorporated into the methyl moiety of ribothymidine. These data indicate that the enzyme uses methylenetetrahydrofolate solely as a 1-carbon donor and employs FADH2 as a reducing agent in vitro according to the following reaction: tRNA(U psi C) + CH2 = THF + FADH2 leads to tRNA(T psi C) + THF + FAD.

Enterococcus faecalis↗
PubMed1980

Formyl-methenyl-methylenetetrahydrofolate synthetase(combined) from yeast. Biochemical characterization of the protein from an ade3 mutant lacking the formyltetrahydrofolate synthetase function.

A protein from Saccharomyces cerevisiae mutant ade3-1050, a formyltetrahydrofolate synthetase-deficient mutant, has been purified to apparent homogeneity. The purified mutant enzyme shows both methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities, but lacks formyltetrahydrofolate synthetase activity. The biochemical characterization of the mutant protein described in this paper is consistent with genetic data which indicate that the 1050 mutation is a point mutation at the ade3 locus of chromosome VII of S. cerevisiae. The molecular weight of the native mutant protein (Mr = 227,000 by exclusion chromatography), as well as the number and size of its subunits are exactly the same as those of the trifunctional wild type enzyme. In addition, both proteins have the same sedimentation behavior in a glycerol density gradient (s20,w = 9.4 S), and their activities and structures are equally affected by exposure to mild tryptic degradation. ATP protects both enzymes from tryptic degradation, but NADP+ does not. Some of the kinetic properties of the activities of both enzymes were also determined and were essentially similar. Although both enzymes require the presence of metals for maximal synthetase and dehydrogenase activities, metals are not necessary to maintain their structures intact.

Aminohydrolases↗