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R Levitz

Publications and source records attributed to R Levitz.

14 recordsLinked to original sources
PubMed1999

Cytotoxic hammerhead ribozymes.

Small catalytic RNA molecules of the hammerhead ribozyme type were found to have cytotoxic effects unrelated to their intended activity. An expression library of ribozyme sequence variants was constructed in a recA-deficient strain of Escherichia coli such that individual library members differed in regions designed to form base pairs with human immunodeficiency virus-1 (HIV-1) tat mRNA. The parental ribozyme and many variants exhibited a bacteriostatic effect. One variant studied in detail was also bactericidal. When its expression was induced, ribozyme-dependent inhibition of bacterial growth was not observed in recA+ or recA+ lexA3 (Ind-) cells, suggesting that the recombination function of the RecA protein, not the absence of the SOS response, is sufficient to alleviate the cytotoxic effect. These data document the need for careful testing for toxic effects during intracellular studies of ribozyme action.

Base Sequence↗
PubMed1998

Degradation of hammerhead ribozymes by human ribonucleases.

Hammerhead ribozymes were used as substrates to examine endoribonucleolytic activities in cell extracts and cultured human cells. Primer-extension analyses showed that ribozymes directed against tumor necrosis factor-alpha mRNA and human immunodeficiency virus type 1 tat mRNA were cleaved at UA and CA dinucleotides by extracts. Preferred cleavage sites were similar to those observed following digestion with RNase A, and cleavage was blocked by RNasin, an inhibitor of pyrimidine-specific ribonucleases. Removal of UA and CA dinucleotides rendered ribozymes more stable when incubated in cell extracts that were not significantly contaminated by extracellular nucleases. Placement of UA dinucleotides adjacent to a ribozyme in mRNA led to excision of the ribozyme from long transcripts during incubation in extracts. UA dinucleotides also made mRNA more labile than a control RNA when expressed from an endogenous plasmid gene in the human myeloid cell line U937. Similarly, UA and CA dinucleotides caused ribozymes to have a shorter half-life when delivered to U937 cells by lipofectin-mediated transformation. Taken together, these data indicate that one or more members of the pyrimidine-specific ribonuclease family is involved in the intracellular degradation of RNA, and they explain the paucity of UA dinucleotides in eukaryotic mRNA. Judicious manipulation of preferred target sequences of pyrimidine-specific ribonucleases may be useful in designing effective hammerhead ribozymes.

Base Sequence↗
PubMed1994

In vivo decay kinetic parameters of hammerhead ribozymes.

Ribozymes offer a potentially important way to inactivate intracellular RNA from almost any gene whose nucleotide sequence is known. Recently, we found that hammerhead ribozymes directed against mRNA of tumour necrosis factor alpha (TNF alpha) and its derivatives, preferentially bind to a cellular protein(s). To better understand the effect of different 3'-terminal hairpins on ribozyme stability as well as their effect on the protein binding to the ribozyme, a mathematical treatment of the decay of three TNF alpha ribozymes that differed at their 3' ends was performed. One ribozyme contained a 3'-terminal hairpin derived from a transcription terminator of bacteriophage T7, another contained the same hairpin but modified to be highly enriched for G+C nucleotides, and a third lacked a hairpin. The TNF alpha ribozyme decay had two kinetic components. The slow component exhibited exponential decay with a half life of approximately 250 h in all cases. The 3'-terminal hairpin has no significant effect on this component. This slow phase accounted for 60-80% of ribozyme decay. The rapid phase also exhibited exponential decay. For this phase, a 3'-terminal hairpin roughly doubled the half-life (1.7-3.4). The slow phase of degradation was about three times faster for a ribozyme directed at the integrase mRNA of human immunodeficiency virus-1 than that seen with the TNF alpha ribozyme. Taken together, these results suggest that the ribozyme population is initially sensitive to degradation, with the presence of a hairpin provides some protection, and indicate that the addition of the hairpin to the ribozyme did not prevent the in vivo additional stabilizing effect of the protein(s).

Base Sequence↗
PubMed1994

HIV-1 integrase blocks infection of bacteria by single-stranded DNA and RNA bacteriophages.

Expression of human immunodeficiency virus-1 integrase in Escherichia coli, at levels that had no effect on bacterial cell growth, blocked plaque formation by bacteriophages having single-stranded genomic DNA (M13) or RNA (R17, Q beta, PRR1). Plaque formation by phages having double-stranded genomic DNA (T4, PR4) was unaffected. Integrase also inhibited infection by the phagemid M13KO7, but it had no effect on production of phage once infection by M13KO7 was established. This result indicated that integrase affects an early stage in infection. Integrase also inhibited phage production following transfection by either single-stranded or double-stranded (replicative form) M13 DNA, it blocked M13 DNA replication, as assayed by incorporation of radioactive nucleotides into DNA, and it failed to affect bacterial pilus function. These data suggest that integrase interacts in vivo with phage nucleic acid, a conclusion supported by studies in which integrase was shown to have a DNA-binding activity in its C-terminal portion. This portion of integrase was both necessary and sufficient for interference of plaque formation by M13 in the present study. Expression of the N-terminal portion of integrase at the same level as intact integrase had little effect on phage growth, indicating that expression of foreign protein in general was not responsible for the inhibitory effect. The simple bacteriophage assay described is potentially useful for identifying integrase mutants that lack single-stranded DNA binding activity.

Antiviral Agents↗
PubMed1993

Construction of a Synechocystis PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes.

The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803 delta rbc, were detected within 8 days when grown under 5% CO2 in air. These transformants were unable to grow in air (0.03% CO2). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO2 in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803 delta rbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.

Amino Acid Sequence↗
PubMed1990

The optional E. coli prr locus encodes a latent form of phage T4-induced anticodon nuclease.

The optional Escherichia coli prr locus restricts phage T4 mutants lacking polynucleotide kinase or RNA ligase. Underlying this restriction is the specific manifestation of the T4-induced anticodon nuclease, an enzyme which triggers the cleavage-ligation of the host tRNALys. We report here the molecular cloning, nucleotide sequence and mutational analysis of prr-associated DNA. The results indicate that prr encodes a latent form of anticodon nuclease consisting of a core enzyme and cognate masking agents. They suggest that the T4-encoded factors of anticodon nuclease counteract the prr-encoded masking agents, thus activating the latent enzyme. The encoding of a tRNA cleavage-ligation pathway by two separate genetic systems which cohabitate E. coli may provide a clue to the evolution of RNA splicing mechanisms mediated by proteins.

Amino Acid Sequence↗
PubMed1987

Bacteriophage T4 anticodon nuclease, polynucleotide kinase and RNA ligase reprocess the host lysine tRNA.

Host tRNAs cleaved near the anticodon occur specifically in T4-infected Escherichia coli prr strains which restrict polynucleotide kinase (pnk) or RNA ligase (rli) phage mutants. The cleavage products are transient with wt but accumulate in pnk- or rli- infections, implicating the affected enzymes in repair of the damaged tRNAs. Their roles in the pathway were elucidated by comparing the mutant infection intermediates with intact tRNA counterparts before or late in wt infection. Thus, the T4-induced anticodon nuclease cleaves lysine tRNA 5' to the wobble position, yielding 2':3'-P greater than and 5'-OH termini. Polynucleotide kinase converts them into a 3'-OH and 5' P pair joined in turn by RNA ligase. Presumably, lysine tRNA depletion, in the absence of polynucleotide kinase and RNA ligase mediated repair, underlies prr restriction. However, the nuclease, kinase and ligase may benefit T4 directly, by adapting levels or decoding specificities of host tRNAs to T4 codon usage.

Base Sequence↗
PubMed1987

Native-valve endocarditis caused by Staphylococcus epidermidis. A histologically confirmed case.

Staphylococcus epidermidis is among the most common organisms isolated from blood cultures. Conversely, it is rarely a well-documented cause of natural-valve endocarditis. However, several authors have reported series of patients with the clinical picture of endocarditis and S. epidermidis bacteremia. Most of these cases have not been confirmed by examination of the valve. The authors present a case of natural-valve endocarditis caused by S. epidermidis with pathologic documentation of the offending agent.

Endocarditis, Bacterial↗
PubMed1985

The effect of the locus pstB on phosphate binding in the phosphate specific transport (PST) system of Escherichia coli.

The periplasmic phosphate binding protein is a product of the phoS gene and is an essential component of the phosphate specific transport (PST) system, which mediates Pi uptake in Escherichia coli. The binding of Pi to periplasmic protein(s) and the kinetic parameters of Pi uptake were studied in phoT and pstB mutants of E. coli. These mutants are impaired in Pi uptake but have a periplasmic Pi-binding protein whose Pi-binding capacity was estimated by the retention kinetics. The Pi-binding activity in two pstB mutants was found to be weaker as compared to phoT9 and the wild type. The KD values for Pi binding to periplasmic protein were determined by equilibrium dialysis. In the pstB mutants the KD value was found to be 9-31 times higher than the values obtained for the wild type and the phoT mutant. The apparent Km values for Pi uptake in one pstB mutant is 14.3 times higher than in the wild type. Vmax of the mutant is 8.3 times lower that of the wild type. The data indicate that pstB, an essential gene of the PST transport system, is promoting the binding capacity of the Pi-binding protein.

Bacterial Proteins↗
PubMed1984

A new locus in the phosphate specific transport (PST) region of Escherichia coli.

PhoS64 is a mutation in the Phosphate Specific Transport (PST) region on the E. coli chromosome which lacks the periplasmic phosphate binding protein. In contrast to other phoS mutations (which have the same phenotype) it complements the mutations in phoT and pstB. A detailed genetic map of the PST region constructed by three point transductional crosses has revealed that phoS64 is located distally from other phoS mutations. The genetic order obtained was phoS64-phoU35-pstB401-phoT-phoS-ilvC. The data indicate that phoS64 belongs to a different complementation unit in the PST region not known hitherto. We propose to name it phoV.

Bacterial Proteins↗
PubMed1979

Isolation and properties of Escherichia coli K-12 mutants impaired in the utilization of gamma-aminobutyrate.

We have isolated mutants of Escherichia coli K-12 CS101B that have lost the ability to utilize gamma-aminobutyrate as a source of nitrogen. One class of mutants, which were not affected in the utilization of other nitrogen sources (proline, arginine, glycine), included many isolates with lesions in gamma-aminobutyrate transport or in its transamination and one mutant completely devoid of succinic semialdehyde dehydrogenase activity and exhibiting low gamma-aminobutyrate transport and transamination. gamma-Aminobutyrate-utilizing revertants of the latter recovered full transport and transamination capacities but remained dehydrogenaseless. Another class of mutants showed pleiotropic defects in nitrogen metabolism. One such mutant was lacking glutamate synthase activity. The genes specifying the synthesis of gamma-aminobutyrate permease, gabP, gamma-aminobutyrate transaminase, gabT, and succinic semialdehyde dehydrogenase, gabD, and the control gene, gabC, that coordinately regulates their expression all form a cluster on the E. coli chromosome, linked to the srl and recA loci (at 57.5 min). The mutations with pleiotropic effects on the metabolism of nitrogenous compounds are not linked to the gab cluster.

4-Aminobutyrate Transaminase↗
PubMed1978

Specificity and regulation of gamma-aminobutyrate transport in Escherichia coli.

A specific gamma-aminobutyrate (GABA) transport system in Escherichia coli K-12 cells with a K(m) of 12 muM and a V(max) of 278 nmol/ml of intracellular water per min is described. Membrane vesicles contained d-lactate-dependent activity of the system. Mutants defective in GABA transport were isolated; they lost the ability to utilize GABA as a nitrogen source, although the activities of glutamate-succinylsemialdehyde transaminase (GSST) (EC 2.6.1.19) and succinylsemialdehyde dehydrogenase (SSDH) (EC 1.2.1.16), the enzymes that catalyze GABA utilization, remained as high as in the parental CS101B strain. The ability to utilize l-ornithine, l-arginine, putrescine, l-proline, and glycine as a nitrogen source was preserved in the mutants. The genetic lesions resulting in the loss of GABA transport, gabP5 and gabP9, mapped in the gab gene cluster in close linkage to gabT and gabD, the structural genes of GSST and SSDH, and to gabC, a gene controlling the utilization of GABA, arginine, putrescine, and ornithine. The synthesis of the GABA transport carrier is subject to dual physiological control by (i) catabolite repression and (ii) nitrogen availability. Experiments with glutamine synthetase (EC 6.3.1.2)-negative and with glutamine synthetase-constitutive strains strongly indicate that this enzyme is the effector in the regulation of GABA carrier synthesis by route (ii).

Aminobutyrates↗