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G Kaufmann

Publications and source records attributed to G Kaufmann.

At least 55 records · Page 3Linked to original sources
PubMed1990

Two DNA polymerases may be required for synthesis of the lagging DNA strand of simian virus 40.

Agents discriminating between DNA polymerase alpha and DNA polymerases of class delta (polymerase delta or epsilon) were used to characterize steps in the synthesis of the lagging DNA strand of simian virus 40 during DNA replication in isolated nuclei. The synthesis of lagging-strand intermediates below 40 nucleotides, termed DNA primers (T. Nethanel, S. Reisfeld, G. Dinter-Gottlieb, and G. Kaufmann, J. Virol. 62:2867-2873, 1988), was selectively inhibited by butylphenyl dGTP or by neutralizing DNA polymerase alpha monoclonal antibodies. The synthesis of longer lagging chains of up to 250 nucleotides (Okazaki pieces) was affected to a lesser extent, possibly indirectly, by these agents. Aphidicolin, which inhibits both alpha- and delta-class enzymes, elicited the opposite pattern: DNA primers accumulated in its presence and were not converted into Okazaki pieces. These and previous data suggest that DNA polymerase alpha primase synthesizes DNA primers, whereas another DNA polymerase, presumably DNA polymerase delta or epsilon, mediates the conversion of DNA primers into Okazaki pieces.

Animals↗
PubMed1989

In vitro reconstitution of anticodon nuclease from components encoded by phage T4 and Escherichia coli CTr5X.

During phage T4 infection of Escherichia coli strains containing the prr locus the host tRNALys undergoes cleavage-ligation in reactions catalyzed by anticodon nuclease, polynucleotide kinase and RNA ligase. Known genetic determinants of anticodon nuclease are prr, which restricts T4 mutants lacking polynucleotide kinase or RNA ligase, and stp, the T4 suppressor of prr encoded restriction. The present communication describes an in vitro anticodon nuclease assay in which the specific cleavage of tRNALys is driven by an extract from E. coli prrr (restrictive) cells infected by phage T4. The in vitro anticodon nuclease reaction requires factor(s) encoded by prr, is stimulated by a synthetic Stp polypeptide and appears to require additional T4 induced factor(s) distinct from Stp.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Nucleotide and deduced amino acid sequence of stp: the bacteriophage T4 anticodon nuclease gene.

Pre-existing host tRNAs are reprocessed during bacteriophage T4 infection of certain Escherichia coli strains. In this pathway, tRNALys is cleaved 5' to the wobble base by anticodon nuclease and is later restored in polynucleotide kinase and RNA ligase reactions. Anticodon nuclease depends on prr, a locus found only in host strains that restrict T4 mutants lacking polynucleotide kinase and RNA ligase; and on stp, the T4 suppressor of prr restriction. stp was cloned and the nucleotide sequences of its wild-type and mutant alleles determined. Their comparison defined an stp open reading frame of 29 codons at 162.8 to 9 kb of T4 DNA (1 kb = 10(3) base-pairs). We suggest that stp encodes a subunit of anticodon nuclease, perhaps one that harbors the catalytic site; while additional subunits, such as a putative prr gene product, impart protein folding environment and tRNA substrate recognition.

Amino Acid Sequence↗
PubMed1988

An Okazaki piece of simian virus 40 may be synthesized by ligation of shorter precursor chains.

It is generally accepted that an aphidicolin-sensitive DNA polymerase elongates the eucaryotic RNA primer (iRNA) into a mature Okazaki piece reaching ca. 200 nucleotides. Yet, as shown here, nascent DNA chains below 40 nucleotides accumulated in simian virus 40 (SV40) DNA replicating in isolated nuclei in the presence of aphidicolin. These products resembled precursors of longer Okazaki pieces synthesized in the absence of aphidicolin (termed here DNA primers) in size distribution, lagging-replication-fork polarity, and content of iRNA. Within the isolated SV40 replicative intermediate, DNA primers could be extended in a reaction catalyzed by the Escherichia coli DNA polymerase I large fragment. This increased their length by an average of 21 deoxyribonucleotide residues, indicating that single-stranded gaps of corresponding length existed 3' to the DNA primers. Incubation with T4 DNA ligase converted most of the extended DNA primers into products resembling long Okazaki pieces. These data led us to propose that the synthesis of an SV40 Okazaki piece could be itself discontinuous and could comprise the following steps: (i) iRNA synthesis by DNA primase, (ii) iRNA extension into a DNA primer by an aphidicolin-resistant activity associated with DNA primase-DNA polymerase alpha, (iii) removal of iRNA moieties between adjacent DNA primers, (iv) "gap filling" between DNA primers by the aphidicolin-sensitive DNA polymerase alpha, and (v) ligation of DNA primer units onto a growing Okazaki piece. Eventually, a mature Okazaki piece is ligated onto a longer nascent DNA chain.

Aphidicolin↗
PubMed1987

Pharmacodynamics of 3-hydroxyquinidine alone and in combination with quinidine in healthy persons.

The relation between serum concentration of 3-hydroxyquinidine (3-OHQ), a major metabolite of quinidine in humans, and the pharmacologic effect alone and in combination with the parent drug was studied. The heart rate-corrected, computer-averaged QT interval (QTc) was used as the pharmacologic endpoint. In a randomized, double-blind study, 5 healthy subjects received, on 3 separate days 1 week apart, either (1) 300 to 400 mg 3-OHQ orally or (2) 150 mg quinidine base intravenously or (3) a combination in the same doses. Blood samples and electrocardiographic recordings were obtained over the following 10 hours. Serum concentrations of 3-OHQ and quinidine were determined by high-pressure liquid chromatography and the free fraction by ultrafiltration. Peak concentrations of 3-OHQ varied between 1,362 and 3,480 ng/ml after oral 3-OHQ ingestion, but were negligible after intravenous quinidine infusion. The free fraction was 49% +/- 4.8 (mean +/- standard deviation) for 3-OHQ and 20% +/- 4.3 for quinidine. In all 5 subjects a statistically significant correlation was found between serum concentration and QTc prolongation for both quinidine and 3-OHQ (largest p value less than 0.025). The mean slope of the regression line was 0.0184 +/- 0.0128 for 3-OHQ and 0.0297 +/- 0.0111 for quinidine. Multiple linear regression revealed in each subject a significant additive effect of 3-OHQ when administered together with quinidine (largest p value less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
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↗
PubMed1986

Phage and host genetic determinants of the specific anticodon loop cleavages in bacteriophage T4-infected Escherichia coli CTr5X.

Anticodon loop cleavages of two host tRNA species occur in bacteriophage T4-infected Escherichia coli CTr5X, a host strain restricting phage mutants deficient in polynucleotide kinase (pnk) or RNA ligase (rli). The cleavage products accumulate with the mutants but are further processed in wt infection through polynucleotide kinase and RNA ligase reactions. Inactivating mutations in stp suppress pnk- or rli- mutations in E. coli CTr5X and, as shown here, also abolish the anticodon nuclease, implicating the stp product with this activity. We show also that there exist other suppressing mutations of a pnk- (pseT2) mutation that appear not to affect the anticodon nuclease and are not in stp. It has been shown that a single locus in E. coli CTr5X, termed prr, determines the restriction of pnk- or rli- mutants. A transductant carrying prr featured upon infection the anticodon nuclease reaction products, suggesting that prr determines the specific manifestation of this activity. However, prr does not encode the tRNA species that are vulnerable to the anticodon nuclease.

Anticodon↗
PubMed1986

Comparison of S9 mix and hepatocytes as external metabolizing systems in mammalian cell cultures: cytogenetic effects of 7,12-dimethylbenzanthracene and aflatoxin B1.

Two external metabolizing systems, S9 mix from Aroclor-induced rat livers and freshly isolated hepatocytes, were used for activation in cultures of human lymphocytes and V79 cells. 7, 12-dimethylbenzanthracene (DMBA) and aflatoxin B1 (AFB1) were employed as indirectly acting reference mutagens. Mutagenic effects were measured by induction of sister chromatid exchange (SCE). With DMBA, SCE-inducing effects were found to be quite similar after activation by S9 mix and activation by hepatocytes. In human lymphocytes nearly the same dose-effect relationships were found with both metabolizing systems; in V79 cells the hepatocyte-mediated induction of SCE was detectable at slightly lower concentrations than the S9-mediated SCE induction. In contrast with AFB1, S9 activation led to a stronger SCE induction than hepatocyte activation in both target cells. The induction of chromosomal aberrations by AFB1 after activation by the two metabolizing systems was also analysed in V79 cells. This experiment again revealed that AFB1 was more efficiently activated by S9 mix than by hepatocytes, and it appeared that AFB1 is a more potent inducer of chromosomal aberrations than of SCE. The different activation capacities of the two metabolizing systems for AFB1 may be due to the maintenance of inactivation mechanisms in hepatocytes or to the Aroclor induction of the S9 fraction. Our experiments have shown that the suitability of hepatocytes as an activation system is not restricted to microbial or eukaryotic point mutation assays, but that hepatocyte metabolism can also be successfully included in cytogenetic tests with short- and long-term cultures of mammalian target cells.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1986

Influence of 1-double bond and 11 beta-hydroxy group on stereospecific microbial reductions of 4-en-3-oxo-steroids.

The yeast Rhodotorula glutinis and the anaerobic bacterium Clostridium paraputrificum were used for stereospecific reductions of 4-chloro-11 beta-hydroxy-17 alpha-methyl-testosterone and the corresponding 1-dehydro compound to prepare 5 alpha- and 5 beta-H derivatives, respectively. C. paraputrificum was able to 5 beta-reduce both substances, whereas the 5 alpha-reduction by R. glutinis was inhibited by the structure elements 1-en and 11 beta-OH so that the substrate with both structure elements was not 5 alpha-reduced. The microbial conversion of the four steroids with and without 1-en and 11 beta-OH was compared in semiquantitative experiments. A number of new substances are described, 11 beta-hydroxy and 11-oxo derivatives of 5 alpha- and 5 beta-dihydro-4-chloro-17 alpha-methyltestosterone including some 3-OH compounds and characterized by NMR, mass spectrometric and further data.

Clostridium↗
PubMed1986

Assignment of anabolic-androgenic and antiandrogenic properties to some chlorine-substituted steroids on the basis of their binding characteristics to the androgen receptor of the rat seminal vesicle.

In this study we investigated the affinity of several 4-chlorinated and 1-ene derivatives of 17 alpha-methyltestosterone (MT) and 17 alpha-methyl-5 alpha-dihydrotestosterone (MDHT) to the androgen receptor, and, additionally, the effect of a few MT-derived steroids on the activity of the 5 alpha-reductase enzyme present in the rat seminal vesicle. From our results we conclude, that delta 1 or/and delta 4 double bonds in ring A counteract the inhibition of receptor-binding caused by chlorine-substitution at C4; the dissociation of myotropic and androgenic effects [= M/A dissociation] of 4-chloro-MT (as compared to MT) is due to its inactivation by 5 alpha-reductase in androgen target organs and/or to the inhibition of the conversion of endogenous testosterone to DHT; the M/A dissociation of 1-ene-MT and 4-chloro-1-ene-MT may be explained by their inability to be activated by 5 alpha-reductase; for the same reason, M/A dissociation can be assigned to the effects of 4 alpha-chloro-1-ene-DHT. We determined the short-term and long-term competition of cyproterone acetate and chlormadinone acetate with [3H]DHT for receptor binding at 0 degrees C and showed, that the complexes formed by these antiandrogens with the androgen receptor have equally reduced stabilities compared to the DHT-receptor complex.

Anabolic Agents↗
PubMed1985

Host transfer RNA cleavage and reunion in T4-infected Escherichia coli CTr5x.

T4 mutants lacking polynucleotide kinase (pnk-) or RNA ligase (rli-) do not grow on E. coli CTr5x. During the abortive infections there accumulate host tRNA fragments that match into two species severed 3' to the anticodon. The CTr5x-specific fragments appear only transiently with wt phage, implicating the affected enzymes in phosphoryl group rearrangement and religation [David et al. (1982) Virol. 123, 480]. In a search for the vulnerable host tRNAs and putative religation products, tRNA ensembles from uninfected E. coli CTr5x or cells infected with various phage strains were fractionated and compared. A tRNA species absent from rli- infected cells but present in uninfected cells or late in wt infection was thus detected. RNase T1 finger prints of this species, isolated before or after wt infection, were compared with that of an in vitro ligated pair of CTr5x-specific fragments. The results indicated that this tRNA is cleaved upon infection and later on restored to it's original or to a very similar form, by polynucleotide kinase and RNA ligase reactions. It is suggested that depletion of such vulnerable host tRNA species underlies the restriction of pnk- or rli- phage on E. coli CTr5x.

Anticodon↗
PubMed1985

A-ring conformational stability and progesterone-receptor binding affinity of 4-en-3-one steroids.

The molecular structure and strain energy of 4-en-3-one steroids in two different A-ring conformations are calculated by means of a molecular mechanics technique. The computations for the isolated molecules provide the following order of increasing stability of the inverted A-ring conformers: 10-methyl, 19-nor, 9-ene compound. This tendency is in agreement with X-ray structure data for single crystals. The normal 1 alpha, 2 beta-half chair conformation of 10-methyl steroids is found to be stabilized by bond angles, mainly at C10, and non-bonded interactions from the 10-methyl group. Pitzer strains favour the inverted 1 beta, 2 alpha-half chair conformation in the case of 4,9-diene-3-one compounds. Binding affinities to the progesterone receptor decrease in the series: 19-nor, 9-ene, 10-methyl compound. In view of this ordering, the calculated relative stabilities of A-ring conformers are in conflict with a conformation-controlled receptor binding. Variations of receptor bond strengths are supposed to be more strongly influenced by a steric hindrance of the 10-methyl group and/or steroid-backbone flexibility.

Animals↗
PubMed1985

Cloning of nascent monkey DNA synthesized early in the cell cycle.

To study the structure and complexity of animal cell replication origins, we have isolated and cloned nascent DNA from the onset of S phase as follows: African green monkey kidney cells arrested in G1 phase were serum stimulated in the presence of the DNA replication inhibitor aphidicolin. After 18 h, the drug was removed, and DNA synthesis was allowed to proceed in vivo for 1 min. Nuclei were then prepared, and DNA synthesis was briefly continued in the presence of Hg-dCTP. The mercury-labeled nascent DNA was purified in double-stranded form by extrusion (M. Zannis-Hadjopoulos, M. Perisco, and R. G. Martin, Cell 27:155-163, 1981) followed by sulfhydryl-agarose affinity chromatography. Purified nascent DNA (ca. 500 to 2,000 base pairs) was treated with mung bean nuclease to remove single-stranded ends and inserted into the NruI site of plasmid pBR322. The cloned fragments were examined for their time of replication by hybridization to cellular DNA fractions synthesized at various intervals of the S phase. Among five clones examined, four hybridized preferentially with early replicating fractions.

Animals↗
PubMed1985

Properties of some monkey DNA sequences obtained by a procedure that enriches for DNA replication origins.

Twelve clones of monkey DNA obtained by a procedure that enriches 10(3)- to 10(4)-fold for nascent sequences activated early in S phase (G. Kaufmann, M. Zannis-Hadjopoulos, and R. G. Martin, Mol. Cell. Biol. 5:721-727, 1985) have been examined. Only 2 of the 12 ors sequences (origin-enriched sequences) are unique (ors1 and ors8). Three contain the highly reiterated Alu family (ors3, ors9, and ors11). One contains the highly reiterated alpha-satellite family (ors12), but none contain the Kpn family. Those remaining contain middle repetitive sequences. Two examples of the same middle repetitive sequence were found (ors2 and ors6). Three of the middle repetitive sequences (the ors2-ors6 pair, ors5, and ors10) are moderately dispersed; one (ors4) is highly dispersed. The last, ors7, has been mapped to the bona fide replication origin of the D loop of mitochondrial DNA. Of the nine ors sequences tested, half possess snapback (intrachain reannealing) properties.

Animals↗