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J Retèl

Publications and source records attributed to J Retèl.

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

By-pass of the major aminofluorene-DNA adduct during in vivo replication of single- and double-stranded phi X174 DNA treated with N-hydroxy-2-aminofluorene.

To examine the effects of aminofluorene-DNA adduct formation on the biological activity of DNA, single-stranded (ss) phi X174 DNA and phi X174 replicative form (RF) DNA were modified to different extents with 3H-labeled N-hydroxy-2-aminofluorene and subsequently transfected to Escherichia coli spheroplasts with different repair capabilities. When the fraction of active ss phi X174 DNA molecules was measured as a function of the mean number of adducts per molecule, exponential survival curves were obtained from which it could be deduced that in wild-type, uvrA- and recA- cells at least 86%, and in uvrC- cells at least 82% of the introduced adducts do not cause inactivation. In the case of RF DNA the survival curves are non-exponential, but they nevertheless show that an exceptionally high number of adducts per RF molecule must be introduced to destroy its biological activity. On average 52 adducts per RF molecule were needed to reduce the survival to 37%, irrespective of whether wild-type, uvrA- or recA- cells were used. On the other hand, the survival of the uvrC- cells was considerably lower, but even in these cells a majority of the adducts is not lethal. By h.p.l.c. analysis of the modified DNA after hydrolysis with trifluoroacetic acid, 81 and 84% of the adducts in ss- and RF DNA, respectively, could be identified as N-(guanin-8-yl)-2-aminofluorene. The results strongly indicate that this type of major modification product is very frequently by-passed during replication of both single- and double-stranded DNA. The results together with the data obtained by sucrose gradient analysis both before and after an alkali treatment and those obtained by h.p.l.c. analysis suggest that inactivation of ssDNA is mainly due to minor modifications such as secondary lesions consisting of chain breaks and alkali-labile sites together with unidentified interaction products.

Bacteriophage phi X 174↗
PubMed1984

Effects of adduct formation on the biological activity of single- and double-stranded øX174 DNA, modified by N-acetoxy-N-acetyl-2-aminofluorene.

In order to establish a good quantitative relationship between the number of acetylaminofluorene adducts and the extent of inactivation of DNA, single-stranded (ss) øX174 DNA and øX174 RF DNA were modified to various extents with 3H-labelled N-acetoxy-N-acetyl-2-aminofluorene (N-AcO-AAF) and subsequently transfected to Escherichia coli spheroplasts having different repair capabilities. Exponential survival curves were obtained. In the case of ssDNA about one adduct per molecule appears to be lethal. On the other hand only 1 out of 10.2 adducts is found to inactivate RF DNA if tested on wild-type E. coli. However, when assayed on strains deficient in excision repair 1 out of 2.3 adducts leads to inactivation of RF DNA. RecA-dependent postreplication repair only has little influence on these figures. Product analysis of the modified DNAs shows that in RF DNA at least 76% of the interaction products is N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (dGuo-C8-AAF) and at least 6% and at most 12% is 3-(deoxyguanosin-N2-yl)-N-acetyl-2-aminofluorene (dGuo-N2-AAF). In ssDNA only dGuo-C8-AAF is formed. No apurinic sites could be detected in the modified DNAs. From these results it can be concluded that in RF DNA most of the dGuo-C8-AAF is removed by excision repair. The remaining damage, consisting probably both of dGuo-N2-AAF and unexcised dGuo-C8-AAF, inactivates RF DNA. Inactivation can be explained by a model which shows that only damage in the minus strand of RF DNA inhibits replication and/or transcription.

2-Acetylaminofluorene↗
PubMed1984

Repair of damage in double-stranded phi X174 (RF) DNA due to radiation-induced water radicals.

Experiments in which the yields of radiation-induced OH and H radicals were varied, showed that both types of water radicals inactivate phi X174 RF DNA to about the same extent as measured by transfection of the (irradiated) DNA to E. coli wild-type spheroplasts. On the other hand, using spheroplasts prepared from E. coli strains, deficient in one of the proteins involved in excision DNA repair (uvrA- or uvrC-) or in post-replication repair (recA-), clear differences between damage originating from OH or H radical attack were found. Part of the radiation damage due to H radicals appeared to be repairable by an uvrA-gene-dependent repair mechanism, whereas this repair pathway does not play an important role in the case of OH radical damage. The reverse applies to uvrC-gene-dependent repair, which only affects OH radical damage (obtained under anoxic conditions), but has no influence on damage due to H radicals. Irradiation of double-stranded phi X174 (RF) DNA in the presence of oxygen however, yields damage--due to OH radicals only--which appeared not to be sensitive to either uvrC- or uvrA-gene-dependent repair. Furthermore, post-replication repair (recA) has only very little effect on the amount of inactivation by H or OH radicals, when irradiation is carried out under anoxic conditions. We did not find significant inactivation due to hydrated electrons, whether the biological activity was determined by use of wild-type spheroplasts or of strains deficient in excision or post-replication repair proteins.

Bacteriophage phi X 174↗
PubMed1978

Genetic organization of the ribosomal transcription units of the yeast Saccharomyces carlsbergensis.

The genetic organization of the multiple ribosomal transcription units (RTUs) on the genome of the yeast Saccharomyces carlsbergensis was studied by electron microscopy of purified ribosomal DNA hybridized to 26S rRNA using the R-loop technique (Thomas, M., White, R.L. and Davis, R.W. (1973) Proc. Natl. Acad. Sci. U.S. 73, 2294-2298). Plasmid pBR 322, the molecular weight of which is known, was used as a standard for converting contour length of double-stranded DNA into molecular weight. The 140 yeast RTUs were found to be arrayed in tandem repeats, each repeat containing at most 0.4 X 10(6) D (about 6% of the length of the RTU) of non-transcribed spacer DNA. The repeats, in turn, are arranged in a number of clusters separated by much longer stretches of non-ribosomal DNA.

Genes↗
PubMed1976

Topographical analysis of yeast ribosomal DNA by cleavage with restriction endonucleases.

Yeast ribosomal DNA (rDNA) was digested with the restriction enzymes Hind III, Hind II and a mixture of Hind II and Hind III. The cleavage products were analyzed by electrophoresis on 1.5% agarose gels. Several distinct bands could be observed, which are derived from the redundant ribosomal transcription units. They are superimposed on a rather broad smear of background DNA, representing the heterogenous 'spacer' sequences. From the restriction maps, together with data obtained by partial digestion, a physical map for the ribosomal transcription unit in yeast could be constructed.

DNA↗
PubMed1976

Topographical analysis of yeast ribosomal DNA by cleavage with restriction endonucleases. II. The physical map of EcoRI fragments.

Yeast ribosomal DNA (rDNA) was digested with the restriction endonuclease EcoRI. Eight distinct fragments were obtained with a molecular weight of 4.35 (1), 1.75 (2), 1.45 (3), 1.07 (4), 0.42 (5), 0.37 (6), 0.26 (7) and 0.22 x 10(6) (8) daltons, respectively. Except for fragment 1 with a molecular weight of 4.35 x 10(6) daltons, all fragments are derived from the multiple ribosomal transcription units. The 'spacer' sequences, on the other hand, gave rise to digestion products which are very heterogeneous in size. By analysis of the partial digestion products which are very heterogeneous in size. By analysis of the partial digestion products, together with the data obtained by digestion with a combination of two restriction enzymes (EcoRI and Hind II or Hind III) and redigestion of the Hind II-and Hind III-fragments with EcoRI, the physical map of the EcoRI cleavage sites in the ribosomal transcription unit could be established.

Base Sequence↗
PubMed1975

Characterization of yeast ribosomal DNA.

High-molecular-weight yeast ribosomal DNA, purified by preparative Hg2+/Cs2SO4 gradient centrifugation, was characterized using RNA-DNA hybridization, CsC1 gradient analysis and denaturation-renaturation experiments. The following conclusions are drawn from the results of these studies. 1. 55--60% of the total yeast rDNA is made up of sequences coding for 42-S ribosomal precursor RNA. 2. The mean length of the 42-S precursor cistrons corresponds to a molecular weight of 6.2 X 10(6). 3. The overall G + C content fo yeast rDNA is 42%. On the hand, both the segments coding for the nonribosomal excess RNA of the 42-S precursor and those specifying 26-S and 17-S rRNA have a higher G + C content of 46.9--47.5%. 4. The 42-S precursor cistrons are closely bound up with sequences rather low in G + C content (34--35%), which account for 40--45% of the total yeast rDNA. In contrast to the multiple 42-S precurosr cistrons which show very little or no base sequence divergence these intervening (A + T)-rich sequences display a considerable heterogeneity in base sequence.

Base Sequence↗
PubMed1975

Structure and transcription specificity of yeast RNA polymerase A.

DNA-dependent RNA polymerase A (Nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was isolated from whole yeast cells and purified to a nearly homogeneous state. The subunit structure as well as the transcription specificity of the purified enzyme were investigated. Polyacrylamide gel electrophoresis under denaturating conditions revealed that yeast polymerase A is made up of two large subunits having mol. wts of 190 000 and 135 000, and five smaller subunits with mol. wts of 54 000, 44 000, 35 000, 25 000 and 16 000, respectively. The molar ratios of all these polypeptides were found to be about unity. The transcription specificity of yeast polymerase A was tested using homologous nuclear DNA as a template. The in vitro synthesized RNA was characterized by determining its degree of self-complementarity and its ability to compete with purified ribosomal RNA in hybridization experiments. It was found that yeast polymerase A is capable of a highly selective transcription in vitro of the rRNA cistrons, provided DNA of high integrity is used as a template.

DNA↗