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J G de Boer

Publications and source records attributed to J G de Boer.

At least 19 recordsLinked to original sources
PubMed1992

Mutations recovered in the Chinese hamster aprt gene after exposure to carboplatin: a comparison with cisplatin.

Platinum-based compounds such as cisplatin and carboplatin are currently used for the treatment of a variety of solid tumors. Their primary mode of action involves the production of cross-links in DNA. These compounds induce mutations in bacterial as well as in mammalian cells. Previously we determined the sequence specificity and mutational outcome in the Chinese hamster aprt gene in mutants isolated after exposure to cisplatin. The second-generation platinum drug, carboplatin, is of clinical relevance because it displays different and less toxic effects. Here we report the mutagenic specificity of this related compound. Mutations recovered after exposure to carboplatin display the same preference for sequences that contain 5'-AGG-3', 5'-AGA-3' and 5'-GAG-3' as was found for cisplatin. We thus conclude that the mutagenic outcome of exposure to carboplatin and cisplatin respectively, is similar, if not identical.

Adenine Phosphoribosyltransferase↗
PubMed1991

Mutational analysis of the structure and function of the adenine phosphoribosyltransferase enzyme of Chinese hamster.

We have analyzed the adenine phosphoribosyltransferase (APRT) enzyme from Chinese hamster ovary cells through the study of mutants that are able to grow in the presence of the toxic adenine analogue 8-azaadenine. The distribution of the amino acid alterations was analyzed in terms of the binding regions for the purine and phosphoribosylpyrophosphate substrates and a comparison was made with mutants known in human APRT and human, mouse and hamster hypoxanthine-guanine phosphoribosyltransferase. A number of mutants were found to cluster in several regions of the amino acid sequence. Residual enzyme activity with adenine was determined and this was correlated with substrate binding regions. A model of the secondary structure features is proposed.

Adenine Phosphoribosyltransferase↗
PubMed1989

Mutational specificity studies of endogenous mammalian cell loci: methodological aspects.

We report here the details of a modified cloning method first described by Seed et al. (1983) for use in an extensive study of mutational specificity at the aprt locus of Chinese hamster ovary cells. The technology depends on homologous recombination between a suppressor probe plasmid and the desired insert in a genomic library constructed in a double amber mutant bacteriophage lambda vector. Recombinant phage form blue plaques on a non-suppressor, lacZ amber host. We have determined the sensitivity of the method. Homologous recombination frequency is in the order of 10(-3), 6-7 orders of magnitude above non-homologous events. Recombination efficiency is unaffected when the target phage is diluted 100,000-fold with parental vector. A background of plaques is observed along with the desired blue plaques. Although the color discrimination permits us to easily avoid these background plaques, we have characterized the frequency and the nature of these events.

Adenine Phosphoribosyltransferase↗
PubMed1989

Sequence specificity of mutation induced by the anti-tumor drug cisplatin in the CHO aprt gene.

Cis-diammine dichloroplatinum (cisplatin) is an effective anti-cancer drug which forms adducts with DNA, in both bacterial and mammalian cells. It is suspected of producing tumors as well. To determine the molecular nature of genetic alterations induced by cisplatin, we cloned and sequenced cisplatin-induced mutants in the adenine phosphoribosyltransferase (aprt) gene of Chinese hamster ovary (CHO) cells. Mutation by cisplatin appears to be targeted as the sites of mutation are consistent with the known binding specificity of cisplatin. Many mutations occur at or proximal to the sequence 5'-AGG-3' and 5'-GAG-3' and include transversions, transitions, frameshifts and short deletions and duplications. Several double changes were also observed. No major rearrangements were recovered in our collection. At several locations, a number of mutants were found to be clustered within a small target region, but unlike traditional hotspots, these represent diverse changes occurring in a localized region of a few base pairs.

Adenine Phosphoribosyltransferase↗
PubMed1988

Base substitutions, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells.

The relative role of point mutations and large genomic rearrangements in ionizing radiation-induced mutagenesis has been an issue of long-standing interest. Recent studies using Southern blotting analysis permit the partitioning of ionizing radiation-induced mutagenesis in mammalian cells into detectable deletions and major genomic rearrangements and into point mutations. The molecular nature of these point mutations has been left unresolved; they may include base substitutions as well as small deletions, insertions, and frame-shifts below the level of resolution of Southern blotting analysis. In this investigation, we have characterized a collection of ionizing radiation-induced point mutations at the endogenous adenine phosphoribosyltransferase (aprt) locus of Chinese hamster ovary cells at the DNA sequence level. Base substitutions represented approximately equal to 2/3 of the point mutations analyzed. Although the collection of mutants is relatively small, every possible type of base substitution event has been recovered. These mutations are well distributed throughout the coding sequence with only one multiple occurrence. Small deletions represented the remainder of characterized mutants; no insertions have been observed. Sequence-directed mechanisms mediated by direct repeats could account for some of the observed deletions, while others appear to be directly attributable to radiation-induced strand breakage.

Adenine Phosphoribosyltransferase↗
PubMed1988

An in vitro assay for frameshift mutations: hotspots for deletions of 1 bp by Klenow-fragment polymerase share a consensus DNA sequence.

The fidelity of in vitro DNA synthesis catalyzed by the large fragment of DNA polymerase I was examined. The templates, specifically designed to detect shifts to the +1 or to the -1 reading frame, are composites of M13mp8 and bacteriophage T4 rIIB DNA and were designed to assist in the identification of the types of frameshifts that are the specific consequence of DNA polymerization errors. In vitro polymerization by the Klenow fragment produced only deletions, rather than the mixture of duplications and deletions characteristic of in vivo frameshifts. The most frequent frameshifts were deletions of 1 bp opposite a template purine base. Hotspots for these deletions occurred when the template purine immediately preceded the template sequence TT. The highest mutation frequencies were seen when the TTPu consensus sequence was adjacent to G:C rich sequences in the 3' direction. The nature of the consensus sequence itself distinguishes this 1-bp deletion mechanism from those operating in DNA repeats and attributed to the misalignment of DNA primers during synthesis. Deletions that were larger than 1 or 2 bp isolated after in vitro replication were consistent with the misalignment of the primer. Deletions of 2 bp and complex frameshifts (the replacement of AA by C) were also found. Mechanisms that may account for these mutations are discussed.

Base Sequence↗
PubMed1977

Synthesis of guanosine 5'-diphosphate, 3'-diphosphate in spot mutants of Escherichia coli.

The synthesis of ppGpp in spoT- mutants of Escherichia coli has been invesitgated. In these mutants the first-order rate constant for ppGpp breakdown is low, and pppGpp is barely detectable. It is shown that the rate of pppGpp, and hence ppGpp, synthesis is strongly reduced compared with that observed in spot+ strains. The low rate of magic spot synthesis satisfactorily explains the low levels of pppGpp in spoT- mutants. The pentaphosphate very probably is the precursor of ppGpp as it is in wild-type, i.e. spoT+, strains.

Escherichia coli↗
PubMed1976

The sequence of ppGpp and pppGpp in the reaction scheme for magic spot synthesis.

The kinetics of ppGpp (guanosine 5'-diphosphate, 3'-diphosphate) ahd pppGpp (guanosine 5'-triphosphate, 3'-diphosphate) synthesis, at the onset of amino acid starvation, and of their decay after inhibiting synthesis, were analysed in Escherichia coli. The pentaphosphate, but not ppGpp, is the first product of the stringent response to amino acid starvation. The pentaphosphate is rapidly converted with first order kinetics (t 1/2 = 6 s) to ppGpp which is broken down less rapidly (t 1/2 = 20 s).

Amino Acids↗