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D Bootsma

Publications and source records attributed to D Bootsma.

At least 73 records · Page 4Linked to original sources
PubMed1990

A presumed DNA helicase encoded by ERCC-3 is involved in the human repair disorders xeroderma pigmentosum and Cockayne's syndrome.

The human gene ERCC-3 specifically corrects the defect in an early step of the DNA excision repair pathway of UV-sensitive rodent mutants of complementation group 3. The predicted 782 amino acid ERCC-3 protein harbors putative nucleotide, chromatin, and helix-turn-helix DNA binding domains and seven consecutive motifs conserved between two superfamilies of DNA and RNA helicases, strongly suggesting that it is a DNA repair helicase. ERCC-3-deficient rodent mutants phenotypically resemble the human repair syndrome xeroderma pigmentosum (XP). ERCC-3 specifically corrects the excision defect in one of the eight XP complementation groups, XP-B. The sole XP-B patient presents an exceptional conjunction of two rare repair disorders: XP and Cockayne's syndrome. This patient's DNA contains a C----A transversion in the splice acceptor sequence of the last intron of the only ERCC-3 allele that is detectably expressed, leading to a 4 bp insertion in the mRNA and an inactivating frameshift in the C-terminus of the protein. Because XP is associated with predisposition to skin cancer, ERCC-3 can be considered a tumor-preventing gene.

Amino Acid Sequence↗
PubMed1990

Molecular cloning of the human DNA excision repair gene ERCC-6.

The UV-sensitive, nucleotide excision repair-deficient Chinese hamster mutant cell line UV61 was used to identify and clone a correcting human gene, ERCC-6. UV61, belonging to rodent complementation group 6, is only moderately UV sensitive in comparison with mutant lines in groups 1 to 5. It harbors a deficiency in the repair of UV-induced cyclobutane pyrimidine dimers but permits apparently normal repair of (6-4) photoproducts. Genomic (HeLa) DNA transfections of UV61 resulted, with a very low efficiency, in six primary and four secondary UV-resistant transformants having regained wild-type UV survival. Southern blot analysis revealed that five primary and only one secondary transformant retained human sequences. The latter line was used to clone the entire 115-kb human insert. Coinheritance analysis demonstrated that five of the other transformants harbored a 100-kb segment of the cloned human insert. Since it is extremely unlikely that six transformants all retain the same stretch of human DNA by coincidence, we conclude that the ERCC-6 gene resides within this region and probably covers most of it. The large size of the gene explains the extremely low transfection frequency and makes the gene one of the largest cloned by genomic DNA transfection. Four transformants did not retain the correcting ERCC-6 gene and presumably have reverted to the UV-resistant phenotype. One of these appeared to have amplified an endogenous, mutated CHO ERCC-6 allele, indicating that the UV61 mutation is leaky and can be overcome by gene amplification.

Animals↗
PubMed1990

Molecular cloning and biological characterization of the human excision repair gene ERCC-3.

In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion (L. H. Thompson, A. V. Carrano, K. Sato, E. P. Salazar, B. F. White, S. A. Stewart, J. L. Minkler, and M. J. Siciliano, Somat. Cell. Mol. Genet. 13:539-551, 1987).

Alkylating Agents↗
PubMed1990

The cellular retinoic-acid-binding protein is expressed in tissues associated with retinoic-acid-induced malformations.

Retinoic acid (RA) is thought to play a role in embryonic pattern formation in vertebrates. A naturally occurring gradient of endogenous RA has been demonstrated in the developing chick limb bud, while local application of RA leads to the formation of additional digits. In mammals, a well-defined spectrum of birth defects has been reported as a result of fetal exposure to excess RA. In analogy to the chick limb bud, it may be speculated that these malformations are the result of disturbance of morphogenetic RA concentration gradients. A candidate gene involved in the regulation of endogenous RA concentrations is the gene encoding cellular RA binding protein (CRABP). We have isolated a partial cDNA clone corresponding to the chicken homolog of CRABP, and performed in situ hybridization experiments on sections of embryos at various stages of development. CRABP expression was detected in the CNS, the craniofacial mesenchyme, ganglia of the peripheral nervous system, the limb bud, and the visceral arch area. Our results indicate that the spatiotemporally specified expression pattern displayed by the CRABP gene exhibits a striking correspondence to the tissues that are affected by exposure of avian or mammalian embryos to RA. We hypothesize that CRABP plays an important role in normal embryogenesis and that embryonic tissues showing high CRABP expression are susceptible to the adverse effects of excess RA.

Abnormalities, Drug-Induced↗
PubMed1990

Molecular genetics of eukaryotic DNA excision repair.

DNA repair plays a key role in the prevention of carcinogenesis and mutagenesis. Defective DNA repair has been implicated in various human hereditary disorders that predispose affected individuals to cancer. This article reviews our current understanding of one of major DNA repair systems--the nucleotide excision repair pathway--with special emphasis on the novel findings that have emerged from molecular genetic analysis of yeast and cultured mammalian cells.

Animals↗
PubMed1989

Establishment and characterization of a melanoma cell line from a xeroderma pigmentosum patient: activation of N-ras at a potential pyrimidine dimer site.

Patients suffering from the genetic disorder xeroderma pigmentosum (XP) display an extreme sensitivity of their skin to sun (UV) exposure and predisposition to skin cancer due to deficiencies in the excision DNA repair pathway. Here we describe the establishment and characterization of the first tumor cell line derived from an XP patient (belonging to complementation group C). The melanoma cell line designated XP44RO(Mel) has retained its tumorigenic and XP phenotype (UV sensitivity, reduced unscheduled DNA synthesis) and showed karyotypic abnormalities characteristic of melanomas. Transfection of XP44RO(Mel) DNA to NIH3T3 cells and oligonucleotide hybridization revealed that the N-ras oncogene was activated by an A.T to T.A or C.G transversion at the third position of codon 61. This mutation occurs at a dipyrimidine site. It is likely initiated by a UV-induced pyrimidine dimer and is of a type rarely observed in mammalian shuttle vector systems and endogenous genes after UV irradiation.

Animals↗
PubMed1989

The cloned human DNA excision repair gene ERCC-1 fails to correct xeroderma pigmentosum complementation groups A through I.

The human DNA excision repair gene ERCC-1 complements the ultraviolet light (UV) and mitomycin C (MMC) sensitivity of CHO mutants of complementation group 1. We have investigated whether ERCC-1 is the mutated gene in cell lines from xeroderma pigmentosum (XP) complementation groups A through I by analyzing the endogenous gene in XP cells and by introduction of the gene followed by repair assays. Our studies show that ERCC-1 is not deleted or grossly rearranged in representative cell lines of 9 XP groups. Furthermore, Northern blot analysis revealed correct transcription of ERCC-1 in all groups. The cloned human ERCC-1 gene was introduced into immortalized XP cells by DNA transfection (groups A, C, D, E and F). The presence of the integrated transfected sequences was verified on Southern blots and by selection for 2 dominant marker genes that flank the ERCC-1 gene on the transfected cos43-34 DNA. ERCC-1 failed to confer a normal UV survival and UV-induced unscheduled DNA synthesis (UDS) to transfected populations. In the case of the remaining XP complementation groups (B, G, H and I), nuclear microinjection was used to introduce an ERCC-1 cDNA construct driven by an SV40 promoter into primary fibroblasts. Coinjection of the SV40 large T gene and analysis of its expression served as a control for the injection. The ERCC-1 cDNA failed to induce increased levels of UDS in the microinjected fibroblasts. We infer from these experiments that ERCC-1 is not the mutated gene in the 9 XP complementation groups examined. From a similar type of experiments we conclude that ERCC-1 is not the defective gene in UV-sensitive Cockayne's syndrome cells.

Blotting, Northern↗
PubMed1989

Preferential expression of cellular retinoic acid binding protein in a subpopulation of neural cells in the developing mouse embryo.

The cellular retinoic acid binding protein is thought to be involved in the retinoic-acid-mediated signal transduction pathway. We have isolated the mouse cellular retinoic acid binding protein cDNA from an embryonal-carcinoma-derived cell line by using differential cDNA cloning strategies. In situ hybridization on sections of mouse embryos of various developmental stages indicated that the cellular retinoic acid binding protein gene, which we localized on mouse chromosome 9, is preferentially expressed in a subpopulation of neurectodermal cells. This restricted expression pattern suggests an important role for cellular retinoic acid binding protein in murine neurogenesis.

Amino Acid Sequence↗
PubMed1989

Conserved pattern of antisense overlapping transcription in the homologous human ERCC-1 and yeast RAD10 DNA repair gene regions.

We report that the genes for the homologous Saccharomyces cerevisiae RAD10 and human ERCC-1 DNA excision repair proteins harbor overlapping antisense transcription units in their 3' regions. Since naturally occurring antisense transcription is rare in S. cerevisiae and humans (this is the first example in human cells), our findings indicate that antisense transcription in the ERCC-1-RAD10 gene regions represents an evolutionarily conserved feature.

Base Sequence↗
PubMed1988

Evolution and mutagenesis of the mammalian excision repair gene ERCC-1.

The human DNA excision repair protein ERCC-1 exhibits homology to the yeast RAD10 repair protein and its longer C-terminus displays similarity to parts of the E. coli repair proteins uvrA and uvrC. To study the evolution of this 'mosaic' ERCC-1 gene we have isolated the mouse homologue. Mouse ERCC-1 harbors the same pattern of homology with RAD10 and has a comparable C-terminal extension as its human equivalent. Mutation studies show that the strongly conserved C-terminus is essential in contrast to the less conserved N-terminus which is even dispensible. The mouse ERCC-1 amino acid sequence is compatible with a previously postulated nuclear location signal and DNA-binding domain. The ERCC-1 promoter harbors a region which is highly conserved in mouse and man. Since the ERCC-1 promoter is devoid of all classical promoter elements this region may be responsible for the low constitutive level of expression in all mouse tissues and stages of embryogenesis examined.

Amino Acid Sequence↗
PubMed1988

Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group-2 mutants.

The human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In order to establish whether the correction by ERCC-1 is confined to CHO mutants of one complementation group, the cloned repair gene, present on cosmid 43-34, was transfected to representative cell lines of the 6 complementation groups that have been identified to date. Following transfection, mycophenolic acid was used to select for transferants expressing the dominant marker gene Ecogpt, also present on cosmid 43-34. Cotransfer of the ERCC-1 gene was shown by Southern blot analysis of DNA from pooled (500-2000 independent colonies) transformants of each mutant. UV survival and UV-induced UDS showed that only mutants belonging to complementation group 2 and no mutants of other groups were corrected by the ERCC-1 gene. This demonstrates that ERCC-1 does not provide an aspecific bypass of excision-repair defects in CHO mutants and supports the assumption that the complementation analysis is based on mutations in different repair genes.

Animals↗
PubMed1988

Genetic complementation analysis of ataxia telangiectasia and Nijmegen breakage syndrome: a survey of 50 patients.

Cultured cells from patients with ataxia telangiectasia (AT) or Nijmegen breakage syndrome (NBS) are hypersensitive to ionizing radiation. After radiation exposure, the rate of DNA replication is inhibited to a lesser extent than in normal cells, whereas the frequency of chromosomal aberrations is enhanced. Both of these features have been used in genetic complementation studies on a limited series of patients. Here we report the results of extended complementation studies on fibroblast strains from 50 patients from widely different origins, using the radioresistant DNA replication characteristic as a marker. Six different genetic complementation groups were identified. Four of these, called AB, C, D, and E (of which AB is the largest), represent patients with clinical signs of AT. Patients having NBS fall into two groups, V1 and V2. An individual with clinical symptoms of both AT and NBS was found in group V2, indicating that the two disorders are closely related. In AT, any group-specific patterns with respect to clinical characteristics or ethnic origin were not apparent. In addition to the radiosensitive ATs, a separate category of patients exists, characterized by a relatively mild clinical course and weak radiosensitivity. It is concluded that a defect in one of at least six different genes may underlie inherited radiosensitivity in humans. To facilitate research on defined defects, a complete list of genetically characterized fibroblast strains is presented.

Ataxia Telangiectasia↗
PubMed1988

DNA repair in human cells: from genetic complementation to isolation of genes.

The genetic disease xeroderma pigmentosum (XP) demonstrates the association between defective repair of DNA lesions and cancer. Complementation analysis performed on XP cell strains and on repair deficient rodent cell lines has revealed that at least nine and possibly more than 13 genes are involved in early steps of the excision of ultraviolet light-induced DNA lesions in mammalian cells. Two of these genes have been cloned and others are in an advanced stage of cloning. One cloned gene, ERCC-1, has been characterized at the molecular level. This human gene is homologous with excision repair genes in yeast and in Escherichia coli. These results indicate that the excision repair system is conserved during evolution. It is expected that the cloning and characterization of prokaryotic and eukaryotic repair genes will pave the way to a deeper understanding of mammalian repair systems and their association with cancer.

Animals↗
PubMed1987

Genomic characterization of the human DNA excision repair gene ERCC-1.

In this report the genomic characterization of the human excision repair gene ERCC-1 is presented. The gene consists of 10 exons spread over approximately 15 kb. By means of a transfection assay the ERCC-1 promoter was confined to a region of +/- 170 bp upstream of the transcriptional start site. Classical promoter elements like CAAT, TATA and GC-boxes are absent from this region. Furthermore, ERCC-1 transcription is not UV-inducible. A possible explanation is provided for the previously reported alternative splicing of exon VIII. Analysis of ERCC-1 cDNA clones revealed the occurrence of differential polyadenylation which gives ERCC-1 transcripts of 3.4 and 3.8 kb in addition to the major 1.1 kb mRNA. Apparent evolutionary conservation of differential polyadenylation of ERCC-1 transcripts suggests a possible role for this mode of RNA processing in the ERCC-1 repair function.

Animals↗
PubMed1987

Unique fusion of bcr and c-abl genes in Philadelphia chromosome positive acute lymphoblastic leukemia.

The Philadelphia (Ph) chromosome, the product of t(9:22), is the cytogenetic hallmark of chronic myelogenous leukemia. The c-abl oncogene on chromosome 9 is translocated to the Ph chromosome and linked to a breakpoint cluster region (bcr), which is part of a large bcr gene. This results in the formation of a bcr-c-abl fusion gene, which is transcribed into an 8.5 kb chimeric mRNA encoding a 210 kd bcr-c-abl fusion protein. The Ph chromosome is also found in acute lymphoblastic leukemia (Ph+ ALL). Although the c-abl is translocated and a new 190 kd c-abl protein has been identified, no breakpoints are observed in the bcr (Ph+bcr- ALL). Here we show that in Ph+bcr- ALL, breakpoints in chromosome 22 occur within the same bcr gene, but more 5' of the bcr. Cloning of a chimeric bcr-c-abl cDNA demonstrates that the fusion gene is transcribed into a 7 kb mRNA, encoding a novel fusion protein.

Base Sequence↗
PubMed1987

Localization of a gene involved in complementation of the defect in xeroderma pigmentosum group A cells on human chromosome 1.

Human, Chinese hamster or Chinese hamster/human hybrid cytoplasts were fused with UV-irradiated xeroderma pigmentosum group A (XP-A) cells. Unscheduled DNA synthesis (UDS) of the XP-A nucleus was measured 0-2 and 2-4 h after seeding of the fused population. Human cytoplasts did correct the defect in the XP-A nucleus immediately after fusion, whereas the chinese hamster cytoplasts did not show this rapid increase in excision repair. The results obtained after fusion of cytoplasts isolated from a panel of 26 Chinese hamster-human hybrids showed that chromosome 1 bears genetic information that is necessary for the rapid correction of the XP-A defect. Furthermore, this genetic information was regionally assigned to 1q42-qter by analysing hybrid cell lines having retained various segments of chromosome 1. Cytoplasts from a Chinese hamster/XP-A hybrid containing chromosome 1 of XP-A origin corrected also the defect with fast kinetics. This result indicate that the correcting factor consists of human and Chinese hamster components. As a consequence, the gene mapped on chromosome 1 may not be the gene which is mutated in XP-A cells.

Cell Fusion↗