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At least 811 records · Page 45Linked to original sources
PubMed2002

Attenuation of the formation of DNA-repair foci containing RAD51 in Fanconi anaemia.

The role of the Fanconi anaemia genes in DNA repair was examined by a quantitative analysis of nuclear DNA repair foci in FA primary fibroblasts after ionising irradiation using antibodies directed against RAD51, MRE11 and BRCA1 for visualisation. IR induced foci detected with anti-RAD51, but not those detected with anti-MRE11, are reduced in fibroblasts of all eight FA complementation groups in comparison to control cells. Correction of FA-A, FA-C and FA-G cells by retroviral cDNA transfer specifically corrected the RAD51-foci response but did not affect formation of foci containing BRCA1 or MRE11. Since all FA cells, except FA-D1, lack the monoubiquitinated FANCD2-L protein, this isoform is likely to be involved in the formation of nuclear foci containing RAD51 in diploid FA cells. FA-D1 cells show the same attenuation in RAD51 foci formation, suggesting that the unknown FANCD1 protein is similarly involved in RAD51 foci formation, either independently or as a subsequent step in the FANCD2 pathway. These findings indicate that Fanconi anaemia cells have an impairment in the RAD51-dependent homologous recombination pathway for DNA repair, explaining their chromosomal instability and extreme sensitivity to DNA cross-linking agents.

BRCA1 Protein↗
PubMed1999

Mouse A9 cells containing single human chromosomes for analysis of genomic imprinting.

To develop an systematic in vitro approach for the study of genomic imprinting, we generated a new library of human/mouse A9 monochromosomal hybrids. We used whole cell fusion and microcell-mediated chromosome transfer to generate A9 hybrids containing a single, intact, bsr-tagged human chromosome derived from primary fibroblasts. A9 hybrids were identified that contained either human chromosome 1, 2, 4, 5, 7, 8, 10, 11, 15, 18, 20, or X. The parental origin of these chromosomes was determined by polymorphic analysis using microsatellite markers, and matched hybrids containing maternal and paternal chromosomes were identified for chromosomes 5, 10, 11 and 15. The imprinted gene KVLQT1 on human chromosome 11p15.5 was expressed exclusively from the maternal chromosome in A9 hybrids, and the parental-origin-specific expression patterns of several other imprinted genes were also maintained. This library of human monochromosomal hybrids is a valuable resource for the mapping and cloning of human genes and is a novel in vitro system for the screening of imprinted genes and for their functional analysis.

Animals↗
PubMed2000

Werner's syndrome protein (WRN) migrates Holliday junctions and co-localizes with RPA upon replication arrest.

Individuals affected by the autosomal recessive disorder Werner's syndrome (WS) develop many of the symptoms characteristic of premature ageing. Primary fibroblasts cultured from WS patients exhibit karyotypic abnormalities and a reduced replicative life span. The WRN gene encodes a 3'-5' DNA helicase, and is a member of the RecQ family, which also includes the product of the Bloom's syndrome gene (BLM). In this work, we show that WRN promotes the ATP-dependent translocation of Holliday junctions, an activity that is also exhibited by BLM. In cells arrested in S-phase with hydroxyurea, WRN localizes to discrete nuclear foci that coincide with those formed by the single-stranded DNA binding protein replication protein A. These results are consistent with a model in which WRN prevents aberrant recombination events at sites of stalled replication forks by dissociating recombination intermediates.

Adenosine Triphosphatases↗
PubMed1997

The Cockayne syndrome B protein, involved in transcription-coupled DNA repair, resides in an RNA polymerase II-containing complex.

Transcription-coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. Here we demonstrate by microinjection of antibodies against CSB and CSA gene products into living primary fibroblasts, that both proteins are required for TCR and for recovery of RNA synthesis after UV damage in vivo but not for basal transcription itself. Furthermore, immunodepletion showed that CSB is not required for in vitro NER or transcription. Its central role in TCR suggests that CSB interacts with other repair and transcription proteins. Gel filtration of repair- and transcription-competent whole cell extracts provided evidence that CSB and CSA are part of large complexes of different sizes. Unexpectedly, there was no detectable association of CSB with several candidate NER and transcription proteins. However, a minor but significant portion (10-15%) of RNA polymerase II was found to be tightly associated with CSB. We conclude that within cell-free extracts, CSB is not stably associated with the majority of core NER or transcription components, but is part of a distinct complex involving RNA polymerase II. These findings suggest that CSB is implicated in, but not essential for, transcription, and support the idea that Cockayne syndrome is due to a combined repair and transcription deficiency.

Cockayne Syndrome↗
PubMed1999

Novel processing in a mammalian nuclear 28S pre-rRNA: tissue-specific elimination of an 'intron' bearing a hidden break site.

Splitting and apparent splicing of ribosomal RNA, both previously unknown in vertebrates, were found in rodents of the genus Ctenomys. Instead of being formed by a single molecule of 4.4 kb, 28S rRNA is split in two molecules of 2.6 and 1.8 kb. A hidden break, mapping within a 106 bp 'intron' located in the D6 divergent region, is expressed in mature ribosomes of liver, lung, heart and spleen, as well as in primary fibroblast cultures. Testis-specific processing eliminates the intron and concomitantly the break site, producing non-split 28S rRNA molecules exclusively in this organ. The intron is flanked by two 9 bp direct repeats, revealing the acquisition by insertion of a novel rRNA processing strategy in the evolution of higher organisms.

Animals↗
PubMed1999

Aging process is accompanied by increase of transglutaminase C.

Crosslinking has been suggested as one of the mechanisms involved in the aging process. Among the various random or enzyme-mediated crosslinking reactions, transglutaminase (TGase)-catalyzed crosslinking activity has been proposed for its possible involvement in cell proliferation, differentiation, carcinogenesis, programmed death, and aging. Moreover, recent findings of TGase C as a putative signal transducer and cell cycle regulator has renewed interest in the study of TGase C in relation to aging phenomena. The ubiquitous presence of TGase C compared to the organ-specific localization of other types of TGases has attracted special attention as a cellular aging device. In the present investigation for in vitro studies, we have compared the pattern of TGase C in young and old human red blood cells, separated by density differentiation, and in early and late-passage or hydrogen peroxide-treated human primary fibroblasts. For in vivo study, we monitored the age-dependent changes of TGase C in the liver and brain tissues of 4, 12, 18, and 24-month-old Sprague-Dawley rats. We obtained evidence that both the activity and protein levels of TGase C were high in old RBC and late-passage or hydrogen peroxide-treated fibroblasts. Similar findings were seen in liver and brain tissue such as age-dependent increases in TGase activity and protein level in an organ-specific pattern. These data suggest that TGase C might play an active role in the cellular process with age.

Adult↗
PubMed2001

The spatial organization of human chromosomes within the nuclei of normal and emerin-mutant cells.

To fully understand genome function, the linear genome map must be integrated with a spatial map of chromosomes in the nucleus. Distinct nuclear addresses for a few human chromosomes have been described. Previously we have demonstrated that the gene-rich human chromosome 19 is located in a more central position in the nucleus than the similarly sized, but gene-poor, chromosome 18. To determine whether these two chromosomes are a paradigm for the organization of chromatin in the human nucleus, we have now analysed the nuclear organization of every human chromosome in diploid lymphoblasts and primary fibroblasts. We find that the most gene-rich chromosomes concentrate at the centre of the nucleus, whereas the more gene-poor chromosomes are located towards the nuclear periphery. In contrast, we find no significant relationship between chromosome size and position within the nucleus. Proteins of the nuclear membrane or lamina are candidates for molecules that might anchor regions of the genome at the nuclear periphery and it has been suggested that disruption of this organization may play a role in some disease pathologies. We show that the intranuclear organization of chromosomes is not altered in cells that lack the integral nuclear membrane protein emerin, from an individual with X-linked Emery--Dreifuss muscular dystrophy. This suggests that emerin is not necessary for localizing chromosomes at the nuclear periphery and that the muscular dystrophy phenotype in such individuals is not due to grossly altered nuclear organization of chromatin.

Cell Nucleus↗
PubMed1997

Structural and functional characterization of the human FMR1 promoter reveals similarities with the hnRNP-A2 promoter region.

Fragile X mental retardation syndrome is associated with an expansion of a CGG repeat within the 5'UTR of the first exon of the FMR1 gene, abnormal methylation of the CpG island in the promoter region, and a transcriptional silencing of this gene. We studied transcriptional regulation of the FMR1 gene using protein footprint analysis of the active and inactive gene in vivo . We identified four footprints within the FMR1 promoter region which correspond to consensus binding sites of known transcription factors, alpha-PAL/NRF1, Sp1, H4TF1/Sp1-like and c-myc. These footprints were present in normal cells with a transcriptionally active FMR1 gene. The same footprints were present in different cell types: primary fibroblasts, lymphoblastoid cells and peripheral lymphocytes. However, for the 1.1 kb region analyzed, no footprints were detected in a variety of cell types derived from patients with fragile X syndrome which have a transcriptionally inactive FMR1 gene. A BLAST nucleotide search identified sequence similarities between the region of the FMR1 gene containing the footprints and an analogous region within the promoter region of the gene for the heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a member of a family of ribonucleoproteins implicated in mRNA processing and nuclear-cytoplasm transport. The nucleotide sequences identified in the hnRNP-A2 promoter region correspond to the same consensus binding sites showing DNA-protein interactions in the FMR1 gene. Our previous functional studies and the studies of others demonstrate that FMR proteins, like hnRNP-A2, are also ribonucleoproteins which appear to be involved in mRNA transport. The results from our footprint studies suggest that the expression of the FMR1 gene is regulated by the binding of specific transcription factors to sequence elements in the 5' region of the gene and that this expression may be regulated by elements in common with the hnRNP-A2 gene. Common regulation of these two genes might play an important role in the cooperative processing and transport of mRNA from the nucleus to the translation machinery.

Female↗
PubMed2026

Reference genomes of Japanese raccoon dog (Nyctereutes viverrinus) and a Japanese red fox (Vulpes vulpes japonica).

We established primary fibroblast cultures from a Japanese raccoon dog (Nyctereutes viverrinus) and a Japanese red fox (Vulpes vulpes japonica) and generated highly contiguous reference genome assemblies using Oxford Nanopore Technologies PromethION long-read sequencing. The Japanese raccoon dog assembly spanned 2.69 Gb in 813 scaffolds, with a scaffold N50 of 52 Mb and a Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness score of 98.2%. The Japanese red fox assembly spanned 2.47 Gb in 903 scaffolds, with a scaffold N50 of 139 Mb and a BUSCO completeness score of 97.5%. Phylogenomic analysis placed the Japanese raccoon dog in a lineage distinct from the continental raccoon dog, supporting its evolutionary differentiation within Nyctereutes. The Japanese red fox formed a distinct lineage within the red fox clade, consistent with its recognized regional differentiation. These genome assemblies and associated fibroblast cultures provide resources for studies of canid systematics, population history, local adaptation, comparative genome evolution, and conservation genetics.

Canidae↗
PubMed1995

Fragile site at Xq21 in Chinese hamster and its implications for the in vitro chromosomal aberration test.

Normal Chinese hamster primary fibroblasts were exposed to methotrexate, an antifolate agent known to induce fragile sites. In a total of 156 banded metaphases prepared from methotrexate-treated cells, 93 breaks and gaps were observed and mapped to the individual hamster chromosomes and their bands. About two-thirds (69%) of all breaks and gaps mapped to the band Xq21, which is significantly more than expected. Some other bands exhibited fragility but the effect was not significant. In 6% of cells, homozygous fragility of Xq21 was observed, suggesting that this is a common fragile site. It has been reported previously that various chemicals induce preferential localized fragility of Xq in Chinese hamster ovary (CHO) and V79 cell lines. Our results suggest that preferential fragility of Xq in the presence of chemical compounds represents examples of fragile-site induction. A tendency of the in vitro chromosomal aberration test to yield positive results in the absence of positive data in other test systems, such as the Ames test and the mouse bone marrow micronucleus test, might be due to fragile-site expression induced by chemical compounds under study.

Animals↗
PubMed1999

Nucleotide excision repair modulates the cytotoxic and mutagenic effects of N-n-butyl-N-nitrosourea in cultured mammalian cells as well as in mouse splenocytes in vivo.

The butylating agent N-n-butyl-N-nitrosourea (BNU) was employed to study the role of nucleotide excision repair (NER) in protecting mammalian cells against the genotoxic effects of monofunctional alkylating agents. The direct acting agent BNU was found to be mutagenic in normal and XPA mouse splenocytes after a single i.p. treatment in vivo. After 25 and 35 mg/kg BNU, but not after 75 mg/ kg, 2- to 3-fold more hprt mutants were detected in splenocytes from XPA mice than from normal mice. Using O6-alkylguanine-DNA alkyltransferase (AGT)-deficient hamster cells, it was found that NER-deficient CHO UV5 cells carrying a mutation in the ERCC-2 gene were 40% more mutable towards lesions induced by BNU when compared with parental NER-proficient CHO AA8 cells. UV5 cells were 1.4-fold more sensitive to the cytotoxic effects of BNU compared with AA8 cells. To investigate whether this increased sensitivity of NER-deficient cells is modulated by AGT activity, cell survival studies were performed in human and mouse primary fibroblasts as well. BNU was 2.7-fold more toxic for mouse XPA fibroblasts compared with normal mouse fibroblasts. Comparable results were found for human fibroblasts. Taken together these data indicate that the role of NER in protecting rodent cells against the mutagenic and cytotoxic effects of the alkylating agent BNU depends on AGT.

Animals↗
PubMed1999

Differences in malsegregation rates obtained by scoring ana-telophases or binucleate cells.

In this work we have applied in situ hybridization with alphoid centromeric probes specific to chromosomes 7 and 11 to ana-telophase cells from human primary fibroblasts. The aim was to visualize the events leading to aneuploidy directly during anaphase, analyse the induction of aneuploidy during this mitotic stage and compare the frequencies of chromosome malsegregation observed in ana-telophases with the estimated malsegregation obtained in binucleate cells after a short cytochalasin B treatment. Significantly higher frequencies of chromosome loss and chromosome non-disjunction were observed in fibroblasts undergoing ana-telophase during recovery from a nocodazole-induced mitotic arrest compared with binucleate cells obtained by a further 30 min incubation with cytochalasin B. Using the same experimental schedule, analysis of hybridization signals in mononucleate cells showed higher frequencies of polyploid nuclei in cytochalasin B-treated cultures, indicating that part of the ana-telophases observed after release from the nocodazole-induced mitotic arrest may give rise to polyploid mononucleate cells instead of binucleate ones. A reduced distance between spindle poles was also measured in cells undergoing ana-telophase in the presence of cytochalasin B. Our study suggests that in nocodazole and cytochalasin B-treated cultures the shorter pole-to-pole distance may favour the reformation of a single membrane around telophase chromosomes, especially when several lagging chromosomes lie between the two future daughter nuclei. This would give rise to polyploid mononucleate cells at the ensuing interphase.

Anaphase↗
PubMed2001

Termini of human chromosomes display elevated rates of mitotic recombination.

The strand-specific in situ hybridization technique of CO-FISH was used to probe telomeres of human mitotic cells in order to determine the spontaneous frequency of crossover. This approach allowed the detection of recombinational crossovers occurring anywhere along the length of individual chromosomes, including reciprocal events taking place between sister chromatids. Although the process of sister chromatid exchange (SCE) is the most prominent type of recombination in somatic mammalian cells, our results show that SCEs accounted for less than a third of the recombinational events revealed by CO-FISH. It is concluded that chromosomal regions near the termini of chromosome arms undergo extraordinarily high rates of spontaneous recombination, producing terminal crossovers whose small size precludes detection by standard cytogenetic methods. That similar results were observed for transformed epithelial cells, as well as primary fibroblasts, suggests that the phenomenon is a common characteristic of human cells. These findings are noteworthy because, although telomeric and subtelomeric DNA is known to be preferentially involved in certain types of recombination, the tips of somatic mammalian chromosomes have not previously been identified as preferred sites for crossover. Implications of these results are discussed in terms of limitations imposed on CO-FISH for its proposed use in directional hybridization mapping.

Chromosomes, Human↗
PubMed1990

Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice.

Transgenic mice suitable for the in vivo assay of suspected mutagens at the chromosome level have been constructed by stable integration of a lambda phage shuttle vector. The shuttle vector, which contains a beta-galactosidase (beta-gal) target gene, can be rescued from genomic DNA with in vitro packaging extracts. Mutations in the target gene are detected by a change in lambda phage plaque color on indicator agar plates. Initial rescue efficiencies of less than 1 plaque forming unit (pfu)/100 micrograms of genomic DNA were too low for mutation analysis. We determined the cause of the low rescue efficiencies by examining primary fibroblast cultures prepared from fetuses of lambda transgenic animals. The rescue efficiency of 5-azacytidine-treated cells increased 50-fold over non-treated controls indicating that methylation was inhibiting rescue. The inhibitory role of methylation was supported by the observation that mcr deficient E. coli plating strains and mcr deficient lambda packaging extracts further improved lambda rescue efficiency. Present rescue efficiencies of greater than 2000 pfu/copy/micrograms of genomic DNA represent a 100,000-fold improvement over initial rescue efficiencies, permitting quantitative mutational analysis. The background mutagenesis rate was estimated at 1 x 10(-5) in two separate lineages. Following treatment with the mutagen N-ethyl-N-nitrosourea (EtNU), a dose dependent increase in the mutation rate was observed in DNA isolated from mouse spleen, with significant induction also observed in mouse testes DNA.

Animals↗
PubMed1993

Mouse Brn-3 family of POU transcription factors: a new aminoterminal domain is crucial for the oncogenic activity of Brn-3a.

The class IV POU domain genes Brn-3a, -b and -c are differentially expressed during neural development and at least Brn-3a also in neuroectodermal tumors. In contrast to Brn-3b and Brn-3c, Brn-3a encodes two protein variants: Brn-3a(l) and Brn-3a(s). Brn-3a(s) lacks 84 aminoterminal residues but is otherwise identical to Brn-3a(l). Outside the well conserved carboxyterminal POU domains all three Brn-3 proteins (-a, -b and -c) diverge until the aminoterminal end where a new domain of about 100 amino acids is identified. This domain is conserved only between Brn-3 proteins and other class IV POU factors. Brn-3a(l) that contains the complete domain but not Brn-3a(s) that lacks 84 amino acids of it is able to tumorigenically transform primary fibroblasts. Brn-3b that lacks 40 amino acids of the new domain does itself not transform, but abolishes the oncogenic potential of Brn-3a(l) when transfected together. This demonstrates not only that Brn3-a(l) is a proto-oncogene and may well be causally involved in the generation of neuroectodermal tumors but also suggests that the intactness of the new aminoterminal domain described here is crucial for oncogenic activity. In addition, our data indicate that Brn-3b acts as an inhibitor of Brn-3a(l) activity.

Amino Acid Sequence↗
PubMed1995

Late induction of human DNA ligase I after UV-C irradiation.

We have studied the regulation of DNA ligase I gene expression in UV-C irradiated human primary fibroblasts. An increase of approximately 6-fold both in DNA ligase I messenger and activity levels was observed 24 h after UV treatment, when nucleotide excision repair (NER) is no longer operating. DNA ligase I induction is serum-independent and is controlled mainly by the steady-state level of its mRNA. The activation is a function of the UV dose and occurs at lower doses in cells showing UV hypersensitivity. No increase in replicative DNA polymerase alpha activity was found, indicating that UV induction of DNA ligase I occurs through a pathway that differs from the one causing activation of the replication machinery. These data suggest that DNA ligase I induction could be linked to the repair of DNA damage not removed by NER.

Cells, Cultured↗
PubMed1998

Selection of primary cell cultures with cre recombinase induced somatic mutations from transgenic mice.

Deletion of genes in defined cell types has been achieved using a combination of gene targeting techniques and the Cre- lox P recombination system. Here we present a method to selectively isolate genetically altered primary cell cultures based on the permanent activation of a drug-resistance gene by the Cre recombinase. Transgenic mice were generated harboring a dormant form of the hygromycin resistance gene. This mouse line was crossed with mice carrying a constitutive Cre gene and an endogenous floxed allele. Primary fibroblasts established from triple transgenic embryos displayed not only hygromycin resistance but also recombination of the endogenous floxed allele. These results prove the potential of this approach.

Animals↗
PubMed2001

UV induces nucleolar translocation of ING1 through two distinct nucleolar targeting sequences.

The ING1 candidate tumor suppressor is downregulated in a variety of primary tumors and established cancer cell lines. Blocking its expression experimentally promotes unregulated growth in vitro and in vivo, using cell and animal models. Alternative splicing products encode proteins that localize to the nucleus, inhibit cell cycle progression and affect apoptosis in different model systems. Here we show that ING1 proteins translocate to the nucleolus 12-48 h after UV-induced DNA damage. When a small 50 amino acid portion of ING1 was fused to green fluorescent protein, the fusion protein was efficiently targeted to the nucleolus, indicating that ING1 possesses an intrinsic nucleolar targeting sequence (NTS). We mapped this activity to two distinct 4 amino acid regions, which individually direct fused heterologous proteins to the nucleolus. Overexpression of ING1 induced apoptosis of primary fibroblasts in the presence and absence of UV exposure. In contrast, NTS mutants of ING1 that were not targeted to the nucleolus did not efficiently induce apoptosis when overexpressed and instead protected cells from UV-induced apoptosis. Taken together, these results indicate that UV induces ING1 to translocate to the nucleolus and that this translocation may facilitate apoptosis.

Amino Acid Sequence↗