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Results for “Homologous Recombination Deficiency”

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At least 433 records · Page 24Linked to original sources
PubMed2000

UV-damage-mediated induction of homologous recombination in Arabidopsis is dependent on photosynthetically active radiation.

Plants are continuously subjected to UV-B radiation (UV-B; 280-320 nm) as a component of sunlight causing damage to the genome. For elimination of DNA damage, a set of repair mechanisms, mainly photoreactivation, excision, and recombination repair, has evolved. Whereas photoreactivation and excision repair have been intensely studied during the last few years, recombination repair, its regulation, and its interrelationship with photoreactivation in response to UV-B-induced DNA damage is still poorly understood. In this study, we analyzed somatic homologous recombination in a transgenic Arabidopsis line carrying a beta-glucuronidase gene as a recombination marker and in offsprings of crosses of this line with a photolyase deficient uvr2-1 mutant. UV-B radiation stimulated recombination frequencies in a dose-dependent manner correlating linearly with cyclobutane pyrimidine dimer (CPD) levels. Genetic deficiency for CPD-specific photoreactivation resulted in a drastic increase of recombination events, indicating that homologous recombination might be directly involved in eliminating CPD damage. UV-B irradiation stimulated recombination mainly in the presence of photosynthetic active radiation (400-700 nm) irrespective of photolyase activities. Our results suggest that UV-B-induced recombination processes may depend on energy supply derived from photosynthesis.

Arabidopsis↗
PubMed1994

Hyperuricemia and urate nephropathy in urate oxidase-deficient mice.

Urate oxidase, or uricase (EC 1.7.3.3), is a purine metabolic enzyme that catalyzes the conversion of uric acid to allantoin in most mammals except humans and certain other primates. The loss of urate oxidase in the human during primate evolution predisposes man to hyperuricemia, a metabolic disturbance that can lead to gouty arthritis and renal stones. To create a mouse model for hyperuricemia and gout, and to address the question of whether urate oxidase is essential in lower mammalian species, we have disrupted the urate oxidase gene in the mouse by homologous recombination in embryonic stem cells. Unlike the human situation, urate oxidase deficiency in mice causes pronounced hyperuricemia and urate nephropathy. More than half of the mutant mice died before 4 weeks of age, indicating that urate oxidase is essential in mice. These mutant mice may also serve as animal models for hyperuricemia and its related nephropathy in humans.

Allopurinol↗
PubMed1996

Isolation and characterization of human hepatoma cells with targeted insertions of a gpt selectable marker in the alpha 1-antitrypsin locus.

The bacterial xanthine-guanine phosphoribosyl transferase (gpt) gene was inserted by homologous recombination into the chromosomal alpha 1-antitrypsin (alpha 1AT) gene of HPRT-deficient human hepatoma cells. These insertions encoded chimeric alpha 1AT-gpt mRNAs that were expressed in the modified cells. Six targeted integrations were obtained, but only two of these harbored simple insertion events. The remaining four homologous insertions contained additional DNA sequences 3' of the gpt coding cassette. Variant cell lines deficient for gpt expression were isolated from transfectants containing either homologous or non-homologous gpt insertions by selection in media containing 6-thioguanine. These variant cell lines expressed alpha 1AT but not alpha 1AT-gpt mRNAs, indicating that they contained expression defects in cis. Genotypic analyses suggested that the predominant mechanism by which the variants were generated was by nondisjunctive loss of chromosomes containing the modified alpha 1AT-gpt alleles. Somatic cell hybrids formed by fusing hepatoma cells containing targeted alpha 1AT-gpt insertions with fibroblasts exhibited extinction of both modified and unmodified alpha 1AT alleles.

Base Sequence↗
PubMed1998

Unequal homologous recombination between LINE-1 elements as a mutational mechanism in human genetic disease.

Unequal homologous recombination between repetitive genetic elements is one mechanism that mediates genome instability. We have characterized a homologous recombination event between two neighboring LINE-1 sequences in the human gene encoding the beta subunit of phosphorylase kinase (PHKB). It has lead to the deletion of 7574 nucleotides of genomic DNA including exon 8 of this gene, giving rise to glycogen storage disease through phosphorylase kinase deficiency. To our knowledge, this is the first example of a mutation due to unequal homologous recombination between LINE-1 elements. The sequence features of the recombining LINE-1 elements and of the recombination junction site, and possible reasons for the more frequent occurrence of unequal homologous recombination between Alu elements are discussed.

Base Sequence↗
PubMed1997

Parthenogenetic development of Mos-deficient mouse oocytes.

Ovarian teratomas develop in Mos-/- mutant mice produced by homologous recombination. These teratomas are probably derived from oocytes that undergo spontaneous parthenogenetic activation within the ovaries. However, it is not clear how the activated eggs develop into teratomas since embryonic development beyond the four-cell stage was not observed either in vitro or in vivo. In this study, Mos-/- parthenotes derived from in vitro-matured oocytes were cultured using a recently developed medium, KSOM/AA, which promotes a high frequency of preimplantation development by normal embryos. In total, 5% of the Mos-/- oocytes developed to the blastocyst stage. Preimplantation-like and early postimplantation-like embryos were observed in the ovaries of 60-63-day-old Mos-/- mice. These observations support the hypothesis that Mos-/- teratomas are derived from parthenogenetically activated oocytes that undergo early embryonic development up to early postimplantation-like stages within the ovaries. Aberrant meiotic divisions commonly observed in Mos-/- oocytes in vitro may adversely affect preimplantation development and reduce the frequency of blastocyst formation even under the best culture conditions.

Animals↗
PubMed2005

Targeting the DNA repair defect of BRCA tumours.

Carriers of heterozygous mutations in BRCA1 or BRCA2 are strongly predisposed to breast and ovarian cancers. Cancers arising in these individuals have consistently lost the wild-type allele during tumour progression, and are therefore deficient in BRCA1 or BRCA2 function. Both BRCA1 and BRCA2 proteins have been implicated in the repair of double-strand DNA breaks by homologous recombination. This functional role in DNA repair could be exploited in the treatment of BRCA-deficient cancers by targeting the tumours with drugs that create DNA damage highly reliant on BRCA1 or BRCA2 for repair.

BRCA1 Protein↗
PubMed2001

Switch junction sequences in PMS2-deficient mice reveal a microhomology-mediated mechanism of Ig class switch recombination.

Isotype switching involves a region-specific, nonhomologous recombinational deletion that has been suggested to occur by nonhomologous joining of broken DNA ends. Here, we find increased donor/acceptor homology at switch junctions from PMS2-deficient mice and propose that class switching can occur by microhomology-mediated end-joining. Interestingly, although isotype switching and somatic hypermutation show many parallels, we confirm that PMS2 deficiency has no major effect on the pattern of nucleotide substitutions generated during somatic hypermutation. This finding is in contrast to MSH2 deficiency. With MSH2, the altered pattern of switch recombination and hypermutation suggests parallels in the mechanics of the two processes, whereas the fact that PMS2 deficiency affects only switch recombination may reflect differences in the pathways of break resolution.

Adenosine Triphosphatases↗
PubMed1991

Molecular cloning and functional characterization of a recA analog from Pseudomonas stutzeri and construction of a P. stutzeri recA mutant.

A recombinant plasmid carrying the Pseudomonas stutzeri recA gene was isolated by complementation of the Escherichia coli recA13 mutation. Subcloning experiments showed that the gene was located on a 1500 bp PvuII-BglII fragment. The cloned gene complements an E. coli recA mutant for resistance to Methylmethanosulphonate (MMS) and UV irradiation. It was also capable of restoring the recombination proficiency in that mutant. The cloned fragment was used to construct a recA deletion mutant of P. stutzeri. This mutant too was shown to be sensitive towards MMS and UV irradiation. The mutant strain was found to be completely deficient in natural transformation with chromosomal DNA, due to the impairment in homologous recombination.

Chromosome Deletion↗
PubMed2005

Expression of a peroxiredoxin-glutaredoxin by Haemophilus influenzae in biofilms and during human respiratory tract infection.

Evidence is mounting that nontypeable Haemophilus influenzae grows as a biofilm in the middle ear of children with otitis media and the airways of adults with chronic obstructive pulmonary disease. To begin to assess antigens expressed by H. influenzae in biofilms, cell envelopes of bacteria grown as a biofilm were compared to those grown planktonically. A approximately 30kDa peroxiredoxin-glutaredoxin was present in greater abundance during growth in biofilms. Mutants deficient in expression of peroxiredoxin-glutaredoxin were constructed by homologous recombination in four clinical isolates. The mutants showed a 25-50% reduction in biofilm formation compared to the corresponding parent strains. To study in vivo expression of peroxiredoxin-glutaredoxin during human respiratory tract infection, paired pre- and post-exacerbation serum from adults with chronic obstructive pulmonary disease and H. influenzae in sputum were assayed using an enzyme-linked immunosorbent assay and purified recombinant peroxiredoxin-glutaredoxin. Eight from 18 (44.4%) paired serum samples showed a significant increase in antibody to peroxiredoxin-glutaredoxin from pre- to post-infection. These results indicate that (1) peroxiredoxin-glutaredoxin is present in greater abundance in H. influenzae biofilms compared to planktonically grown bacteria; (2) peroxiredoxin-glutaredoxin is involved in biofilm formation by H. influenzae and the degree of involvement varies among strains; and (3) peroxiredoxin-glutaredoxin is expressed by H. influenzae during infection of the human respiratory tract and is recognized by the human immune system.

Adult↗
PubMed1995

Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice.

The receptor tyrosine kinase Flk-1 (ref. 1) is believed to play a pivotal role in endothelial development. Expression of the Flk-1 receptor is restricted to endothelial cells and their embryonic precursors, and is complementary to that of its ligand, vascular endothelial growth factor (VEGF), which is an endothelial-specific mitogen. Highest levels of flk-1 expression are observed during embryonic vasculogenesis and angiogenesis, and during pathological processes associated with neovascularization, such as tumour angiogenesis. Because flk-1 expression can be detected in presumptive mesodermal yolk-sac blood-island progenitors as early as 7.0 days postcoitum, Flk-1 may mark the putative common embryonic endothelial and haematopoietic precursor, the haemangioblast, and thus may also be involved in early haematopoiesis. Here we report the generation of mice deficient in Flk-1 by disruption of the gene using homologous recombination in embryonic stem (ES) cells. Embryos homozygous for this mutation die in utero between 8.5 and 9.5 days post-coitum, as a result of an early defect in the development of haematopoietic and endothelial cells. Yolk-sac blood islands were absent at 7.5 days, organized blood vessels could not be observed in the embryo or yolk sac at any stage, and haematopoietic progenitors were severely reduced. These results indicate that Flk-1 is essential for yolk-sac blood-island formation and vasculogenesis in the mouse embryo.

Animals↗
PubMed1992

Osteoclasts express high levels of pp60c-src in association with intracellular membranes.

Deletion of the c-src gene in transgenic mice by homologous recombination leads to osteopetrosis, a skeletal defect characterized by markedly deficient bone resorption (Soriano, P., C. Montgomery, R. Geske, and A. Bradley. 1991. Cell. 64:693-702), demonstrating a critical functional role of pp60c-src in osteoclast activity. Since decreased bone resorption could result from a defect either within the osteoclast or within other cells present in its environment, indirectly affecting osteoclast functions, we determined which cell(s) in bone expressed high levels of pp60c-src Measuring pp60c-src protein and kinase activities in osteoclasts and immunolocalizing pp60c-src in bone, we find that expression of pp60c-src is nearly as high in osteoclasts as in brain and platelets. In contrast, other bone cells contain only very low levels of the protein. In addition, expression of the c-src gene product increases when bone marrow cells are induced to express an osteoclast-like phenotype by 1,25-dihydroxy-vitamin D3, further suggesting that high expression of pp60c-src is part of the osteoclast phenotype. Three other src-like kinases, c-fyn, c-yes, and c-lyn, are also expressed in osteoclasts at ratios to pp60c-src similar to what is found in platelets. These src-related proteins do not, however, compensate for the absence of pp60c-src in the src- mice, thereby suggesting that pp60c-src may have a specific function in osteoclasts. Although further work is necessary to elucidate what the critical role of pp60c-src in osteoclasts is, our observation that the protein is associated mostly with the membranes of intracellular organelles suggests the possibility that this role might be at least in part related to the targeting or fusion of membrane vesicles.

Animals↗
PubMed1995

Mouse ubiquitous mitochondrial creatine kinase: gene organization and consequences from inactivation in mouse embryonic stem cells.

Individual members of the creatine kinase isoenzyme family (CK; EC 2.7.3.2), which play a prominent role in energy homeostasis, are encoded by four separate nuclear genes. We have isolated and characterized the complete mouse UbCKmit gene, the product of which is ubiquitously expressed and is located in the intermembrane space of mitochondria. Transcription of this gene is initiated at multiple adjacent positions and the region immediately upstream of these sites shares many features with genes encoding housekeeping proteins. These include a high G/C content, absence of TATA and CCAAT motifs, and presence of SP1 and AP2 recognition sequences. In addition, a binding site for HIP1, hormone-responsive elements, and three Mt-motifs, known as boxes shared between nuclear genes encoding mitochondrial proteins, were identified. To study the functional role of the UbCKmit protein, we have inactivated both UbCKmit alleles in mouse embryonic stem (ES) cells. UbCKmit-deficient cells, obtained by consecutive rounds of gene targeting using homologous recombination and drug selection-driven gene conversion events, show no obvious growth disadvantage or abnormal differentiation potential. Activities of mitochondrial cytochrome c oxidase and citrate synthase, as well as the rate of pyruvate oxidation, showed values equal to wild-type cells, indicating a normal aerobic metabolism. Mitochondria of in vivo differentiated knock-out cells were structurally intact, as demonstrated by electron microscopy. Approaches to study the role of the UbCKmit gene further are discussed.

Animals↗
PubMed1997

The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis.

The transcriptional programs that regulate blood vessel formation are largely unknown. In this paper, we examine the role of the zinc finger transcription factor LKLF in murine blood vessel morphogenesis and homeostasis. By in situ hybridization and immunohistochemistry, we show that LKLF is expressed as early as embryonic day 9.5 (E9.5) in vascular endothelial cells throughout the developing mouse embryo. To better understand the function of LKLF, we used homologous recombination in embryonic stem (ES) cells to generate LKLF-deficient (LKLF-/-) mice. Both angiogenesis and vasculogenesis were normal in the LKLF-/- mice. However, LKLF-/- embryos died between E12.5 and E14.5 from severe intra-embryonic and intra-amniotic hemorrhaging. This bleeding disorder was associated with specific defects in blood vessel morphology. Umbilical veins and arteries in the LKLF-/- embryos displayed an abnormally thin tunica media and aneurysmal dilatation before rupturing into the amniotic cavity. Similarly, vascular smooth muscle cells in the aortae from the LKLF-/- animals displayed a cuboidal morphology and failed to organize into a compact tunica media. Consistent with these findings, electron microscopic analyses demonstrated endothelial cell necrosis, significant reductions in the number of vessel-wall pericytes and differentiating smooth muscle cells, and decreased deposition of extracellular matrix in the LKLF-/- vessels. Despite these defects, in situ hybridization demonstrated normal expression of platelet-derived growth factor B, Tie1, Tie2, transforming growth factor beta, and heparin-binding epidermal growth factor in the vasculature of the LKLF-/- embryos. Therefore, LKLF defines a novel transcriptional pathway in which endothelial cells regulate the assembly of the vascular tunica media and concomitant vessel wall stabilization during mammalian embryogenesis.

Animals↗
PubMed1995

Targeted disruption of the murine VCAM1 gene: essential role of VCAM-1 in chorioallantoic fusion and placentation.

Vascular cell adhesion molecule-1 (VCAM-1) is expressed on vascular endothelium in a variety of inflammatory conditions and mediates leukocyte recruitment from blood into tissues. In this study we report a novel role for VCAM-1 in the formation of the umbilical cord and placenta during development. The murine VCAM1 gene was disrupted by targeted homologous recombination, and a distinct phenotype was found in VCAM-1-deficient embryos. At 8.5 days of gestation, the allantois failed to fuse to the chorion, resulting in abnormal placental development and embryonic death within 1-3 days. In addition, a role for VCAM-1 in early placental formation after chorioallantoic fusion was observed. In a minority of VCAM-1-deficient embryos, the allantois was able to fuse with the chorion, but the allantoic mesoderm was abnormally distributed over the chorionic surface. A small number of VCAM-1-deficient embryos survived, presumably by circumventing the placentation defects. They became viable and fertile adult mice with lack of VCAM-1 expression, normal organs, and an elevated number of circulating blood mononuclear leukocytes.

Allantois↗
PubMed2000

Protein S secretion differences of missense mutants account for phenotypic heterogeneity.

To elucidate the molecular background for the heterogeneity in protein S plasma concentrations observed in protein S deficient individuals, the in vitro synthesis of recombinant protein S missense mutants was investigated. Six different naturally occurring mutations identified in the protein S gene (PROS1) of thrombosis patients were reproduced in protein S cDNA by site directed mutagenesis. Two mutants, G441C and Y444C (group A), were associated with low total plasma concentration of protein S. Modestly low protein S was found in families with R520G and P626L (group B) mutants. T57S and I518M (group C), which was associated with marginally low protein S, did not segregate with protein S deficiency in the respective families, raising doubts as to whether they were causative mutations or rare neutral variants. The 6 protein S mutants were transiently expressed in COS 1 cells. The Y444C mutant showed the lowest level of secretion (2.5%) followed by the G441C mutant (40%). Group B demonstrated around 50% reduction in secretion, whereas group C mutants showed normal secretion. Pulse-chase experiments demonstrated impaired protein S processing with intracellular degradation and decreased secretion into the culture media of group A and B mutants. Interestingly, there was a good correlation between in vitro secretion and the concentration of free protein S in the plasma of heterozygous carriers. These results demonstrate impaired protein S secretion to be an important mechanism underlying hereditary protein S deficiency and that variations in protein secretion is a major determinant of the phenotypic heterogeneity observed in protein S deficiency. (Blood. 2000;95:173-179)

Amino Acid Sequence↗
PubMed1987

[Directed mutagenesis of chromosomal genes of Escherichia coli in vivo: construction of RecA- and HtpR- mutants].

The deletions in Escherichia coli chromosomal genes recA and htpR were constructed using the site-directed mutagenesis techniques. The obtained RecA- mutants are UV-sensitive and have a phenotype defective for the homologous DNA recombination. HtpR- mutant is temperature sensitive for growth and deficient in intracellular proteolysis. As a result a HtpR- mutant seems to be a preferable candidate for attempting to synthesize efficiently any alien protein in Escherichia coli cells.

Chromosomes, Bacterial↗
PubMed1996

Molecular basis of a hereditary type I protein S deficiency caused by a substitution of Cys for Arg474.

The molecular basis for a hereditary type I protein S (PS) deficiency was investigated. DNA sequence analysis in the proband showed a novel missense mutation substituting Cys (TGT) for Arg474 (CGT) that is a highly conserved amino acid residue among the related proteins. This missense mutation cosegregated with the type I PS deficiency in this family. Transient expression studies showed that the secretion of the recombinant Cys-mutant PS was markedly decreased compared with that of the recombinant wild-type PS, reproducing the observed phenotype of type I deficiency. Stable expression and pulse-chase experiments demonstrated an intracellular degradation and an impaired secretion of the recombinant Cys-mutant PS. Furthermore, the substitution of Arg474 by Ala or Glu, but not by Lys, markedly reduced the secretion of the recombinant PS mutants in transient expression studies, suggesting that a positively charged basic amino acid might be needed at residue 474 and might play a key role in the protein structure and conformation of the sex hormone binding globulin-homology domain of the PS molecule. We postulate that the loss of the highly conserved Arg474 might be responsible for the type I PS deficiency inherited in this family.

Androgen-Binding Protein↗
PubMed1999

DNA binding of Xrcc4 protein is associated with V(D)J recombination but not with stimulation of DNA ligase IV activity.

Mammalian cells are protected from the effects of DNA double-strand breaks by end-joining repair. Cells lacking the Xrcc4 protein are hypersensitive to agents that induce DNA double-strand breaks, and are unable to complete V(D)J recombination. The residual repair of broken DNA ends in XRCC4-deficient cells requires short sequence homologies, thus possibly implicating Xrcc4 in end alignment. We show that Xrcc4 binds DNA, and prefers DNA with nicks or broken ends. Xrcc4 also binds to DNA ligase IV and enhances its joining activity. This stimulatory effect is shown to occur at the adenylation of the enzyme. DNA binding of Xrcc4 is correlated with its complementation of the V(D)J recombination defects in XRCC4-deficient cells, but is not required for stimulation of DNA ligase IV. Thus, the ability of Xrcc4 to bind to DNA suggests functions independent of DNA ligase IV.

Animals↗