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Results for “Homologous Recombination Deficiency”

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PubMed1996

Functional significance of the Fas molecule in naive lymphocytes.

The Fas molecule mediates apoptotic signal in many cell types. Mouse mutations (lpr, lprcg, gld), which impair the function of Fas, cause spontaneous autoimmune disease. We generated Fas-deficient (Fas-/-) mice by homologous recombination. In embryonic stem cells Fas-/- mice developed lpr-like disease, confirming that the abnormality of Fas is causal in the lpr phenotype. We also made Fas-/- chimeric mice composed of a mixture of Fas+/+ and Fas-/- cells. The chimeric mice also showed the lpr phenotype. In Fas-/-, chimeric mice, the Fas-deficient population expanded progressively among mature T and B lymphocytes. The expansion of Fas-deficient lymphocytes occurred at the naive, pre-primed, lymphocyte stage. These results suggest that the Fas molecule functions not only after antigenic stimulation, as previously hypothesized, but also at the naive lymphocyte stage.

Animals↗

PubMed2000

Poly(ADP-ribose) polymerase-1: what have we learned from the deficient mouse model?

Poly (ADP-ribose) polymerase (113 kDa; PARP-1) is a constitutive factor of the DNA damage surveillance network developed by the eukaryotic cell to cope with the numerous environmental and endogenous genotoxic agents. This enzyme recognizes and is activated by DNA strand breaks. This original property plays an essential role in the protection and processing of the DNA ends as they arise in DNA damage that triggers the base excision repair (BER) pathway. The generation, by homologous recombination, of three independent deficient mouse models have confirmed the caretaker function of PARP-1 in mammalian cells under genotoxic stress. Unexpectedly, the knockout strategy has revealed the instrumental role of PARP-1 in cell death after ischemia-reperfusion injury and in various inflammation process. Moreover, the residual PARP activity found in PARP-1 deficient cells has been recently attributed to a novel DNA damage-dependent poly ADP-ribose polymerase (62 kDa; PARP-2), another member of the expanding PARP family that, on the whole, appears to be involved in the genome protection. The present review summarizes the recent data obtained with the three PARP knockout mice in comparison with the chemical inhibitor approach.

Amino Acid Sequence↗

PubMed1993

Two types of sites required for meiotic chromosome pairing in Caenorhabditis elegans.

Previous studies have shown that isolated portions of Caenorhabditis elegans chromosomes are not equally capable of meiotic exchange. These results led to the proposal that a homolog recognition region (HRR), defined as the region containing those sequences enabling homologous chromosomes to pair and recombine, is localized near one end of each chromosome. Using translocations and duplications we have localized the chromosome I HRR to the right end. Whereas the other half of chromosome I did not confer any ability for homologs to pair and recombine, deficiencies in this region dominantly suppressed recombination to the middle of the chromosome. These deletions may have disrupted pairing mechanisms that are secondary to and require an HRR. Thus, the processes of pairing and recombination appear to utilize at least two chromosomal elements, the HRR and other pairing sites. For example, terminal sequences from other chromosomes increase the ability of free duplications to recombine with their normal homologs, suggesting that telomere-associated sequences, homologous or nonhomologous, play a role in facilitating meiotic exchange. Recombination can also initiate at internal sites separated from the HRR by chromosome rearrangement, such as deletions of the unc-54 region of chromosome I. When crossing over was suppressed in a region of chromosome I, compensatory increases were observed in other regions. Thus, the presence of the HRR enabled recombination to occur but did not determine the distribution of the crossover events. It seems most likely that there are multiple initiation sites for recombination once homolog recognition has been achieved.

Animals↗

PubMed2004

Requirement for TRAF3 in signaling by LMP1 but not CD40 in B lymphocytes.

CD40, a member of the tumor necrosis factor receptor family, and the Epstein-Barr virus-encoded oncoprotein latent membrane protein 1 (LMP1) share several tumor necrosis factor receptor-associated factor (TRAF) adaptor proteins for signaling. Among these, TRAF3 was the first identified to directly bind both receptors, yet its role remains a mystery. To address this, we generated B cell lines deficient in TRAF3 by homologous recombination. We found that CD40 signals were normal in the absence of TRAF3, with the exception of moderately enhanced c-Jun NH2-terminal kinase (JNK) activation and antibody secretion. In sharp contrast, LMP1 signaling was markedly defective in TRAF3-/- B cells. LMP1-induced activation of JNK and nuclear factor kappaB, up-regulation of CD23 and CD80, and antibody secretion were substantially affected by TRAF3 deficiency. Reconstitution of TRAF3 expression decreased CD40-induced JNK activation and antibody secretion, and fully restored LMP1 signaling. Although TRAF2 is widely believed to be important for LMP1 function, LMP1 signaling was intact in TRAF2-/- B cells. Our data reveal that CD40 and LMP1 unexpectedly use TRAF3 in different ways, and that TRAF3 is required for LMP1-mediated activation of B cells.

Animals↗

PubMed1997

The Bacillus subtilis chromatin-associated protein Hbsu is involved in DNA repair and recombination.

The Bacillus subtilis hbs gene encodes an essential chromatin-associated protein termed Hbsu. Hbsu, the counterpart of the Escherichia coli HU protein, binds DNA in a non-specific way but has a clear preference for bent, kinked or altered DNA sequences. To investigate the role of Hbsu in DNA repair and DNA recombination we have constructed a series of site-directed mutants in the hbs gene and used these mutant genes to substitute the wild-type chromosomal hbs gene. The hbs47 mutation, which codes for a mutant protein in which residue Phe-47 has been replaced by Trp, does not cause any discernible phenotype. Additional substitution of residue Arg-55 by Ala (hbs4755 mutation) rendered cells deficient in DNA repair, homologous recombination and beta protein-mediated site-specific recombination. We have also tested the effect on DNA repair of the hbs4755 mutation in combination with mutations in different functions of homologous DNA recombination (recA, recF, recG, recH and addAB). The hbs4755 mutation did not modify the sensitivity of recH and addAB cells to the DNA-damaging agents methylmethane sulphonate (MMS) or 4-nitroquinoline-1-oxide (4NQO), and it only marginally affected recF and recG cells. The hbs4755 mutation blocked intermolecular recombination in recH cells and markedly reduced it (20- to 50-fold) in recF and recG cells, but had no effect on addAB cells. Taken together, these data indicate that the Hbsu protein is required for DNA repair and for homologous DNA recombination.

Amino Acid Sequence↗

PubMed1983

Novel use of synthetic oligonucleotide insertion mutants for the study of homologous recombination in mammalian cells.

Thymidine kinase-deficient mouse L cells have been transformed with plasmid DNAs carrying 8-base-pair Xho I linker insertion mutations in the coding region of the herpes simplex virus type 1 thymidine kinase gene. When the mutant plasmids are introduced individually into LTK- cells, transformation efficiencies are greatly reduced relative to the wild type. However, when two mutant plasmids are cotransferred into the same LTK- recipients, significantly higher frequencies of transformation are observed (30-300 times). Here we demonstrate the usefulness of linker insertions for the study of homologous recombination in detecting the existence of normal thymidine kinase gene sequences (i.e., sequences lacking the insertions after recombination are substantiated by DNA . DNA hybridization). In addition, the frequencies of recombination in the various "crosses" are consistent with the known positions of the mutations.

Animals↗

PubMed2001

The prostaglandin E2 EP1 receptor mediates pain perception and regulates blood pressure.

The lipid mediator prostaglandin E2 (PGE2) has diverse biological activity in a variety of tissues. Four different receptor subtypes (EP1-4) mediate these wide-ranging effects. The EP-receptor subtypes differ in tissue distribution, ligand-binding affinity, and coupling to intracellular signaling pathways. To identify the physiological roles for one of these receptors, the EP1 receptor, we generated EP1-deficient (EP1-/-) mice using homologous recombination in embryonic stem cells derived from the DBA/1lacJ strain of mice. The EP1-/- mice are healthy and fertile, without any overt physical defects. However, their pain-sensitivity responses, tested in two acute prostaglandin-dependent models, were reduced by approximately 50%. This reduction in the perception of pain was virtually identical to that achieved through pharmacological inhibition of prostaglandin synthesis in wild-type mice using a cyclooxygenase inhibitor. In addition, systolic blood pressure is significantly reduced in EP1 receptor-deficient mice and accompanied by increased renin-angiotensin activity, especially in males, suggesting a role for this receptor in cardiovascular homeostasis. Thus, the EP1 receptor for PGE2 plays a direct role in mediating algesia and in regulation of blood pressure.

Animals↗

PubMed2006

A novel gross deletion caused by non-homologous recombination of the PDHX gene in a patient with pyruvate dehydrogenase deficiency.

We report here the molecular analysis of a pyruvate dehydrogenase E3-binding protein (PDH-E3BP) deficiency in a new patient, born to first cousin parents. She has initially presented with a non-progressive and unspecific encephalopathy, followed by an acute neurological deterioration at 14 years of age. E3BP subunit was undetectable on Western blot. The sequence of exons 1-9 and exon 11 of the PDHX gene were normal, but exon 10 was impossible to amplify with standard PCR. Long-range PCR including exons 9-11 (11.5 kb) was performed. The patient's sample displayed a unique PCR product of 7.5 kb, whereas the parents' samples displayed two bands (11.5 and 7.5 kb). The deletion breakpoints were determined by restriction analysis followed by direct sequencing. The homozygous deletion covered the end of intron 9, exon 10 and the beginning of intron 10 and was found to be 3913 bp long. The cDNA sequencing confirmed the deletion of exon 10. The most probable mechanism for this gross deletion appears to be a slipped mispairing mediated by an exact direct repeat CCACTG. It is the first time that a non-homologous recombination is reported in the PDHX gene causing pyruvate dehydrogenase complex (PDHc) deficiency.

Adolescent↗

PubMed2002

Deficiency in BRCA2 leads to increase in non-conservative homologous recombination.

The BRCA2 tumor suppressor has been implicated in the maintenance of genomic integrity through a function in cellular responses to DNA damage. The BRCA2 protein directly associates with Rad51, that is essential for repair of double-strand breaks (DSBs) by homologous recombination (HR). In this report, we study the BRCA2-defective Chinese hamster cell mutant V-C8 for its ability to perform homology-directed repair (HDR) between repeated sequences. V-C8 cells were recently shown to be defective in Rad51 foci formation in response to DNA damage. Strikingly, we find that these BRCA2 mutant cells exhibit a strong stimulation of HDR activity compared to the V79 parental cells, which harbor a wild-type BRCA2. Furthermore, molecular characterization of the HDR products shows that loss of BRCA2 in V-C8 cells leads to significant reduction in Rad51-dependent gene conversion but strong enhancement of Rad51-independent single-strand annealing (SSA) events frequency. These data imply that, when HDR by conservative gene conversion is impaired, DSBs usually repaired by this pathway are instead resolved by other non-conservative HDR subpathways. Therefore, high chromosomal instability in BRCA2-deficient cells presumably results from enhancement of error-prone repair mechanisms, such as SSA.

Animals↗

PubMed2001

Anuria, omphalocele, and perinatal lethality in mice lacking the CD34-related protein podocalyxin.

Podocalyxin is a CD34-related sialomucin that is expressed at high levels by podocytes, and also by mesothelial cells, vascular endothelia, platelets, and hematopoietic stem cells. To elucidate the function of podocalyxin, we generated podocalyxin-deficient (podxl(-/)-) mice by homologous recombination. Null mice exhibit profound defects in kidney development and die within 24 hours of birth with anuric renal failure. Although podocytes are present in the glomeruli of the podxl(-/)- mice, they fail to form foot processes and slit diaphragms and instead exhibit cell--cell junctional complexes (tight and adherens junctions). The corresponding reduction in permeable, glomerular filtration surface area presumably leads to the observed block in urine production. In addition, podxl(-/)- mice frequently display herniation of the gut (omphalocele), suggesting that podocalyxin may be required for retraction of the gut from the umbilical cord during development. Hematopoietic and vascular endothelial cells develop normally in the podocalyxin-deficient mice, possibly through functional compensation by other sialomucins (such as CD34). Our results provide the first example of an essential role for a sialomucin in development and suggest that defects in podocalyxin could play a role in podocyte dysfunction in renal failure and omphalocele in humans.

Animals↗

PubMed2001

Expression of the fibroblast activation protein during mouse embryo development.

Human Fibroblast Activation Protein (FAP), a member of the serine prolyl oligopeptidase family, is a type II cell surface glycoprotein that acts as a dual-specificity dipeptidyl-peptidase (DPP) and collagenase in vitro. Its restricted expression pattern in embryonic mesenchyme, in wound healing and in reactive stromal fibroblasts of epithelial cancers, has suggested a role for the FAP protease in extracellular matrix degradation or growth factor activation in sites of tissue remodeling. The FAP homologue in Xenopus laevis has been reported to be induced in the thyroid hormone-induced tail resorption program during tadpole metamorphosis supporting a role for FAP in tissue remodeling processes during embryonic development. However, Fap-deficient mice show no overt developmental defects and are viable. To study the expression of FAP during mouse embryogenesis, a second Fap-deficient mouse strain expressing beta-Galactosidase under the control of the Fap promoter was generated by homologous recombination (Fap-/- lacZ mice). FAP deficiency was confirmed by the absence of FAP-specific dipeptidyl-peptidase activity in detergent-soluble extracts isolated from 17.5 d.p.c. Fap-/- lacZ embryos. We report that Fap-/- lacZ mice express beta-Galactosidase at regions of active tissue remodeling during embryogenesis including somites and perichondrial mesenchyme from cartilage primordia.

Animals↗

PubMed1996

Cyp1a2(-/-) null mutant mice develop normally but show deficient drug metabolism.

Cytochrome P450 1A2 (CYP1A2) is a predominantly hepatic enzyme known to be important in the metabolism of numerous foreign chemicals of pharmacologic, toxicologic, and carcinogenic significance. CYP1A2 substrates include aflatoxin B1, acetaminophen, and a variety of environmental arylamines. To define better the developmental and metabolic functions of this enzyme, we developed a CYP1A2-deficient mouse line by homologous recombination in embryonic stem cells. Mice homozygous for the targeted Cyp1a2 gene, designated Cyp1a2(-/-), are completely viable and fertile; histologic examination of 15-day embryos, newborn pups, and 3-week-old mice revealed no abnormalities. No CYP1A2 mRNA was detected by Northern blot analysis. Moreover, mRNA levels of Cyp1a1, the other gene in the same subfamily, appear unaffected by loss of the Cyp1a2 gene. Because the muscle relaxant zoxazolamine is a known substrate for CYP1A2, we studied the Cyp1a2(-/-) genotype by using the zoxazolamine paralysis test: the Cyp1a2(-/-) mice exhibited dramatically lengthened paralysis times relative to the Cyp1a2(+/+) wild-type animals, and the Cyp1a2(+/-) heterozygotes showed an intermediate effect. Availability of a viable and fertile CYP1A2-deficient mouse line will provide a valuable tool for researchers wishing to define the precise role of CYP1A2 in numerous metabolic and pharmacokinetic processes.

Animals↗

PubMed2001

Mesodermal patterning defect in mice lacking the Ste20 NCK interacting kinase (NIK).

We have previously shown that the Drosophila Ste20 kinase encoded by misshapen (msn) is an essential gene in Drosophila development. msn function is required to activate the Drosophila c-Jun N-terminal kinase (JNK), basket (Bsk), to promote dorsal closure of the Drosophila embryo. Later in development, msn expression is required in photoreceptors in order for their axons to project normally. A mammalian homolog of msn, the NCK-interacting kinase (NIK) (recently renamed to mitogen-activated protein kinase kinase kinase kinase 4; Map4k4), has been shown to activate JNK and to bind the SH3 domains of the SH2/SH3 adapter NCK. To determine whether NIK also plays an essential role in mammalian development, we created mice deficient in NIK by homologous recombination at the Nik gene. Nik(-/-) mice die postgastrulation between embryonic day (E) 9.5 and E10.5. The most striking phenotype in Nik(-/-) embryos is the failure of mesodermal and endodermal cells that arise from the anterior end of the primitive streak (PS) to migrate to their correct location. As a result Nik(-/- )embryos fail to develop somites or a hindgut and are truncated posteriorly. Interestingly, chimeric analysis demonstrated that NIK has a cell nonautonomous function in stimulating migration of presomitic mesodermal cells away from the PS and a second cell autonomous function in stimulating the differentiation of presomitic mesoderm into dermomyotome. These findings indicate that despite the large number of Ste20 kinases in mammalian cells, members of this family play essential nonredundant function in regulating specific signaling pathways. In addition, these studies provide evidence that the signaling pathways regulated by these kinases are diverse and not limited to the activation of JNK because mesodermal and somite development are not perturbed in JNK1-, and JNK2-deficient mice.

Animals↗

PubMed2001

The REC41 gene of Saccharomyces cerevisiae: isolation and genetic analysis.

Recombination-deficient strains have been proven useful for the understanding of the genetic control of homologous recombination. As the genetic screens used to isolate recombination-deficient (rec(-)) yeast mutants have not been saturated, we sought to develop a simple colony color assay to identify mutants with low or elevated rates of recombination. Using this system we isolated a collection of rec(-) mutants. We report the characterization of the REC41 gene identified in this way. REC41 is required for normal levels of interplasmid recombination and gamma-ray induced mitotic interchromosomal recombination. The rec41-1 mutant failed to grow at 37 degrees C. Microscopic analysis of plated cells showed that 45-50% of them did not form visible colonies at permissive temperature. Haploid cells of the rec41 mutant show the same gamma-ray sensitivity as wild type ones. However, the diploid rec41 mutant shows gamma-ray sensitivity which is comparable with heterozygous REC41/rec41-1 diploid cells. This fact indicates semidominance of the rec41-1 mutation. Diploid strains homozygous for the rec41 rad52 mutations had the same gamma-ray sensitivity as single rad52 diploids and exhibited dramatically decreased growth rate. The expression of the HO gene does not lead to inviability of rec41 cells. The rec41 mutation has an effect on meiosis, likely meiotic recombination, even in the heterozygous state. We cloned the REC41 gene. Sequence analysis revealed that the REC41 gene is encoded by ORF YDR245w. Earlier, this ORF was attributed to MNN10, BED1, SLC2, CAX5 genes. Two multicopy plasmids with suppressers of the rec41-1 mutation (pm21 and pm32) were isolated. The deletion analysis showed that only DNA fragments with the CDC43 and HAC1 genes can partially complement the rec41-1 mutation.

Cell Division↗

PubMed1996

Investigation of a phage resistant Serratia entomophila strain (BC4B), establishment of generalised transduction and construction of S. entomophila RecA mutants.

A recA clone was isolated from a cosmid library of Serratia entomophila constructed in the Escherichia coli strain HB101. Subcloning and transposon mutagenesis were used to identify a 1.36 kb fragment containing the recA gene. A cloned recA mutation, generated by transposon mutagenesis and the replacement of a portion of the recA gene with an antibiotic resistance cassette, was introduced into the chromosome via a marker exchange technique. The recA strains created were deficient in DNA repair, homologous recombination and both the spontaneous and UV induction of prophages. S. entomophila recA strains showed continued pathogenicity towards the New Zealand grass grub, Costelytra zealandica. Simple procedures for further construction of S. entomophila recA strains have been demonstrated.

Bacteriophages↗

PubMed1995

Molecular analysis of the regulation of muscarinic receptor expression and function.

Several systems are being used to determine the molecular and cellular basis for the regulation of expression and function of the muscarinic receptors. Treatment of chick heart cells in culture results in decreased levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription which requires concomitant mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. Site-directed mutagenesis was used to demonstrate that the single tyrosine residue in the carboxyl-terminal cytoplasmic tail of the m2 receptor is involved in agonist-induced down-regulation but not sequestration. Activation of heterologous receptors in chick heart cells can also regulate mAChR mRNA levels. A cAMP-regulated luciferase reporter gene, has been used to demonstrate that the m4 receptor preferentially couples to Gi alpha-2 or Go alpha over Gi alpha-1 or Gi alpha-3 to mediate inhibition of adenylyl cyclase activity. Finally, in order to determine the role of individual receptor subtypes in muscarinic-mediated responses in vivo, we are beginning to use the method of targeted gene disruption by homologous recombination to generate mice deficient in specific receptor subtypes.

Adenylate Cyclase Toxin↗

PubMed2006

Genetic analysis of ionizing radiation-induced mutagenesis in Saccharomyces cerevisiae reveals TransLesion Synthesis (TLS) independent of PCNA K164 SUMOylation and ubiquitination.

Ionizing radiation-induced mutagenesis (IR-IM) underlies a basis for radiation associated carcinogenesis as well as resistance to radiation therapy. This process was examined in Saccharomyces cerevisiae using an array of isogenic DNA repair deficient mutants. Mutations inactivating homologous recombination (rad51, 52, 54) or nucleotide excision repair (rad1, rad10, rad4) caused elevated IR-IM whereas inactivation of TransLesion Synthesis (TLS: rad6) caused severely defective IR-IM. Of the mutations inactivating TLS polymerases, rev3 and rev1 caused equally severe defects in IR-IM whereas rad30 did not significantly affect the process. The effects of the rev3, rev1, and rad6 mutations on IR-IM were epistatic, suggesting the requirement of both polymerase zeta and Rev1p in IR-IM related TLS. Although PCNA K164 SUMOylation/ubiquitination is a proposed prerequisite for TLS, the IR-IM defect of a rev3 or a rad6 mutant was worse than and epistatic to the pol30K164R mutant, a mutant in which the PCNA had been mutated to abolish such modifications. These results suggested that IR-IM related TLS occurs in the absence of PCNA K164 modification. Further analysis of a mutant simultaneously defective in SUMOylation and mono-ubiquitination (rad18 siz1) revealed that these modifications redundantly affected TLS as well as NHEJ. A genetic model based on these observations is proposed.

DNA Repair↗

PubMed1997

Regulation of muscarinic receptor expression and function in cultured cells and in knock-out mice.

We have investigated the molecular and cellular basis for the regulation of expression and function of the muscarinic acetylcholine receptors. Treatment of cultured chick cardiac cells with the agonist carbachol results in decreased levels of mRNA encoding the m2 and m4 receptors. Treatment of chick embryos in ovo with carbachol results in decreased levels of mRNA encoding the potassium channel subunits GIRK1 and GIRK4 as well as the m2 receptor. There are thus multiple pathways for the regulation of mAChR responsiveness by long-term agonist exposure. Immunoblot, immunoprecipitation, and solution hybridization analyses have been used to quantitate the regulation of mAChR expression in chick retina during embryonic development. The m4 receptor is the predominant subtype expressed early in development, while the expression of the m3 and m2 receptors increases later in development. A cAMP-regulated luciferase reporter gene has been used to demonstrate that the m2 and m4 receptors have distinct specificities for coupling to G-protein subtypes to mediate inhibition of adenylyl cyclase. This system has also been used to demonstrate that beta-arrestin1 and beta-adrenergic receptor kinase-1 act synergistically to promote receptor desensitization. We have isolated the promoter region for the chick m2 receptor gene, identified regions of the promoter required to drive high level expression in cardiac and neural cells, and have identified a region which confers sensitivity of gene expression to neurally active cytokines. Finally, in order to determine the role of individual receptor subtypes in muscarinic-mediated responses in vivo, we have used the method of targeted gene disruption by homologous recombination to generate mice deficient in the m1 receptor.

Animals↗