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PubMed1995

Effects of disruption of the plasminogen gene on thrombosis, growth, and health in mice.

BACKGROUND: Circumstantial evidence suggests that the plasminogen/plasmin system plays a role in many biological processes, including hemostasis, cell migration, and development. METHODS AND RESULTS: The in vivo function of the plasminogen/plasmin system was studied by generation of plasminogen-deficient (Plg-/-) mice. Inactivation of the murine plasminogen-gene (Plg) was achieved by replacing, via homologous recombination in embryonic stem cells, genomic sequences encoding the exons containing the catalytic site amino acids His605 and Asp648 with a neomycin phosphotransferase expression cassette. Germline transmission of the mutated allele, as determined by Southern blot hybridization and polymerase chain reaction, was obtained via blastocyst injection. Mendelian inheritance of the inactivated plasminogen allele was observed, and homozygous-deficient mice (Plg-/-) displayed normal viability but retarded growth up to at least 12 weeks of age. At 8 weeks of age, body weight was 21.8 +/- 1.2 g (n = 10) for wild-type (Plg+/+) mice, 21.0 +/- 1.1 g (n = 16) for heterozygous-deficient (Plg+/-) mice, and 17.4 +/- 1.3 g (n = 12) for Plg-/- mice; P < .05 versus Plg+/+ or Plg+/-. None of 36 Plg+/+ or 65 Plg+/- mice but 7 of 37 Plg-/- mice (19%) developed rectal prolapse at 7.4 +/- 0.6 weeks of age (P = .03 versus Plg+/+ and P = .003 versus Plg+/-); 4 of 37 Plg-/- mice (11%) became runted and apathic at 5.3 +/- 0.3 weeks of age (P = .041 versus Plg+/-); and 6 of 37 Plg-/- mice (16%) died prematurely at 8.8 +/- 1.7 weeks of age (P = .057 versus Plg+/+ and P = .029 versus Plg+/-). Although male and female Plg-/- mice were able to sire offspring, the fertility of Plg-/- female mice was reduced, possibly owing to their impaired health. Levels of plasminogen-related antigen in plasma, measured by ELISA, were 84 +/- 8 micrograms/mL (n = 4) in Plg+/+, 35 +/- 2 micrograms/mL (n = 3) in Plg+/-, and 0.076 +/- 0.032 microgram/mL (n = 6) in Plg-/- mice (P < .001 versus Plg+/- and Plg+/+). Plasmin activity generated by urokinase activation was unmeasurable in Plg-/- mice (< 5% of Plg+/+ mice). Plasminogen-specific immunoreactivity was observed in hepatocytes from Plg+/+ mice but not from Plg-/- mice (< 10% of Plg+/+ mice). Neither native nor variant plasminogen mRNA nor translation products could be identified by Northern or Western blot of liver extracts from Plg-/- mice. Spontaneous lysis within 24 hours of a 125I-fibrin-labeled pulmonary plasma clot was 85 +/- 5% (n = 5) in Plg+/+ mice, 62 +/- 7% (n = 3) in Plg+/- mice, and -2 +/- 1% (n = 3) in Plg-/- mice (P < .001 versus Plg+/- and Plg+/+). Delayed clot lysis within 72 hours was 33 +/- 1% (n = 3) in tPA-/- mice and 26 +/- 2% (n = 3) in Plg-/- mice (P = .054). Histological examination of several organs revealed fibrin deposition in the liver; lung; and in the stomach, associated with gastric ulcers, in 6- to 12-week-old Plg-/- mice but not in Plg+/+ or Plg+/- littermates. CONCLUSIONS: Plasminogen-deficient mice survive embryonic development but develop spontaneous fibrin deposition due to impaired thrombolysis and suffer retarded growth and reduced fertility and survival. The Plg-/- phenotype is reminiscent of the combined tPA-/-:uPA-/- phenotype, which suggests that there is no significant additional pathway for physiological plasminogen activation in mice.

Animals↗
PubMed1995

MARCKS deficiency in mice leads to abnormal brain development and perinatal death.

The MARCKS protein is a widely distributed cellular substrate for protein kinase C. It is a myristoylprotein that binds calmodulin and actin in a manner reversible by protein kinase C-dependent phosphorylation. It is also highly expressed in nervous tissue, particularly during development. To evaluate a possible developmental role for MARCKS, we disrupted its gene in mice by using the techniques of homologous recombination. Pups homozygous for the disrupted allele lacked detectable MARCKS mRNA and protein. All MARCKS-deficient pups died before or within a few hours of birth. Twenty-five percent had exencephaly and 19% had omphalocele (normal frequencies, < 1%), indicating high frequencies of midline defects, particularly in cranial neurulation. Nonexencephalic MARCKS-deficient pups had agenesis of the corpus callosum and other forebrain commissures, as well as failure of fusion of the cerebral hemispheres. All MARCKS-deficient pups also displayed characteristic lamination abnormalities of the cortex and retina. These studies suggest that MARCKS plays a vital role in the normal developmental processes of neurulation, hemisphere fusion, forebrain commissure formation, and formation of cortical and retinal laminations. We conclude that MARCKS is necessary for normal mouse brain development and postnatal survival.

Animals↗
PubMed2004

Strand invasion involving short tract gene conversion is specifically suppressed in BRCA2-deficient hamster cells.

The BRCA2 tumour suppressor protein is involved in maintaining genetic stability through its role in homologous recombination (HR), where it mediates RAD51-dependent strand invasion. Here, we show that BRCA2-defective cells are not completely impaired in HR by strand invasion although the spontaneous HR rate is 10-fold lower than that in wild-type cells. Furthermore, a DNA double-strand break (DSB) triggers HR repair by strand invasion also in BRCA2-defective cells, but less efficiently. Thus, either the strand invasion pathway(s) in which BRCA2 operates is still operative in the absence of a functional BRCA2, albeit at a reduced frequency, or there is a separate pathway for strand invasion still functional in BRCA2-deficient cells. Consistent with the latter hypothesis, we show that HR events occurring in BRCA2-defective cells differ from HR events in wild-type cells. These data suggest that BRCA2-defective hamster cells are impaired in short tract gene conversion but maintain proficiency in sister chromatid exchange.

Animals↗
PubMed2000

Functional link of BRCA1 and ataxia telangiectasia gene product in DNA damage response.

BRCA1 encodes a familial breast cancer suppressor that has a critical role in cellular responses to DNA damage. Mouse cells deficient for Brca1 show genetic instability, defective G2-M checkpoint control and reduced homologous recombination. BRCA1 also directly interacts with proteins of the DNA repair machinery and regulates expression of both the p21 and GADD45 genes. However, it remains unclear how DNA damage signals are transmitted to modulate the repair function of BRCA1. Here we show that the BRCA1-associated protein CtIP becomes hyperphosphorylated and dissociated from BRCA1 upon ionizing radiation. This phosphorylation event requires the protein kinase (ATM) that is mutated in the disease ataxia telangiectasia. ATM phosphorylates CtIP at serine residues 664 and 745, and mutation of these sites to alanine abrogates the dissociation of BRCA1 from CtIP, resulting in persistent repression of BRCA1-dependent induction of GADD45 upon ionizing radiation. We conclude that ATM, by phosphorylating CtIP upon ionizing radiation, may modulate BRCA1-mediated regulation of the DNA damage-response GADD45 gene, thus providing a potential link between ATM deficiency and breast cancer.

Animals↗
PubMed1999

DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal effects of chemotherapeutic alkylating agents.

We have generated mice deficient in O6-methylguanine DNA methyltransferase activity encoded by the murine Mgmt gene using homologous recombination to delete the region encoding the Mgmt active site cysteine. Tissues from Mgmt null mice displayed very low O6-methylguanine DNA methyltransferase activity, suggesting that Mgmt constitutes the major, if not the only, O6-methylguanine DNA methyltransferase. Primary mouse embryo fibroblasts and bone marrow cells from Mgmt -/- mice were significantly more sensitive to the toxic effects of the chemotherapeutic alkylating agents 1,3-bis(2-chloroethyl)-1-nitrosourea, streptozotocin and temozolomide than those from Mgmt wild-type mice. As expected, Mgmt-deficient fibroblasts and bone marrow cells were not sensitive to UV light or to the crosslinking agent mitomycin C. In addition, the 50% lethal doses for Mgmt -/- mice were 2- to 10-fold lower than those for Mgmt +/+ mice for 1,3-bis(2chloroethyl)-1-nitrosourea, N-methyl-N-nitrosourea and streptozotocin; similar 50% lethal doses were observed for mitomycin C. Necropsies of both wild-type and Mgmt -/mice following drug treatment revealed histological evidence of significant ablation of hematopoietic tissues, but such ablation occurred at much lower doses for the Mgmt -/- mice. These results demonstrate the critical importance of O6-methylguanine DNA methyltransferase in protecting cells and animals against the toxic effects of alkylating agents used for cancer chemotherapy.

Alkylating Agents↗
PubMed1982

Initiation of recombination during transformation of Bacillus subtilis requires no extensive homologous sequences.

Lysates obtained shortly after entry of transforming DNA to Bacillus subtilis contain donor-recipient DNA complexes, in which the donor moiety is associated with the recipient DNA in an unstable way. The complexes could be artificially stabilized by crosslinking with 4,5',8-trimethylpsoralen. The unstable complexes dissociated upon helix-destabilizing treatments, such as heating at 70 degrees C, and CsCl gradient centrifugation at pH 11.2, but remained stable during CsCl gradient centrifugation at pH 10. Donor-recipient DNA complexes were not formed after entry of heterologous pUB110 DNA. These observations suggest that base-pairing is involved in the unstable association. The donor moiety of the unstable complexes was completely, or almost completely, digestible by nuclease S1, indicating that the donor and recipient base-sequences are only paired over very short distances. The unstable donor-recipient DNA complexes are true recombination intermediates because (i) strain 7G224 (recE4) was impaired in the formation of the unstable complexes, and (ii) the unstable complexes were rapidly converted to stable complexes in recombination proficient strains, whereas their conversion was delayed in the recombination deficient strain 7G84. Unstable complexes were also formed with Escherichia coli donor DNA, but to a lesser extent. Apparently a limited degree of base-sequence homology is sufficient to initiate recombination.

Bacillus subtilis↗
PubMed2001

Hypersensitivity to camptothecin in MSH2 deficient cells is correlated with a role for MSH2 protein in recombinational repair.

DNA mismatch repair (MMR) corrects DNA polymerase insertion errors that have escaped proofreading in order to avoid the accumulation of deleterious mutations. While the role of MMR in the correction of replication errors is well established, its involvement in the processing of DNA damage induced by chemical and physical agents is less clear. A role for some of the MMR proteins, such as MSH2, in the repair of double strand break (DSBs) through recombination has also been envisaged. Why MMR- deficient cells are sensitive to agents causing replication fork stalling and thus DSBs remains unclear. To verify a possible role of MSH2 in homologous recombinational repair, we have treated cells from knockout mice for the MSH2 gene and mouse colorectal carcinoma cells also defective for MSH2 with different doses of camptothecin, an agent known to interfere with DNA replication. In the absence of MSH2, we found a reduced survival rate accompanied by higher levels of chromosomal damage and SCE induction. Furthermore, MSH2(-/-) cells displayed an elevated spontaneous RAD51 focus-forming activity and a higher induction of RAD51 foci following camptothecin treatment. Thus, the absence of MSH2 could result in both spontaneous DNA damage and uncontrolled recombination events leading to the observed higher yield of chromosomal damage and the higher induction of RAD51 foci following CPT treatment. Therefore, our results suggest an involvement of MSH2 in the early events leading to correct RAD51 relocalization after the formation of DSBs specifically produced at the blocked replication fork.

Animals↗
PubMed2001

Gene targeting in hemostasis. Alpha2-antiplasmin.

Apha2-antiplasmin (AP), the main physiological plasmin inhibitor in mammalian plasma, is a 70 kDa single chain serpin (serine proteinase inhibitor) with reactive site peptide bond Arg-Met. It inhibits plasmin very rapidly (second-order inhibition rate constant of = 2 x 107 M-1.s-1) following formation of an inactive 1:1 stoichiometric complex. The high reaction rate requires the presence of a free active site and free lysine-binding site(s) in plasmin. The pathophysiologic relevance of AP is suggested by the finding that homozygous deficient patients show a bleeding tendency; heterozygotes, in contrast, frequently have no or only mild bleeding complications. Inactivation of the AP gene in mice was achieved by replacing, via homologous recombination in embryonic stem cells, a 7 kb genomic sequence encoding the entire murine protein with the neomycin resistance expression cassette. Homozygous AP deficient mice display normal fertility, viability and development. They have an enhanced endogenous fibrinolytic capacity without overt bleeding; this is reflected by a higher spontaneous lysis rate of experimental pulmonary emboli, by a reduced fibrin deposition in the kidneys following challenge with endotoxin, by more limited photochemically induced arterial thrombosis, and by reduced infarct size following induction of focal cerebral ischemia by ligation of the left middle cerebral artery. In a vascular injury restenosis model, AP deficiency has no significant effect on smooth muscle cell migration and neointima formation. These data suggest that, at least in the murine system, the main role of alpha2-antiplasmin is in regulating plasmin activity in the circulating blood and in controlling intravascular fibrinolysis.

Animals↗
PubMed2003

Reduced apoptotic response to camptothecin in CHO cells deficient in XRCC3.

Eukaryotic cells respond to DNA damage by activation of DNA repair, cell-cycle arrest and apoptosis. Several reports suggest that such responses may be coordinated by communication between damage repair proteins and proteins signalling other cellular responses. The Rad51-guided homologous recombination (HR) repair plays an important role in recognition and repair of DNA double-strand breaks (DSBs) and cells deficient in this repair pathway become hypersensitive to agents that induce DSBs. In the work reported here we investigated the possible role of the Rad51-like HR proteins XRCC2, XRCC3 and Rad51C in apoptosis following the induction of DSBs by camptothecin. We show that a hamster cell line (irs1SF) deficient in the HR repair gene XRCC3 exhibits altered death and cell-cycle checkpoint responses following treatment with growth inhibitory concentrations of camptothecin. In contrast, hamster cells defective in XRCC2 (irs1) or Rad51C (irs3) treated with equally toxic doses of this agent exhibit a rapid induction of apoptosis similar to that seen in the parental cell line or mutant cells corrected for the HR defect. These results suggest that XRCC3 activity may be necessary for efficient entry into apoptosis in response to DSBs.

Animals↗
PubMed1990

Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors.

The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 10/mug of DNA to 10/mug of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination.

Journal Article↗
PubMed1994

Isolation and characterization of the Rickettsia prowazekii recA gene.

The recA gene has been isolated from Rickettsia prowazekii, an obligate intracellular bacterium. Comparison of the amino acid sequence of R. prowazekii RecA with that of Escherichia coli RecA revealed that 62% of the residues were identical. The highest identity was found with RecA of Legionella pneumophila, in which 69% of the residues were identical. Amino acid residues of E. coli RecA associated with functional activities are conserved in rickettsial RecA, and the R. prowazekii recA gene complements E. coli recA mutants for UV light and methyl methanesulfonate sensitivities as well as recombinational deficiencies. The characterized region upstream of rickettsial recA did not contain a sequence homologous to an E. coli LexA binding site (SOS box), suggesting differences in the regulation of the R. prowazekii recA gene.

Amino Acid Sequence↗
PubMed1999

RecD function is required for high-pressure growth of a deep-sea bacterium.

A genomic library derived from the deep-sea bacterium Photobacterium profundum SS9 was conjugally delivered into a previously isolated pressure-sensitive SS9 mutant, designated EC1002 (E. Chi and D. H. Bartlett, J. Bacteriol. 175:7533-7540, 1993), and exconjugants were screened for the ability to grow at 280-atm hydrostatic pressure. Several clones were identified that had restored high-pressure growth. The complementing DNA was localized and in all cases found to possess strong homology to recD, a DNA recombination and repair gene. EC1002 was found to be deficient in plasmid stability, a phenotype also seen in Escherichia coli recD mutants. The defect in EC1002 was localized to a point mutation that created a stop codon within the recD gene. Two additional recD mutants were constructed by gene disruption and were both found to possess a pressure-sensitive growth phenotype, although the magnitude of the defect depended on the extent of 3' truncation of the recD coding sequence. Surprisingly, the introduction of the SS9 recD gene into an E. coli recD mutant had two dramatic effects. At high pressure, SS9 recD enabled growth in the E. coli mutant strain under conditions of plasmid antibiotic resistance selection and prevented cell filamentation. Both of these effects were recessive to wild-type E. coli recD. These results suggest that the SS9 recD gene plays an essential role in SS9 growth at high pressure and that it may be possible to identify additional aspects of RecD function through the characterization of this activity.

Escherichia coli↗
PubMed2006

Hand, an evolutionarily conserved bHLH transcription factor required for Drosophila cardiogenesis and hematopoiesis.

The Hand gene family encodes highly conserved basic helix-loop-helix (bHLH) transcription factors that play crucial roles in cardiac and vascular development in vertebrates. In Drosophila, a single Hand gene is expressed in the three major cell types that comprise the circulatory system: cardioblasts, pericardial nephrocytes and lymph gland hematopoietic progenitors, but its function has not been determined. Here we show that Drosophila Hand functions as a potent transcriptional activator, and converting it into a repressor blocks heart and lymph gland formation. Disruption of Hand function by homologous recombination also results in profound cardiac defects that include hypoplastic myocardium and a deficiency of pericardial and lymph gland hematopoietic cells, accompanied by cardiac apoptosis. Targeted expression of Hand in the heart completely rescued the lethality of Hand mutants, and cardiac expression of a human HAND gene, or the caspase inhibitor P35, partially rescued the cardiac and lymph gland phenotypes. These findings demonstrate evolutionarily conserved functions of HAND transcription factors in Drosophila and mammalian cardiogenesis, and reveal a previously unrecognized requirement of Hand genes in hematopoiesis.

Animals↗
PubMed2005

[Expression of HBV preS2/S gene in mammalian cells transferred with adenoviral vector].

OBJECTIVE: To study HBV preS2/S gene expression effects in mammalian cells transferred with recombinant adenoviral vector. METHODS: The replication-deficient recombinant adenoviral vector (Ad-HBs) carrying HBV preS2/S gene were constructed by homologous recombination in bacteria. The 293 cells, Vero cells, HepG2 cells and mesenchymal stem cells (MSCs) were infected with adenoviruses. The expressions of enhanced green fluorescent protein (EGFP) were observed with fluorescence microscope and the expressions of HBsAg were detected by RT-PCR and ELISA in vitro. RESULTS: More than 90% of 293 cells, Vero cells, HepG2 cells or MSCs expressed EGFP after transfection at the MOI of 20 and the titers of HBsAg were more than 3.229 (A value) in culture supernatant. CONCLUSION: The HBV preS2/S gene was not only expressed efficiently in immortalized cells, but also expressed efficiently in stem cells with the recombinant adenoviruses vector.

Adenoviridae↗
PubMed1997

[Cytokine network involved in inflammatory reactions].

A cytokine, present in a minute amount in vivo, generally exhibits multiple functions shared by other cytokines. The recent progress in molecular biology and protein chemistry have facilitated identification of the structures and functions of cytokines and their receptors. This has resulted to the clinical application of several cytokines, such as granulocyte colony-stimulating factor and erythropoietin. However, the elucidation of the pathophysiological roles of cytokines in various diseases, has been hindered by a complicated network where cytokines regulate the production and/or functions of others. The utilization of homologous recombination and ES cells has led to the generation of mice that are deficient in a particular cytokine or cytokine receptor gene. Analyses of these deficient mice have given us invaluable information to unravel the roles of each cytokine or cytokine receptor. Here, we described our studies on the pathophysiological roles of pro-inflammatory cytokines using cytokine or cytokine receptor gene-deficient mice to provide examples of the usefulness of these mice in clarifying the cytokine network.

Animals↗
PubMed2006

Functions of multiple exonucleases are essential for cell viability, DNA repair and homologous recombination in recD mutants of Escherichia coli.

Heterotrimeric RecBCD enzyme unwinds and resects a DNA duplex containing blunt double-stranded ends and directs loading of the strand-exchange protein RecA onto the unwound 3'-ending strand, thereby initiating the majority of recombination in wild-type Escherichia coli. When the enzyme lacks its RecD subunit, the resulting RecBC enzyme, active in recD mutants, is recombination proficient although it has only helicase and RecA loading activity and is not a nuclease. However, E. coli encodes for several other exonucleases that digest double-stranded and single-stranded DNA and thus might act in consort with the RecBC enzyme to efficiently promote recombination reactions. To test this hypothesis, I inactivated multiple exonucleases (i.e., exonuclease I, exonuclease X, exonuclease VII, RecJ, and SbcCD) in recD derivatives of the wild-type and nuclease-deficient recB1067 strain and assessed the ability of the resultant mutants to maintain cell viability and to promote DNA repair and homologous recombination. A complex pattern of overlapping and sometimes competing activities of multiple exonucleases in recD mutants was thus revealed. These exonucleases were shown to be essential for cell viability, DNA repair (of UV- and gamma-induced lesions), and homologous recombination (during Hfr conjugation and P1 transduction), which are dependent on the RecBC enzyme. A model for donor DNA processing in recD transconjugants and transductants was proposed.

Cell Survival↗
PubMed2006

Role for RAD18 in homologous recombination in DT40 cells.

RAD18 is an E3 ubiquitin ligase that catalyzes the monoubiquitination of PCNA, a modification central to DNA damage bypass and postreplication repair in both yeast and vertebrates. Although current evidence suggests that homologous recombination provides an essential backup in vertebrate rad18 mutants, we show that in chicken DT40 cells this is not the case and that RAD18 plays a role in the recombination reaction itself. Gene conversion tracts in the immunoglobulin locus of rad18 cells are shorter and are associated with an increased frequency of deletions and duplications. rad18 cells also exhibit reduced efficiency of gene conversion induced by targeted double-strand breaks in a reporter construct. Blocking an early stage of the recombination reaction by disruption of XRCC3 not only suppresses immunoglobulin gene conversion but also prevents the aberrant immunoglobulin gene rearrangements associated with RAD18 deficiency, reverses the elevated sister chromatid exchange of the rad18 mutant, and reduces its sensitivity to DNA damage. Together, these data suggest that homologous recombination is toxic in the absence of RAD18 and show that, in addition to its established role in postreplication repair, RAD18 is also required for the orderly completion of gene conversion.

Animals↗
PubMed2000

Structural evidence of genomic exon-deletion mediated by Alu-Alu recombination in a human case with heme oxygenase-1 deficiency.

We previously reported a family affected by heme oxygenase-1 (HO-1) deficiency [Yachie et al., 1999]. The proband was a compound heterozygote for a complete loss of exon 2 (the maternal allele) and a two-nucleotide deletion within exon 3 (the paternal allele). In this report, we describe a large genomic deletion (1730 bp) including entire exon 2 in this family as a specific mechanism generating exon-2 absence observed in the HO-1 mRNA. Analysis of the deletion junction demonstrated fusion of a 5' portion of Alu-Sx element with a 3' portion of Alu-Sq element. The junction contained sequences with high homology to the recombinogenic Alu "core" sequence. These structural features of the HO-1 gene suggest homologous recombination associated with Alu element. This study presents the initial characterization of the HO-1 gene defect causing a human case of HO-1 deficiency and provides the molecular basis for understanding this genetic disease.

Alleles↗