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At least 307 records · Page 17Linked to original sources
PubMed1994

Structure of REC2, a recombinational repair gene of Ustilago maydis, and its function in homologous recombination between plasmid and chromosomal sequences.

Mutation in the REC2 gene of Ustilago maydis leads to defects in DNA repair, recombination, and meiosis. Analysis of the primary sequence of the Rec2 protein reveals a region with significant homology to bacterial RecA protein and to the yeast recombination proteins Dmc1, Rad51, and Rad57. This homologous region in the U. maydis Rec2 protein was found to be functionally sensitive to mutation, lending support to the hypothesis that Rec2 has a functional RecA-like domain essential for activity in recombination and repair. Homologous recombination between plasmid and chromosomal DNA sequences is reduced substantially in the rec2 mutant following transformation. The frequency can be restored to a level approaching, but not exceeding, that observed in the wild-type strain if transformation is performed with cells containing multiple copies of REC2.

Amino Acid Sequence↗
PubMed1999

Use of a recombination reporter insert to define meiotic recombination domains on chromosome III of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, meiotic recombination is initiated by DNA double-strand breaks (DSBs). DSBs usually occur in intergenic regions that display nuclease hypersensitivity in digests of chromatin. DSBs are distributed nonuniformly across chromosomes; on chromosome III, DSBs are concentrated in two "hot" regions, one in each chromosome arm. DSBs occur rarely in regions within about 40 kb of each telomere and in an 80-kb region in the center of the chromosome, just to the right of the centromere. We used recombination reporter inserts containing arg4 mutant alleles to show that the "cold" properties of the central DSB-deficient region are imposed on DNA inserted in the region. Cold region inserts display DSB and recombination frequencies that are substantially less than those seen with similar inserts in flanking hot regions. This occurs without apparent change in chromatin structure, as the same pattern and level of DNase I hypersensitivity is seen in chromatin of hot and cold region inserts. These data are consistent with the suggestion that features of higher-order chromosome structure or chromosome dynamics act in a target sequence-independent manner to control where recombination events initiate during meiosis.

Argininosuccinate Lyase↗
PubMed1985

Plasmid recombination intermediates generated in a Saccharomyces cerevisiae cell-free recombination system.

We have developed an assay utilizing Saccharomyces cerevisiae cell extracts to catalyze recombination in vitro between homologous plasmids containing different mutant alleles of the tet gene. Electrophoretic analysis of product DNA indicated that a number of novel DNA species were formed during the reaction. These species migrated through agarose gels as distinct bands with decreased electrophoretic mobility compared with the substrate DNA. The DNA from each individual band was purified and shown to be enriched 5- to 100-fold for tetracycline-resistant recombinants by using a transformation assay. The structure of the DNA molecules present in these bands was determined by electron microscopy. Recombination between circular substrates appeared to involve the formation and processing of figure-eight molecules, while recombination between circular and linear substrates involved the formation of molecules in which a circular monomer had a monomer-length linear tail attached at a region of homology.

Alleles↗
PubMed1999

Efficient generation of recombinant adenoviral vectors by Cre-lox recombination in vitro.

BACKGROUND: Although recombinant adenovirus vectors are attractive for use in gene expression studies and therapeutic applications, the construction of these vectors remains relatively time-consuming. We report here a strategy that simplifies the production of adenoviruses using the Cre-loxP system. MATERIALS AND METHODS: Full-length recombinant adenovirus DNA was generated in vitro by Cre-mediated recombination between loxP sites in a linearized shuttle plasmid containing a transgene and adenovirus genomic DNA. RESULTS: After transfection of Cre-treated DNA into 293 cells, replication-defective viral vectors were rapidly obtained without detectable wild-type virus. CONCLUSION: This system facilitates the development of recombinant adenoviral vectors for basic and clinical research.

Adenoviridae↗
PubMed1976

Age trends in human chiasma frequencies and recombination fractions. II. Method for analyzing recombination fractions and applications to the ABO:nail-patella linkage.

A new method is presented for studying the relationship between human recombination fractions and parental age at the time of conception. Assuming the sex specific recombination fraction to be a linear function of age, a feasible computer algorithm is described whereby the likelihood of multigenerational families can be calculated. Using this method and the likelihood ratio test, it is found that for the ABO:nail-patella linkage age (P= .17)is more significant than sex (p= .23) in its effect on the recombination fraction. The age effect, if it is real, appears to be limited to males: the paternal recombination fraction decreases by .0062(+/- .0036) per year.

ABO Blood-Group System↗
PubMed2004

Evolutionary recombination hotspot around GSDML-GSDM locus is closely linked to the oncogenomic recombination hotspot around the PPP1R1B-ERBB2-GRB7 amplicon.

PPP1R1B-STARD3-TCAP-PNMT-PERLD1-ERBB2-MGC14832-GRB7 locus at human chromosome 17q12 is frequently amplified in human gastric cancer and breast cancer. Here, we compared human GSDML-GSDM locus with rodent genomes by using bioinformatics. Rodent ortholog of human GSDML was not identified. Rat Gsdm gene was identified within rat genome clone CH230-28N16 (AC119462.4), and was mapped to rat chromosome 10q31. Rat Gsdm gene, consisting of 12 exons, encoded a 446-amino-acid protein, which showed 86.3% and 32.3% total-amino-acid identities with human GSDM and GSDML, respectively. Mouse Gsdm-like 1 (Gsdml1) and Gsdml2 genes were identified within mouse genome clone RP23-438D7 (AL591125.20). Gsdml1 and Gsdml2 genes were found to encode 456- and 443-amino-acid proteins, respectively. Mouse 2200001G21Rik cDNA (AK008613.1) was a partial cDNA derived from mouse Gsdml2 gene. Mouse Gsdml1 and Gsdml2 were also more homologous to human GSDM than to human GSDML. Mouse Gsdml1, Gsdml2 and Gsdm genes, existing in the tandem homologous gene cluster, was mapped to mouse chromosome 11D. Mouse Gsdml1-Gsdml2-Gsdm gene cluster was predicted to be generated due to triplication of mouse Gsdm gene, while GSDML gene was predicted to be generated due to duplication of GSDM gene. Evolutionary recombination hotspot around the GSDML-GSDM locus was closely linked to the oncogenomic recombination hotspot around the PPP1R1B-ERBB2-GRB7 amplicon. The evolutionary recombination hotspot and oncogenomic recombination hotspot might be clustered around the fragile sites within the human genome.

Amino Acid Sequence↗
PubMed2004

[Combination immunization with EIAV Env protein expressed by recombinant baculovirus and recombinant vaccinia virus containing env gene].

AIM: To develop a novel vaccine candidate of Equine infectious anemia virus(EIAV). METHODS: env genes of EIAV Chinese donkey leukocyte attenuated strain (EIAV DLV) and its parental virus strain (EIAV LN) were expressed using the BAC-To-BAC system, and Env proteins were confirmed by SDS-PAGE and Western blot. BALB/c mice were immunized with recombinant vaccinia viruses containing env genes of EIAV alone or boosted with Env proteins expressed by recombinant baculovirus. Both protective humoral and cellular immune responses were detected. RESULTS: Recombinant baculovirus could express complete Env proteins and the titer of neutralizing antibody in the prime-boost group was found to be 5 to 9 folds higher than that in the rVV group. CONCLUSION: Combination of protein antigen and recombinant vaccinia virus could induce high titer of neutralizing antibody against EIAV. This work facilitates development of a novel genetically engineered vaccine of EIAV as a model for future development of an HIV vaccine.

Animals↗
PubMed1992

Reactions between half- and full-FLP recombination target sites. A model system for analyzing early steps in FLP protein-mediated site-specific recombination.

The FLP recombination target (FRT) can be cut in half so that only one FLP protein binding site is present (a "half site"). FLP protein binds the half sites and joins them into dimeric, asymmetric head-to-head complexes held together chiefly by strong noncovalent interactions. These complexes react with full (normal) FRT sites to generate a variety of products. Analysis of these DNA species reveals that the reaction follows a well-defined reaction pathway that generally parallels the normal reaction pathway. The system is useful in analyzing early steps in recombination, since the identity of the products in a given recombination event unambiguously pinpoints the order in which the cleavage and strand exchange reactions occur. Two conclusions are derived from the present study: (i) Formation of the dimeric head-to-head complex of half sites is a prerequisite to further steps in recombination. (ii) The identity of the base pairs at positions 6 and -6 within the FRT site has a subtle effect in directing the first strand exchange event in the reaction to predominantly one of two possible cleavage sites. In addition, results are presented that suggest that a DNA-DNA pairing intermediate involving only two base pairs of the core sequence is formed prior to the first cleavage and strand exchange. DNA-DNA interactions may therefore not be limited to the isomerization step that follows the first strand exchange.

Base Sequence↗
PubMed1988

Growth of clonogenic myeloblastic leukemic cells in the presence of human recombinant erythropoietin in addition to various human recombinant hematopoietic growth factors.

The effects of human recombinant erythropoietin (rEpo) in the presence of other stimulators on the growth of clonogenic leukemic blast cells from ten Japanese patients with acute myeloblastic leukemia were studied with an in vitro leukemic blast colony assay in methylcellulose culture. With the addition of rEpo alone, no leukemic blast colony formation was stimulated in any of the cases examined. However, when rEpo and phytohemagglutinin lymphocyte-conditioned medium (PHA-LCM) were added to the culture simultaneously, in contrast to results with PHA-LCM alone, the number of leukemic blast colonies formed was significantly increased in two of the ten cases (P less than .01). These two cases were classified as M1 according to the French-American-British (FAB) classification. This enhancing effect of rEpo was observed with human recombinant granulocyte/macrophage colony-stimulating factor (rGM-CSF) or human recombinant interleukin-3 (rIL-3) but was not observed with human recombinant granulocyte CSF (rGCSF).

Cell Division↗
PubMed1986

Recombinant IL 2 but not recombinant interferon-gamma stimulates both proliferation and IgM secretion in a Ly-1+ clone of neoplastic murine B cells (BCL1).

We have used a lymphokine-responsive clone (3B3) of B leukemia cells (BCL1) to examine the effects of several recombinant and purified lymphokines. Cells from BCL1-3B3 were induced to secrete IgM in the presence of recombinant interleukin 2 (rIL 2) (10 to 50 U/ml); a concomitant increase in proliferation was observed. Recombinant interferon-gamma (rIFN-gamma) was a potent inhibitor of proliferation. In addition, rIFN-gamma did not induce an increase in IgM secretion and, when added to rIL 2-stimulated BCL1-3B3 cells, completely blocked IgM secretion at a concentration of 10 U/ml. Purified and recombinant IL 1 (rIL 1) had no significant effect on differentiation either alone or in combination with rIL 2 and/or rIFN-gamma. However, rIL 1 was able to synergize with rIL 2 in enhancing the proliferation of BCL1-3B3. The ability of cells to respond to rIL 2 was limited to the in vitro (Ly-1+) clones of BCL1 cells since the in vivo derived (Ly-1-) BCL1 cells did not differentiate in response to IL 2. Consistent with their functional response to rIL 2, cells from the in vitro clone (3B3) are IL 2-receptor-positive (IL-2R+) and the in vivo derived BCL1 cells are IL-2R-. A second set of neoplastic B cell clones derived from the AKR 225 lymphoma did not respond to rIL 2 even though they expressed receptors for IL 2 and could be induced by T cell supernatant to secrete IgM, thus indicating that expression of IL 2R is not the sole requirement for IL 2 responsiveness. The monoclonal anti-IL 2R antibody (7D4) mimicked IL 2 in its ability to stimulate differentiation of BCL1-3B3 cells. These data suggest that rIL 2 and the monoclonal anti-IL-2R antibody are capable of inducing a differentiative response in the Ly-1+ BCL1-3B3 cells that is functionally equivalent to the response evoked by the previously described lymphokine B cell differentiation factor for IgM (BCDF mu). Thus, two distinct lymphokines appear to be providing a similar signal to a clonal neoplastic B cell population. Furthermore, rIL 2 is capable of providing both a proliferative and a differentiative signal.

Animals↗
PubMed1988

Synergistic antitumor effects of combination immunotherapy with recombinant interleukin-2 and a recombinant hybrid alpha-interferon in the treatment of established murine hepatic metastases.

We investigated the antitumor effects of combined immunotherapy with recombinant human interleukin-2 (rhIL-2) and the recombinant human interferon-alpha (rhIFN-alpha) A/D hybrid in the treatment of established single or multiple murine hepatic metastases. Mice bearing either weakly immunogenic MCA-106 or nonimmunogenic MCA-102 were treated with rhIL-2 alone, rhIFN-alpha alone, or the combination of lymphokines. Therapy was initiated on Day 3 or 10 and continued for 3-4 consecutive days. In the treatment of 3- and 10-day multiple MCA-106 liver metastases, significant reductions in the number of metastases, often more than 90%, were observed with the combination of rhIL-2 and rhIFN-alpha at doses of each lymphokine which had no effect when given alone. This decrease in the number of metastases resulted in a survival benefit that was seen in the combination therapy groups in a dose-dependent manner. Similarly, substantial reductions in tumor weight were seen when the combination of rhIL-2 and rhIFN-alpha was administered to mice with single large hepatic metastases. The decreases in both single and multiple metastatic tumor deposits by the combination of lymphokines were more than that predicted by the additive effect of each treatment alone. With the nonimmunogenic tumor, MCA-102, however, no benefit was derived from the addition of rhIFN-alpha to rhIL-2 therapy. Immunotherapy with recombinant murine interferon-gamma and rhIL-2 was directly compared to therapy with rhIL-2 and rhIFN-alpha. The combination of rhIL-2 and rhIFN-alpha again was found to be effective while recombinant murine interferon-gamma added toxicity but no therapeutic benefit to immunotherapy with rhIL-2 alone. The synergy between rhIL-2 and rhIFN-alpha was shown to be dependent on the host's intact immune system since mice immunosuppressed by sublethal irradiation prior to inoculation of tumor did not respond to the combined treatment. Possible mechanisms of the in vivo synergy between rhIL-2 and rhIFN-alpha are discussed.

Animals↗
PubMed1984

The use of recombinant DNA plasmids for the determination of DNA-repair and recombination in cultured mammalian cells.

Using the recombinant plasmid pSV2gpt and DNA transfer techniques, cell mediated DNA ligation and recombination of plasmid DNA have been demonstrated in four human cell lines. Data suggesting the involvement of a possible defect in the cellular equilibrium between ligation and exonuclease digestion of double strand DNA scissions in an ataxia-telangiectasia (A-T) cell line is discussed. The same A-T line was grossly proficient in DNA recombination but it will be necessary to distinguish between recombination in coding and non-coding plasmid sequences.

Ataxia Telangiectasia↗
PubMed1993

Lymphoid V(D)J recombination. Functional analysis of the spacer sequence within the recombination signal.

The V(D)J recombination reaction is directed by a pair of signal sequences, each consisting of a palindromic heptamer, an A/T-rich nonamer, and an intervening spacer sequence of 12 or 23 base pairs. The spacer sequence previously has not been analyzed for a functional role. In this study, numerous simultaneous sequence changes have been made in the spacer of each signal to test their functional importance. All of the AT base pairs in each signal were changed to GC base pairs. This particular change is of interest because it markedly increases the energy that would be required to melt out the two strands of each signal to permit the intersignal base pairing proposed in a commonly invoked model for signal-signal interaction in V(D)J recombination. We find that changing 6 to 12 AT base pairs in the 12-signal to GC does not affect V(D)J recombination, nor does changing 11 of 23 AT base pairs in the 23-signal. Substrates with all-GC spacer sequences in both the 12- and the 23-signal also recombine at efficiencies that are not significantly reduced. These studies demonstrate that the sequences at these particular positions are not recognized by the recombinase. In addition, the data do not support models invoking signal-signal base pairing.

Animals↗
PubMed1996

Reconstitution of recombinant bovine A1 adenosine receptors in Sf9 cell membranes with recombinant G proteins of defined composition.

We investigated the coupling of A1 adenosine receptors to recombinant G proteins. Recombinant baculoviruses were used to express bovine A1 adenosine receptors in Sf9 insect cells that lack endogenous adenosine receptors. Binding parameters for recombinant receptors expressed in Sf9 cell membranes using the antagonist radioligand [125I]BW-A844U ([125I]8-cyclopentyl-3-iodoaminophenethyl-1-propylxanthine) are Bmax = 2-5 pmol/mg of protein and K(D) = 0.53 +/- 0.12 nM. In competition assays, the potency order of agonists is (R)-phenylisopropyladenosine > (S)-phenylisopropyladenosine > 5'-N-ethylcarboxamidoadenosine, properties characteristic of native bovine A1 adenosine receptors. The agonist radioligand 125I-N6-4-aminobenzyladenosine binds to two affinity states of the recombinant A1 adenosine receptors with K(D) values of 0.09 and 10.4 nM. The high affinity binding site represents <10% of total sites and is increased 7-fold on reconstitution with both alpha and betagamma G protein subunits but not with either subunit alone; thus, exogenous alpha and betagamma subunits do not functionally interact with endogenous Sf9 betagamma and alpha subunits, respectively. Four different alpha subunits (alpha i1, alpha i2, alpha i3, and alpha o) and six different beta gamma subunits (beta1gamma1, beta1gamma2, beta1gamma3, beta2gamma2, beta2gamma3, and bovine brain betagamma)) increased GTP-sensitive, high affinity agonist binding. The results indicate that bovine A1 adenosine receptors couple equally well to G protein alpha i and alpha o subunits in combination with betagamma subunits containing the beta1 or beta2 subunits and gamma2 or gamma3 subunits. G protein heterotrimers that contain the beta1gamma1 dimer couple with similar potency but reduced efficacy to A1 adenosine receptors.

Animals↗
PubMed1998

Recombinant fowlpox viruses coexpressing chicken type I IFN and Newcastle disease virus HN and F genes: influence of IFN on protective efficacy and humoral responses of chickens following in ovo or post-hatch administration of recombinant viruses.

We have constructed recombinant (r) fowl pox viruses (FPVs) coexpressing chicken type I interferon (IFN) and/or hemagglutinin-neuraminidase (HN) and fusion (F) proteins of Newcastle disease virus (NDV). We administered rFPVs and FPV into embryonated chicken eggs at 17 days of embryonation or in chickens after hatch. Administration of FPV or rFPVs did not influence hatchability and survival of hatched chicks. In ovo or after hatch vaccination of chickens with the recombinant viruses resulted in protection against challenge with virulent FPV and NDV. Chickens vaccinated with FPV or FPV-NDV recombinant had significantly lower body weight 2 weeks following vaccination. This loss in body weight was not detected in chickens receiving FPV-IFN and FPV-NDV-IFN recombinants. Chickens vaccinated with FPV coexpressing IFN and NDV genes produced less antibodies against NDV in comparison with chickens vaccinated with FPV expressing NDV genes.

Animals↗
PubMed1995

On the possibility of coherently stimulated recombination and cosmological structure generation: recombination instability.

Possible instabilities during cosmological recombination may produce an epoch of nonlinear density growth and fractal-like structural patterns out to the horizon scale at that epoch (approximately 200 Mpc today). With this motivation, we examine the consequences of the change in effective radiative recombination reaction rate coefficients produced by intense stimulated emission. The proton-electron recombination is considered as a natural laser, leading to the formation of spatially nonuniform distributions of neutral matter earlier than the recombination epoch.

Journal Article↗
PubMed1998

Comparative analysis of humoral immune responses to HIV type 1 envelope glycoproteins in mice immunized with a DNA vaccine, recombinant Semliki Forest virus RNA, or recombinant Semliki Forest virus particles.

The Semliki Forest virus (SFV) system seems to be a useful new approach for generating effective immune responses against HIV-1 in animal models. We evaluated this system by comparing the humoral immune responses raised in mice immunized against the HIV-1 envelope with the SFV system, a DNA vaccine, and a recombinant Env glycoprotein. gp160 ELISA antibody titers (204,800) were highest in the sera from mice immunized with recombinant Semliki Forest virus particles. These sera contained antibodies to the CD4-binding site and recognized linear epitopes on gp120 and gp41 that were also recognized by a pool of sera from HIV1-infected individuals. This demonstrates that the HIV-1 envelope produced in vivo by the SFV system does not fold aberrantly. A low level of neutralizing antibodies against the HIV-1LAI strain was also detected in the serum of one mouse immunized with recombinant SFV particles, suggesting that booster injections should be given to achieve a more effective immune response. SFV recombinant particles induced the strongest humoral responses to the HIV-1 envelope of all the potential HIV env vaccines tested.

AIDS Vaccines↗
PubMed2005

Recombinant adenovirus vector vaccine induces stronger cytotoxic T-cell responses than recombinant vaccinia virus vector, plasmid DNA, or a combination of these.

The efficiency of prime-boost vaccinations on the induction of T-cell responses to Sin Nombre virus nucleocapsid protein expressed by recombinant vaccinia virus, replication-deficient adenovirus, and plasmid DNA in mice was quantitated by the number of epitope-specific interferon-gamma-producing T cells and cytotoxic T-lymphocyte activity induced. In prime-boost immunizations, all combinations that included the recombinant adenovirus induced a much higher number of epitope-specific interferon-gamma-producing T cells than did other combinations. A single immunization of the recombinant adenovirus was able to induce similarly high levels of epitope-specific interferon-gamma-producing cells, despite the fact that the recombinant adenovirus produces less amount of the Sin Nombre virus nucleocapsid protein.

Adenoviridae↗