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PubMed2004

The recombination genes addAB are not restricted to gram-positive bacteria: genetic analysis of the recombination initiation enzymes RecF and AddAB in Rhizobium etli.

Single-strand gaps (SSGs) and double-strand breaks (DSBs) are the major initiation sites for recombination. In bacteria, the SSGs are repaired by RecFOR, while the DSBs are processed by RecBCD in gram-negative bacteria and AddAB in gram-positive bacteria. Unexpectedly, instead of recBCD genes, the addAB genes were found in members of the alpha-proteobacteria group (gram negative). Taking Rhizobium etli as a model, the role of recF and addAB genes in homologous recombination and repair of damaged DNA was evaluated. Inactivation of either recF or addA provoked strong sensitivity to UV radiation and mitomycin C, while an additive effect was observed in the recF-addA mutant. The DSBs generated by nalidixic acid caused low viability only in the addA mutant. The recombination frequency of large and small plasmids was reduced in the recF mutant (24- and 36-fold, respectively), whereas a slight decrease (threefold) in the addA mutant was observed. Moreover, an additive effect (47- and 90-fold, respectively) was observed in the double mutant, but it was not as dramatic as that in a recA mutant. Interestingly, the frequency of deletion and Campbell-type recombination was slightly affected in either single or double mutants. These results suggest that another pathway exists that allows plasmid and Campbell-type recombination in the absence of recF and addA genes.

Amino Acid Sequence↗
PubMed1993

Herpes simplex virus type 1 variant a sequence generated by recombination and breakage of the a sequence in defined regions, including the one involved in recombination.

A herpes simplex virus type 1 clone, GN29, having exclusively the variant a sequence was isolated. This a sequence was composed of unique (U) and directly repeated (DR) elements DR1, Ub, (DR2)14, Ucd, Ubd, (DR2)5, DR4n2, and Uc and was assumed to be generated by recombination between sites in Ub and Uc. Unusual DNA fragments containing parts of the a sequence, present in the DNA preparations of GN29, were molecularly cloned. Almost all termini of the cloned unusual DNA fragments were situated in defined regions assumed to be recombinogenic: (i) a site in the inverted repeat of the L component, (ii) DR1, (iii) DR2, (iv) the DR4 stretch, and (v) the novel recombination stretch in the variant a sequence of GN29. The termini of unusual DNA fragments, possibly produced by strand breaks, can serve as free DNA ends to initiate recombination of the a sequence. These results support the model of double-strand-break repair for recombination of the a sequence. Sequence-specific enhancement of the recombination of the a sequence probably depends on the presence of recombinogenic elements apt to break, such as DR2 repeats and the DR4 stretch.

Base Sequence↗
PubMed1994

Herpes simplex virus type 1 recombination: the Uc-DR1 region is required for high-level a-sequence-mediated recombination.

The a sequences of herpes simplex virus type 1 are believed to be the cis sites for inversion events that generate four isomeric forms of the viral genome. Using an assay that measures deletion of a beta-galactosidase gene positioned between two directly repeated sequences in plasmids transiently maintained in Vero cells, we had found that the a sequence is more recombinogenic than another sequence of similar size. To investigate the basis for the enhanced recombination mediated by the a sequence, we examined plasmids containing direct repeats of approximately 350 bp from a variety of sources and with a wide range of G+C content. We observed that all of these plasmids show similar recombination frequencies (3 to 4%) in herpes simplex virus type 1-infected cells. However, recombination between directly repeated a sequences occurs at twice this frequency (6 to 10%). In addition, we find that insertion of a cleavage site for an a-sequence-specific endonuclease into the repeated sequences does not appreciably increase the frequency of recombination, indicating that the presence of endonuclease cleavage sites within the a sequence does not account for its recombinogenicity. Finally, by replacing segments of the a sequence with DNA fragments of similar length, we have determined that only the 95-bp Uc-DR1 segment is indispensable for high-level a-sequence-mediated recombination.

Animals↗
PubMed2003

Presence of replicating virus in recombinant hepadnavirus stocks results from recombination and can be eliminated by the use of a packaging cell line.

Mutant hepatitis B viruses are useful tools to study the viral life cycle and viral pathogenesis. Furthermore, recombinant hepatitis B viruses are candidate vectors for liver-directed gene therapy. Because wild-type viruses present in recombinant or mutant virus stocks may falsify experimental results and are detrimental for a viral vector, we investigated whether and to what extent wild-type virus is present in recombinant virus stocks and where it originates from. We took advantage of the duck model of hepatitis B virus infection which allows very sensitive detection of replication-competent viruses by infection of primary duck hepatocytes or of ducklings in vivo. Recombinant hepatitis B virus stocks contained significant amounts of wild-type viruses, which were most probably generated by homologous recombination between plasmids containing homologous viral sequences. In addition, replication-competent viral genomes were reconstituted from plasmids which contained replication-deficient but redundant viral sequences. Using a stable cell line for packaging of deficient viral genomes, no wild-type virus was detected, neither by infection of primary hepatocytes nor in vivo.

Animals↗
PubMed1997

The composition of coding joints formed in V(D)J recombination is strongly affected by the nucleotide sequence of the coding ends and their relationship to the recombination signal sequences.

V(D)J recombination proceeds in two stages. Precise cleavage at the border of the conserved recombination signal sequences (RSSs) and the coding ends results in flush double-stranded signal ends and coding ends terminating in hairpins. In the second stage, the signal and coding ends are processed into signal and coding joints. Coding ends containing certain nucleotide homopolymers affect the efficiency of V(D)J recombination. In this study, we have tested the effect of small changes in coding-end nucleotide composition on the frequency of coding- and signal joint formation. Furthermore, we have determined the sequences of coding joints resulting from recombination of coding ends with different compositions. We found that the presence of two T nucleotides 5' of both RSSs, but not a single T, reduces the frequency of signal joint formation, i.e., interferes with the cleavage stage of V(D)J recombination. However, coding-joint processing is sensitive even to a single T. Both the sequence of the coding ends and the particular RSS (12-mer or 23-mer) with which the coding end is associated affect the final composition of the coding joints. Thus, the presence of P nucleotides, the conservation of one undeleted coding end, the formation of joints without any deletions, and the template-dependent insertion of nucleotides are strongly influenced by the coding-end nucleotide composition and/or RSS association. The implications of these results with respect to the processing of coding ends are discussed.

Animals↗
PubMed2026

Pervasive HIV recombination limits the utility of circulating recombinant form nomenclature.

The naming of HIV-1 circulating recombinant forms (CRFs)-descendent viruses from the same intersubtype recombination events, is along with the designation of 'subtypes' and 'groups', routinely used to track HIV-1 diversity. However, we argue that continuing to designate all detected CRFs as distinct entities is biologically unjustified, as many represent recombinants of limited epidemiological significance. Indeed, the mechanistic underpinning of HIV-1 recombination highlights the arbitrary nature of naming these incidental recombinants, the majority of which are rarely detected again. This underlines the need to prioritise taxonomically meaningful clades, with a focus on biological significance such as emergence events associated with significant epidemiological spread, phenotypic properties or transmission advantage.

HIV-1↗
PubMed2004

Construction of recombinant vaccinia viruses using leporipoxvirus-catalyzed recombination and reactivation of orthopoxvirus DNA.

Poxvirus DNA is not infectious because the initiation of the infective process requires proteins encapsidated along with the virus genome. However, infectious virus can be produced if purified poxvirus DNA is transfected into cells previously infected with another poxvirus. This process is termed heterologous reactivation if the infecting virus is different from the transfected virus. We describe a method in which the high-frequency recombination and replication reactions catalyzed by the Leporipoxvirus, Shope fibroma virus (SFV), can be coupled with SFV-promoted reactivation reactions to rapidly construct recombinant vaccinia viruses in high yields (25-100% recombinant progeny). The reactivated vaccinia viruses are easily purified free of the SFV helper virus by plating mixed populations of virus on cells that support only the growth of vaccinia virus. These heterologous reactivation reactions can be used to manipulate the structure of virus genomes and produce viruses that express recombinant proteins at high levels. We illustrate the method by polymerase chain reaction (PCR) cloning the gene encoding green fluorescent protein (GFP), then using double-strand break repair reactions to produce a recombinant virus that expresses high levels of GFP.

Animals↗
PubMed1999

Pharmacokinetics of recombinant factor VIII (recombinate) using one-stage clotting and chromogenic factor VIII assay.

In a study designed to demonstrate the safety and pharmacokinetics of a recombinant factor VIII (Recombinate) manufactured in Andover, MA and Thousand Oaks, CA, two different methods of factor VIII assay (one-stage clotting and Chromogenic substrate) were compared in vivo. The study was performed in four centres in the UK: London, Oxford, Cardiff and Manchester. Two pharmacokinetic studies, at least one week apart, were performed in 30 patients with severe haemophilia A (VIII:C < 2 IU/dl). A dose of 50 IU/kg was administered with sampling pre-infusion, and +0.25, 0.5, 1, 3, 6, 9, 12 and 24 h post-infusion. The aggregate 60 pharmacokinetic study showed a half-life of 12.7 and 13.0 h (p = 0.28) and recovery of 127 and 161 IU/dl (p = 0.0001) using one-stage clotting or chromogenic substrate respectively. In a supplementary experiment, 20 post-infusion samples were re-assayed by 1-stage and chromogenic assay using two plasma (20th British plasma standard and an "in-house" pooled normal plasma) and two concentrate standards, derived from the same type, but different batch of infused concentrate (Recombinate) and pre-diluted in either individual pre-infusion sample or in pooled commercial haemophilic plasma. The use of the Recombinate concentrate standard overcame the significant difference in FVIII levels between 1-stage and chromogenic assay methods when a plasma standard was used (p <0.0001). It is concluded that where potency dosing designation is carried out by an assay system different to that used in the clinical situation, the use of the recombinant concentrate as a standard in post-infusion plasma samples is likely to give more reliable and reproducible results.

Biological Assay↗
PubMed1992

Pharmacokinetic properties of recombinant factor VIII compared with a monoclonally purified concentrate (Hemofil M). The Recombinate Study Group.

A recombinant FVIII preparation, Recombinate, was compared with a high-purity plasma-derived concentrate, Hemofil M, in 47 hemophilia A patients in a cross-over evaluation of pharmacokinetic properties. The recombinant material showed a significantly lower clearance, volume of distribution, and higher in vivo recovery, but a similar half-life to the plasma-based product. In a comparison with reported data from other standard concentrates, the recombinant preparation exhibited potentially better pharmacokinetic properties in that its clearance was slower and its half-life was longer. We conclude that the recombinant DNA method of preparation does not adversely affect the biological and pharmacological characteristics of the factor VIII molecule.

Adolescent↗
PubMed1991

Recombination of exogenous interleukin 2 receptor gene flanked by immunoglobulin recombination signal sequences in a pre-B cell line and transgenic mice.

We have constructed a plasmid, pLTR100, which contains human interleukin 2 receptor light (IL-2R L) chain cDNA in the inverted orientation relative to the upstream SV40 promoter. The cDNA segment is flanked by the immunoglobulin gene recombination signal sequences so that the cDNA segment can invert and the human IL-2R L chain is subsequently expressed under the control of the SV40 promoter. A murine pre-B cell line, 38B9, transfected with pLTR100 began to express the human IL-2R L chain on the cell surface. The frequency of human IL-2R L chain positive cells increased almost linearly up to 50% for 60 days of culture after transfection. Southern blot analysis and sequencing of the DNA fragments at the recombination junction confirmed that the cDNA segment was inverted in a signal sequence-dependent manner by the variable-diversity-joining recombination process. Transgenic mice bearing the recombination substrate DNA similar to pLTR100 expressed the human IL-2 L chain in the spleen, thymus, and bone marrow, but not in the other tissues examined at the detectable level. Both IgM- and CD3-positive cells expressed the human IL-2R L chain, indicating that this artificial DNA can serve as a substrate for recombination both in B- and T-cells and that another DNA segment may be necessary to confer the cell-type specificity on the substrate DNA.

Animals↗
PubMed1988

In vivo antitumor activity of multiple injections of recombinant interleukin 2, alone and in combination with three different types of recombinant interferon, on various syngeneic murine tumors.

We have used several transplantable experimental murine tumors to evaluate the potentiation of antitumor activity by a combination of human recombinant interleukin 2 (rHIL2) and recombinant interferons (rIFNs). The combination of rHIL2 and either human hybrid recombinant alpha-interferon A/D (rIFN-alpha A/D) or mouse recombinant beta-interferon (rIFN-beta) induced the s.c. adenocarcinoma 755, which had been established for 8 days, to regress, although rHIL2 or the rIFNs alone hardly inhibited the tumor's growth. Eight injections of the rHIL2-rIFN-alpha A/D combination cured 38% of the tumor-bearing mice. The rHIL2-rIFN-beta combination achieved a complete cure only when given in more than 13 injections. The administration of rHIL2 and mouse recombinant gamma-interferon (rIFN-gamma) markedly inhibited tumor growth of the s.c. established adenocarcinoma 755, but did not cure any of the mice. Other tumors, B16-F10 melanoma, and colon tumors 38 and 26 responded almost as well to a rHIL2-rIFN-alpha A/D or -beta combination, but not to a rHIL2-rIFN-gamma combination. The growth of Lewis lung carcinoma was inhibited to a lesser extent by all combinations, for which there were no long-term survivors. The combination therapy of rHIL2 and rIFN-beta produced a marked regression of the tumor in beige mice which have low natural killer activity, suggesting the activated natural killer cells not to be responsible for the therapeutic effect. And T-cell immunity may be important in the regression of s.c. established tumors, because of the lesser potentiation of antitumor activity in athymic mice. These results demonstrate that combination therapies of rHIL2 and rIFN-alpha A/D or -beta can function synergistically in the various s.c. established murine tumor systems and give further evidence in support of their clinical potential.

Animals↗
PubMed1989

Effect of recombinant GM-CSF and recombinant G-CSF on colony formation of blast progenitors in acute myeloblastic leukemia.

The colony-promoting activities of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) and recombinant granulocyte colony-stimulating factor (rG-CSF) on primary and secondary colony formation by blast progenitors (leukemic colony-forming units [L-CFU]) from 21 patients with acute myeloblastic leukemia (AML) were examined using blast colony assay and compared to colony promotion stimulated by phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). Recombinant GM-CSF stimulated blast colonies in 13 out of 20 cases examined (1 case not done). The magnitude of stimulation by rGM-CSF varied significantly according to the type of AML, but in general was lower than that of PHA-LCM. Blast cells of type M1 did not form any colonies with rGM-CSF, although numerous colonies were produced with PHA-LCM. Type M4 blasts formed fairly large numbers of colonies, though slightly less than those stimulated by PHA-LCM. Blasts of type M2 and M5 formed colonies with the stimulation of rGM-CSF, but the numbers were considerably smaller than type M4 and those stimulated with PHA-LCM. Recombinant G-CSF stimulated blast colonies in only 5 out of 21 cases, 3 of them being type M2. The number of cases responding to rG-CSF was significantly smaller than that responding to rGM-CSF, and even in cases in which colonies were formed, the magnitude of stimulation was minimal. From these results it seems likely that blast cells of different types of AML require a different kind of CSF for their optimal growth; type M4 blasts responded to the stimulation of rGM-CSF well, but blasts of other types of AML responded poorly. Thus, except for type M4, CSF(s) other than rGM-CSF seems to be required for the sufficient growth of L-CFU. Recombinant G-CSF is not likely to play an essential role in the proliferation of leukemic blasts of most types. Previous exposure to rGM-CSF and rG-CSF did not alter the self-renewal capacity, cellular phenotype, and morphology of colony cells, indicating that the direction and degree of differentiation of L-CFU stimulated by rGM-CSF or rG-CSF were not different from those stimulated with PHA-LCM.

Colony-Stimulating Factors↗
PubMed1988

Studies on recombination within the mouse H-2 complex: IV. Characterization of new recombinant haplotypes H-2t7, H-2t8, H-2as2, and H-2as3.

Four new intra-H-2 recombinants were characterized serologically and functionally. In two of these recombinants, B10.ASR1 and B10.ASR7, crossing over occurred between the A alpha and E alpha subregions, very probably in E beta since most intra-I region recombinants thus far investigated at the DNA level appear to involve recombination within the E beta gene. In the other two recombinants, B10.ASR2 and B10.ARS8, crossing over occurred between the S and D subregions. B10.ASR2 and B10.ASR8, crossing over occurred between the S and D subregions. B10.ASR7, which is serologically indistinguishable from B10.BASR1 and B10.S(8R), slightly stimulates and strongly responds to both of these strains in MLR. The probable location of the B10.S(8R) stimulatory product is E beta. The H-2 composition of B10.ASR1 is closest to that of B10.S(9R) and B10.HTT; therefore, precise definition of the cross-over point at the DNA level will be of particular interest.

Animals↗
PubMed1977

[Genetic control of recombination processes in Aspergillus nidulans. I. Effect of uvs-mutations on the frequency of spontaneous and ultraviolet ray-induced intergenic recombination].

Effect of 3 uvs mutations (uvs 12, 19 and 25) on recombination processes in Aspergillus nidulans is studied. All the mutations are found either to affect the fertility of carp bodies and germination ability of askospores, or result in complete inability of heterokaryons to form cleistocarpia. Two mutations change the frequency of spontaneous meitotic crossing-over at pro-paba region of the chromosome I and do not affect the rate of mitotic recombination at w-centromeric region of the chromosome II: uvs 12 mutation increases, and uvs 19 mutation decreases the frequency of meiotic recombination. One mutation (uvs 25) decreases the rate of spontaneous mitotic crossing-over. All uvs mutations decrease the frequency of VU light induced mitotic recombination at w-centromeric region of the chromosome II. The data obtained, together with earlier reported characteristics of uvs mutants, suggest that recombination mechanisms in yeast participate in reparation processes more actively than in prokariotes. Different effects of the same uvs mutations on spontaneous frequency of meiotic and mitotic crossing-over draw to the conclusion that genetic control and molecular mechanisms of these processes in A. nidulans are not identical.

Aspergillus nidulans↗
PubMed1995

Admixture of a recombinant vaccinia virus containing the gene for the costimulatory molecule B7 and a recombinant vaccinia virus containing a tumor-associated antigen gene results in enhanced specific T-cell responses and antitumor immunity.

At least two signals are required for the activation of naive T cells by antigen-bearing target cells: an antigen-specific signal, delivered through the T-cell receptor, and a costimulatory signal delivered through the T-cell surface molecule CD28 by its natural ligand B7-1. The immunological benefit of coexpression of B7 with target antigen has been demonstrated with the use of several retroviral systems to transfect antigen-bearing cells. Although engineering recombinant constructs with genes for two or more antigens can mediate the dual expression of those antigens, disadvantages of this approach include the time for construction of each desirable combination and the inability to control differential expression levels of each gene product. An alternative approach would utilize separate constructs that could be admixed appropriately before administration. In this report we describe the functional consequences of the admixture of recombinant vaccinia murine B7-1 (rV-B7) to recombinant vaccinia expressing the human carcinoembryonic antigen gene (rV-CEA). Coinfection of cells resulted in high levels of cell surface expression of both the CEA and B7 molecules. Immunization of mice with various ratios (1:3, 1:1, 3:1) of rV-CEA and rV-B7 demonstrated that an admixture of rV-CEA and rV-B7 at a 3:1 ratio resulted in the generation of optimal CEA-specific T-cell responses. Next, we examined the efficacy of this admixture on antitumor activity. Typically, injection of murine carcinoma cells expressing CEA leads to the death of the host. One immunization of C57BL/6 mice with rV-CEA:rV-B7 (3:1) resulted in no tumor establishment. In contrast, administration of rV-CEA or rV-B7 alone had little or no antitumor effects. These studies demonstrate the advantages of the use of recombinant vaccinia viruses to deliver B7 molecules in combination with a tumor-associated antigen. The availability of the rV-B7 single construct and the ability to alter the B7 ratio could also have potential utility when coinfecting rV-B7 with recombinant vaccinia viruses containing genes for infectious agents or other tumor-associated antigen genes.

Animals↗
PubMed1993

[The characteristics of homologous DNA recombination in somatic cells. II. The characteristics of the extrachromosomal recombination of plasmid DNA in cultured cells differing in their tumor potential].

The frequencies of homologous extrachromosomal recombination, following plasmid transfection into cell lines differing in their tumour potential were measured. A 10-fold increase in extrachromosomal plasmid recombination has been reported in tumour cells (Ra-2, Jf-1) relative to immortal (Rat-2) and normal (REF). The differences between the normal and tumour cells are preserved in the in vitro recombination experiments (the recombination in the cell nuclei extracts). However, the recombination activity of nuclear extracts from the immortality transformed cells (Rat-2) is similar to that of nuclear extracts from the tumour cells.

Animals↗
PubMed1995

Identification of a recombinational breakpoint at the BAT5 locus in three intra-H-2 recombinant inbred mouse strains.

We have analyzed the S/D region in 14 inbred mouse strains by restriction fragment length polymorphism (RFLP) using human BAT2 and BAT5 genes as probes. In all recombinant strains, the recombinational breakpoint mapped centromeric to Bat-2 (D17H6S51E). In recombinant strains B6.R4, B6.AK1 and B10.BYR1, recombination occurred within or close to the Bat-5 (D17H6S82E) locus. The immunogenicity of B10.BYR1 and B6.R4 bone marrow cell (BMC) grafts differs from that of both sets of parents, as if genes at or just centromeric to Bat-5 are involved. The phenotype of B6.AK1 BMCs (Kb Dk) is similar to that of the H2k parent suggesting no changes occurred. However, RNA blot analysis has shown that the Bat-5 gene is expressed well in bone marrow cells of B10.BR mice but not in B6.AK1 marrow cells. Analysis of a limited number of tumor cells of hemopoietic origin identified a single transcript for Bat-5. Our present data identify a recombinational hot spot at the Bat-5 locus. The expression of Bat-5 or a nearby gene may influence the immunogenicity of BMCs.

Animals↗
PubMed1996

Automated specific IgE assay with recombinant allergens: evaluation of the recombinant Aspergillus fumigatus allergen I in the Pharmacia Cap System.

BACKGROUND: We report the results of a study comparing the recombinant Aspergillus fumigatus allergen 1 (rAsp f I) to commercial A. fumigatus extracts in serological assays. Pharmacia CAP System and skin tests. OBJECTIVE: The study was designed to test the feasibility of using recombinant allergens in an automated serology system for determination of allergen-specific IgE. METHODS: Patients with allergic bronchopulmonary aspergillosis (ABPA), asthmatics with A. fumigatus allergy and control subjects, who included allergic asthmatics without allergy to A. fumigatus and healthy subjects, were investigated. All subjects were characterized with respect to their total IgE level, radio allergosorbent test to A. fumigatus and skin test reactivity to both commercial A. fumigatus extracts and recombinant rAsp f I protein. RESULTS: All patients with ABPA (n = 30) showed positive skin test reactions with commercial A. fumigatus extracts, and 24 were sensitized to rAsp f I by the same criterion. The 10 patients with asthma and A. fumigatus allergy showed positive skin reactions to at least one commercial extract, and five reacted to rAsp f I. All control subjects (n = 19) scored negatively in skin tests to A. fumigatus extracts and rAsp f I and showed no detectable rAsp f I-specific IgE. ImmunoCAP carrying immobilized rAsp f I were evaluated using sera from all individuals described and the results compared with those obtained with the rAsp f I-specific ELISA for IgE. The data obtained with the two rAsp f I-specific detection systems correlated closely (r = 0.997) and were in perfect agreement with the skin test results. CONCLUSION: The data show that rAsp f I can be used as immobilized allergen in the Pharmacia CAP System indicating the feasibility of using recombinant allergens for an automated serological diagnosis of allergic diseases. However, every recombinant allergen needs to be evaluated individually for its performance if applied to a new diagnostic technology.

Adult↗