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PubMed1985

Intramolecular recombination of linear DNA catalyzed by the Escherichia coli RecE recombination system.

Transformation of different Escherichia coli strains by linear dimers of pBR322 containing different tet alleles was investigated. Linear dimers transformed wild-type strains 0.1 to 1% as efficiently as circular dimers. In contrast, linear dimers transformed recBrecCsbcA strains, where the RecE recombination system is functional, as efficiently as circular dimers. The transformants contained plasmids that had a single recombinant monomer genotype, indicating that transformation was mediated by a recombination-dependent cyclization reaction. Altering the position of the double-strand break changed the frequency of recovering different recombination products, but had no effect on the frequency of transformation. Both the frequency of transformation and the production of Tcr recombinants were decreased by recE mutations, while recA and recF mutations were slightly stimulatory (twofold). Several recombination models consistent with these results are presented.

DNA Restriction Enzymes↗
PubMed1983

Recombination in adenovirus: DNA sequence analysis of crossover sites in intertypic recombinants.

The nucleotide sequence of the adenovirus type 5 genome has been determined for a 620-bp region that spans the C terminus of the pVI gene and the N terminus of the hexon gene, and compared to the adenovirus type 2 DNA sequence: 25 base changes have been identified, most of which do not lead to alterations in the amino acid sequence and regulatory signals in the region. Crossover sites in three intertypic recombinants have been previously located in this region of the genome by fine restriction mapping. A sequence determination for the three recombinants, and the four ts mutants used in generating the ts+ recombinants, was carried out. The crossovers were in each case located in a small region of complete sequence homology (from 45 to 156 nucleotides long) flanked on either side by sequences derived from each parent. These structures are compatible with a reciprocal crossing over model of generalised recombination, where a recombinant joint has resolved in a region of high DNA homology. For the recombinants considered here, this region abutts onto a neighbouring region of much lower sequence homology, and it is possible that the position of the crossover is determined at least in part by the termination of branch migration at a heterologous boundary.

Adenoviruses, Human↗
PubMed1990

Recombination sites in cauliflower mosaic virus DNAs: implications for mechanisms of recombination.

Pairs of mutant cauliflower mosaic virus (CaMV) DNAs readily recombine in plants. Five plasmid clones of CaMV DNAs resulting from infection of turnips with pairs of mutant DNAs from DNAs resulting from infection of turnips with pairs of mutant DNAs from different isolates were obtained. Restriction analysis and nucleotide sequencing identified deletions in two cloned recombinants, VR1249 and VR244B. The sequence missing in the former was consistent with its deletion by splicing of an RNA intermediate. These DNAs were not infectious in turnips. VR1243, VR244A, and VR246 induced in turnips disease symptoms that were mixtures of those produced by the parental isolates. Junctions between sequences of the parental isolates were identified by restriction fragment analysis. Three cloned chimeras resulted from multiple recombination events. Nucleotide sequencing identified more precisely the junctions in the five cloned chimeras and in three chimeras previously characterized. Consistent with a model in which reverse transcription plays a major role in generating recombinants, six chimeras had junctions at or near the site for initiation of DNA(-) strand synthesis, three had junctions near the initiation site of 35 S RNA transcription, and one junction was found near the initiation site of 19 S mRNA transcription. Junctions were also found in regions not bearing any obvious relation to DNA (-) strand synthesis by reverse transcription, suggesting that recombination of double-stranded DNAs may also generate CaMV DNA recombinants.

Base Sequence↗
PubMed2002

Comparison of stimulation with clomiphene citrate in combination with recombinant follicle-stimulating hormone and recombinant luteinizing hormone to stimulation with a gonadotropin-releasing hormone agonist protocol: a prospective, randomized study.

OBJECTIVE: To compare IVF-ET outcome with a new stimulation protocol using clomiphene citrate (CC) with recombinant FSH and LH to stimulation with the standard long GnRH-a protocol. DESIGN: Prospective randomized study. SETTING: Outpatient infertility clinic in Vienna, Austria. PATIENT(S): Two hundred ninety-four infertile women undergoing IVF-ET; 154 IVF cycles stimulated with CC + recombinant FSH + recombinant LH (group A) and 140 cycles with long GnRH-a suppression + recombinant FSH (group B). INTERVENTION(S): Controlled ovarian hyperstimulation, egg retrieval, and ET. MAIN OUTCOME MEASURE(S): Cycle parameters (number of oocytes, fertilization, number of embryos) and outcome (pregnancy rate, cancellation rate, ovarian hyperstimulation syndrome [OHSS]). RESULT(S): Pregnancy rate per ET was 42.9% (implantation rate, 21.3%) in group A and 36.6% (17.4%) in group B. Cancellation rates were similar. The OHSS occurred in four cases (3%) in group A and 12 cases (10%) in group B. CONCLUSION(S): Stimulation with CC + recombinant FSH + recombinant LH leads to comparable pregnancy rates vs. the long protocol. With this new stimulation, less gonadotropins are used and there is less need for monitoring (lower cost for patient and clinic). The risk of OHSS is reduced as well. Therefore, this protocol should be regarded as the first-line treatment.

Adult↗
PubMed2002

Recombination analysis of the human minisatellite MsH42 suggests the existence of two distinct pathways for initiation and resolution of recombination at MsH42 in rat testes nuclear extracts.

We have previously described a GC-rich human minisatellite, termed MsH42, which exists in two allelic forms, long and short. Here, we have identified a third allele of medium length and localized the MsH42 locus in the chromosome 15q25.1 inside an intron belonging to a gene of unknown function. The recombinogenic potential of the three alleles was assayed in vitro incubating pBR322-based constructs containing two copies of the minisatellite MsH42 with its flanking sequences, in the presence of rat testes nuclear extracts. This assay system was configured to monitor only reciprocal exchange type events and not gene conversion. All MsH42 allelic sequences enhanced intramolecular homologous recombination promoting high rates (approximately 76%) of equal crossover, the long allele showing the highest recombinogenic activity. Removal of the MsH42 long allele flanking sequences, which are identical in the three alleles, provoked a decrease in the enhancement of recombination and in the frequency of equal crossovers, suggesting that these sequences are important for the recombinogenic activity and for the correct pairing between homologous sequences. The occurrence of some complex recombination events within the minisatellite MsH42 suggests the existence of processes related to polymerase slippage and unwinding with reinvasion during the repair synthesis. Our findings point toward the existence of two distinct biochemical pathways for initiation and resolution of recombination at the minisatellite MsH42. Finally, the in vitro recombination system employed in this study could provide an approach to dissect processes of repetitive DNA instability and recombination.

Alleles↗
PubMed1997

Barriers to recombination between closely related bacteria: MutS and RecBCD inhibit recombination between Salmonella typhimurium and Salmonella typhi.

Previous studies have shown that inactivation of the MutS or MutL mismatch repair enzymes increases the efficiency of homeologous recombination between Escherichia coli and Salmonella typhimurium and between S. typhimurium and Salmonella typhi. However, even in mutants defective for mismatch repair the recombination frequencies are 10(2)- to 10(3)-fold less than observed during homologous recombination between a donor and recipient of the same species. In addition, the length of DNA exchanged during transduction between S. typhimurium and S. typhi is less than in transductions between strains of S. typhimurium. In homeologous transductions, mutations in the recD gene increased the frequency of transduction and the length of DNA exchanged. Furthermore, in mutS recD double mutants the frequency of homeologous recombination was nearly as high as that seen during homologous recombination. The phenotypes of the mutants indicate that the gene products of mutS and recD act independently. Because S. typhimurium and S. typhi are approximately 98-99% identical at the DNA sequence level, the inhibition of recombination is probably not due to a failure of RecA to initiate strand exchange. Instead, these results suggest that mismatches act at a subsequent step, possibly by slowing the rate of branch migration. Slowing the rate of branch migration may stimulate helicase proteins to unwind rather than extend the heteroduplex and leave uncomplexed donor DNA susceptible to further degradation by RecBCD exonuclease.

Adenosine Triphosphatases↗
PubMed1995

Role of partner homology in DNA recombination. Complementary base pairing orients the 5'-hydroxyl for strand joining during Flp site-specific recombination.

Absolute homology between partner substrates within the strand exchange region is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp. Using combinations of specially designed half- and full-site Flp substrates, we demonstrate that the strand joining step of recombination is exquisitely sensitive to spacer homology. At each exchange point, 2-3 spacer nucleotides adjacent to the nick within the cleaved strand of one substrate must base pair with the corresponding segment of the un-nicked strand from the second substrate for efficient strand joining in the recombinant mode. In accordance with the "cis-activation/trans-nucleophilic attack" model for each of the two transesterification steps of Flp recombination (strand cleavage and strand joining), we propose that the limited strand pairing orients the DNA-nucleophile (5'-hydroxyl) for attack on its target diester (3'-phosphotyrosyl-Flp). During one round of recombination, 4-6 terminal base pairs of the spacer (2-3 base pairs at each spacer end) must unpair, following strand cleavage, within a DNA substrate and pair with the partner substrate prior to strand union. In this model, the extent of branch migration of the covalently closed Holliday intermediate is limited to the central core of the spacer. The templated positioning of reactive nucleic acid groups (which is central to the model) may be utilized by other recombination systems and by RNA splicing reactions.

Base Composition↗
PubMed1992

Biased distribution of recombination sites within S regions upon immunoglobulin class switch recombination induced by transforming growth factor beta and lipopolysaccharide.

We have characterized extrachromosomal circular DNAs from adult mouse spleen cells that were induced to switch to immunoglobulin A (IgA) with bacterial lipopolysaccharide (LPS) and transforming growth factor beta (TGF-beta), and identified breakpoints of S mu/S gamma 3, S mu/S gamma 2, S mu/S alpha, S gamma 3/S alpha, and S gamma 2/S alpha recombinants. The S mu recombination donor sites clustered in the 3' half of the S mu region, while the S alpha recombination acceptor sites clustered in the 5' half of the S alpha region. In addition, donor and acceptor sites of S gamma regions also clustered in the 3' and 5' parts, respectively. These site preferences are in sharp contrast to the dispersed distribution of S mu/S gamma 1 breakpoints within both S mu and S gamma 1 regions upon IgG1 switch induced by LPS and interleukin 4. Our results support the hypotheses that TGF-beta increases the frequency of switch recombination events to IgA and that the switch recombination to IgA often proceeds by successive recombination of S mu/S gamma and S gamma/S alpha.

Animals↗
PubMed1989

Genetic and molecular analysis of recombination events in Saccharomyces cerevisiae occurring in the presence of the hyper-recombination mutation hpr1.

The hyper-recombination mutation hpr1 specifically increases mitotic intrachromatid crossovers, with no effect on other mitotic recombination events such as unequal sister chromatid exchange and plasmid-chromosome recombination, and no effect on meiotic recombination and a lesser effect on intrachromosomal gene conversion. The excision repair RAD1 gene is partially required for the expression on the hpr1 phenotype. The simplest hypothesis to account for some of the hpr1 stimulated recombination events is that a heteroduplex DNA intermediate and localized gene conversion are involved. hpr1 stimulated crossover events are independent of intrachromosomal gene conversion events stimulated by the hyper-gene conversion mutation hpr5. This result suggests that different intrachromosomal recombination processes are affected in each mutant strain. We propose that HPR1 may function to inhibit intrachromatid crossovers.

Crossing Over, Genetic↗
PubMed1989

The genetic control of direct-repeat recombination in Saccharomyces: the effect of rad52 and rad1 on mitotic recombination at GAL10, a transcriptionally regulated gene.

We have previously shown direct-repeat recombination events leading to loss of a plasmid integrated at the GAL10 locus in Saccharomyces cerevisiae are stimulated by transcription of the region. We have examined the role of two recombination- and repair-defective mutations, rad1 and rad52, on direct repeat recombination in transcriptionally active and inactive sequences. We show that the RAD52 gene is required for transcription-stimulated recombination events leading to loss of the integrated plasmid. Similarly, Gal+ events between the duplicated repeats that retain the integrated plasmid DNA (Gal+ Ura+ replacement events) are reduced 20-fold in the rad52 mutant in sequences that are constitutively expressed. In contrast, in sequences that are not expressed, the rad52 mutation reduces plasmid loss events by only twofold and Gal+ Ura+ replacements by fourfold. We also observe an increase in disome-associated plasmid loss events in the rad52 mutant, indicative of chromosome gain. This event is not affected by expression of the region. Plasmid loss events in rad1 mutant strains are reduced only twofold in transcriptionally active sequences and are not affected in sequences that are repressed. However, the rad1 and rad52 double mutant shows a decrease in plasmid loss events greater than the sum of the decreases in the rates of this event displayed by either single mutant in both constitutive and repressed DNA, indicating a synergistic interaction between these two genes. The synergism is limited to recombination since the rad1 rad52 double mutant is no more sensitive when compared with either single mutant in its ability to survive radiation damage. Finally, the recombination pathway that remains in the double mutant is positively affected by transcription of the region.

DNA Repair↗
PubMed1976

The evolutionary advantage of recombination. II. Individual selection for recombination.

Based on the FISHER-MULLER theory of the evolution of recombination, an argument can be constructed predicting that a recessive allele favoring recombination will be favored, if there are either favorable or deleterious mutants occurring at other loci. In this case there is no clear distinction between individual and group selection. Computer simulation of populations segregating for recessive or dominant recombination alleles showed selection favoring recombination, except in the case of a dominant recombination allele with deleterious background mutants. The relationship of this work to parallel investigations by WILLIAMS and by STROBECK, MAYNARD SMITH, and CHARLESWORTH is explored. All seem to rely on the same phenomenon. There seems no reason to assume that the evolution of recombination must have occurred by group selection.

Alleles↗
PubMed1999

An extrachromosomal switch recombination substrate reveals kinetics and substrate requirements of switch recombination in primary murine B cells.

Ig class switch recombination occurs in B lymphocytes upon activation, and is targeted to distinct switch (S) regions by cytokine-mediated induction of switch transcripts spanning the entire S region and the adjacent constant region gene segments. Using a novel type of switch recombination substrate, constructed according to the intron-exon structure of the IgH locus, but with heterologous elements, we here have tested the structural requirements for targeting and the kinetics of switch recombination in activated primary murine B cells. When transfected at various times after activation, up to 10% of the transfected B cells perform recombination of the substrate within 12 h. Switch recombination in primary B cells is restricted to the first 72 h after onset of activation, then rapidly decreases to background levels, as obtained in plasmacytoma cells or with substrates carrying no S region sequences. In terms of structural requirements, switch recombination is targeted to any transcription unit that contains an intronic S region and depends on processing of the primary transcript by splicing.

Animals↗
PubMed2005

A universal BMV-based RNA recombination system--how to search for general rules in RNA recombination.

At present, there is no doubt that RNA recombination is one of the major factors responsible for the generation of new RNA viruses and retroviruses. Numerous experimental systems have been created to investigate this complex phenomenon. Consequently, specific RNA structural motifs mediating recombination have been identified in several viruses. Unfortunately, up till now a unified model of genetic RNA recombination has not been formulated, mainly due to difficulties with the direct comparison of data obtained for different RNA-based viruses. To solve this problem, we have attempted to construct a universal system in which the recombination activity of various RNA sequences could be tested. To this end, we have used brome mosaic virus, a model (+)RNA virus of plants, for which the structural requirements of RNA recombination are well defined. The effectiveness of the new homomolecular system has been proven in an experiment involving two RNA sequences derived from the hepatitis C virus genome. In addition, comparison of the data obtained with the homomolecular system with those generated earlier using the heteromolecular one has provided new evidence that the mechanisms of homologous and non-homologous recombination are different and depend on the virus' mode of replication.

Bromovirus↗
PubMed1980

Recombination in adenovirus. I. Analysis of recombinant viruses under non-selective conditions.

A method is described, based on an approach previously employed for the mapping of adenovirus ts mutants (Grodzicker et al. 1975), for the identification of recombinant viruses in the progeny of mixed infections of human HeLa cells with wild-type adenovirus types 2 and 5 under non-selective conditions. Differences in restriction enzyme sites for the endonuclease HpaI could be used to detect new recombinant DNA fragments in a yield of 3 to 6% after single mixed infections and yields up to 25% following three successive mixed infections. The recombinant nature of the new fragments was proved by the clonal isolation of recombinant viruses in similar yields. Analysis of one cloned recombinant pointed to the occurrence of multiple crossover events at different sites along the virus chromosome. The results are discussed in terms of a biochemical approach towards the study of the mechanism of general genetic recombination in human cells as well as with respect to the evolution of adenoviruses.

Adenoviruses, Human↗
PubMed1991

Characterization of recombination intermediates from DNA injected into Xenopus laevis oocytes: evidence for a nonconservative mechanism of homologous recombination.

Homologous recombination between DNA molecules injected into Xenopus laevis oocyte nuclei is extremely efficient if injected molecules have overlapping homologous ends. Earlier work demonstrated that ends of linear molecules are degraded by a 5'----3' exonuclease activity, yielding 3' tails that participate in recombination. Here, we have characterized intermediates further advanced along the recombination pathway. The intermediates were identified by their unique electrophoretic and kinetic properties. Two-dimensional gel electrophoresis and hybridization with oligonucleotide probes showed that the intermediates had heteroduplex junctions within their homologous overlaps in which strands ending 3' were full length and those ending 5' were shortened. Additional characterization suggested that these intermediates had formed by the annealing of complementary 3' tails. Annealed junctions made in vitro were rapidly processed to products, indicating that they are on the normal recombination pathway. These results support a nonconservative, single-strand annealing mode of recombination. This recombination mechanism appears to be shared by many organisms, including bacteria, fungi, plants, and mammals.

Animals↗
PubMed1994

Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication.

To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments. The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH10B after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.

Animals↗
PubMed2006

An improved recombineering approach by adding RecA to lambda Red recombination.

Recombineering is the use of homologous recombination in Escherichia coli for DNA engineering. Of several approaches, use of the lambda phage Red operon is emerging as the most reliable and flexible. The Red operon includes three components: Redalpha, a 5' to 3' exonuclease, Redbeta, an annealing protein, and Redgamma, an inhibitor of the major E. coli exonuclease and recombination complex, RecBCD. Most E. coli cloning hosts are recA deficient to eliminate recombination and therefore enhance the stability of cloned DNAs. However, loss of RecA also impairs general cellular integrity. Here we report that transient RecA co-expression enhances the total number of successful recombinations in bacterial artificial chromosomes (BACs), mostly because the E. coli host is more able to survive the stresses of DNA transformation procedures. We combined this practical improvement with the advantages of a temperature-sensitive version of the low copy pSC101 plasmid to develop a protocol that is convenient and more efficient than any recombineering procedure, for use of either double- or single-stranded DNA, published to date.

Animals↗
PubMed1997

[Construction of recombinant vaccinia virus expressing HSV-2 gD gene as live recombinant vaccine strain].

We had reported that the recombinant vaccinia virus expressing glycoprotein D of herpes simplex virus type 2 (HSV-2 gD) protected mice against lethal HSV-2 challenge. Following the succeed in animal model, we continue the research to construct the recombinant vaccinia virus expressing HSV-2 gD gene as live recombinant vaccine strain in strict accordance with the guideline for human vaccine research. A PCR-modified HSV-2 gD gene was inserted into the plasmid pJSB1175, under the control of P7.5K early/late promoter of vaccinia virus. The recombinant plasmid was used, with lipofectin reagent, to transfect 2BS cells which had been infected by wild type of TK+ vaccinia virus (Tian Tan 761 strain). The recombinant vaccinia virus harboring HSV-2 gD gene was selected out by using in situ hybridization employing 32P-1a-belled HSV-2 gD fragment as probe, together with three cycles of plaque purification. Dot and Southern blot confirmed HSV-2 gD gene had been integrated into the TK region of vaccinia virus genome, as expected. Indirect immunofluorescent assay using anti-HSV-2 gD monoclonal antibody showed HSV-2 gD was expressed effectively in the recombinant virus infected cells.

Animals↗