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At least 181 records · Page 10Linked to original sources
PubMed1986

Bacteriophage P1 Cre-loxP site-specific recombination. Site-specific DNA topoisomerase activity of the Cre recombination protein.

Site-specific recombination in bacteriophage P1 occurs between two loxP sites in the presence of the Cre recombination protein. The structure of the 34-base pair loxP site consists of two 13-base pair inverted repeats separated by an 8-base pair spacer region. A mutation in the loxP site has been constructed which deletes one of the internal bases of the spacer region at the axis of dyad symmetry. This mutant loxP site shows a 10-fold reduction in recombination activity with a wild-type site both in vivo and in vitro. This low level of intramolecular recombination between a wild-type loxP site and the mutant loxP501 site is observed in vitro only when the DNA substrate is supercoiled. The majority of the supercoiled substrate is relaxed by the Cre protein, and on longer incubations, single-stranded nicks accumulate in the DNA. We have determined that these nicks occur in both the wild-type and the mutant sites. The positions of these nicks correspond to the positions of cleavage found during recombination of two wild-type sites, suggesting that the Cre protein is attempting to carry out recombination with the mutant site but most of the time this reaction is abortive. We have determined that the Cre protein relaxes a supercoiled topoisomer of a DNA substrate containing one wild-type site and one mutant site to yield a distribution of topoisomers whose linking numbers differ by steps of one, indicating that Cre can act as a type I topoisomerase.

Base Sequence↗
PubMed1985

[Plasmid recombination stimulated by restriction endonuclease EcoRI in vivo: formation of recombinant plasmids in recA+-cells of E. coli].

The possible participation of restriction endonuclease EcoRI in recombination of compatible nonhomologous plasmids in E. coli cells has been studied. To study the process, plasmids RP4 and R245 have been transferred by conjugation into the recipient cells of E. coli harbouring one of isogenic plasmids, pSA14 and pSA25, different for the genes coding restriction endonuclease EcoRI. The genetic analysis of transconjugant phenotypes, coded by the plasmids, has permitted to register the recombinant plasmids after compatibility of parent plasmids in E. coli cells. Recombination of plasmid RP4 with the plasmid pSA14, carrying EcoRI genes, has been registered in E. coli cells, producing the restriction endonuclease, while plasmid recombination has not been found in the cells harbouring plasmid pSA25, isogenic for all genes, except for EcoRI genes, with plasmid pSA14. Restriction endonuclease EcoRI is concluded to stimulate site specific recombination of nonhomologous compatible plasmids in vivo. EcoRI-mediated recombination of plasmid R245 with plasmid pSA14 is discussed.

DNA Restriction Enzymes↗
PubMed1995

[Homology of the recombinant DNA of plasmid pDJH2 with the recombinant DNA probe of L. alstoni and analysis of its expression in Escherichia coli].

Dig-labeled recombinant DNA probe of L. alstoni which contains the entire structural OmpL1 gene was hybridized with the recombinant DNA of the plasmid named pDJH2 of the gene library of L. interrogans serovar lai strain 017. The result showed a high degree of homology among them; expression of recombinant DNA of pDJH2 was achieved by beta-D-galactosidase (IPTG) induction in E. coli. The molecular weight of this product is 68kd. Then they were treated with proteinase K and subjected to SDS-PAGE. The results showed it is a protein in nature. Using the specific monoclonal antibody E4B7D5 on immunoblotting and specific polyclonal antibody on dot-ELISA assay, we investigated the immune reaction and noticed that protein 68kd might be an antigen in character. E. coli which contains the recombinant plasmid pDJH2 were injected into BALB/c mice. Then the mice were challenged by leptospires of the strong virulence strain 017, but all the infected mice survived. In this paper, we first report the expression of recombinant DNA of L. interrogans serovar lai strain 017 in E. coli when injected with IPTG, and immunoprotection of BALB/c mice which were injected with the expression against the infection of L. interrogans serovar lai strain 017. pDJH2 may be the first recombinant for which the gene has been cloned and its expression product 68kd may be the immunoprotective antigens.

Animals↗
PubMed2026

Identification of a novel HIV-1 circulating recombinant form (CRF209_cpx) and its descendant unique recombinant form (URF) CRF209_cpx/B among MSM in Guangdong, southern China.

BACKGROUND: The epidemic of human immunodeficiency virus type 1 (HIV-1) continues to pose a significant global health challenge, with increasing genetic diversity. The co-circulation of multiple subtypes among the local population facilitates the emergence of unique or circulating recombinant forms (URFs or CRFs). In China, the predominant strains include CRF07_BC, CRF01_AE, CRF55_01B, and subtype B. This study characterizes a novel CRF209_cpx and its descendant recombinant CRF209_cpx/B among men who have sex with men (MSM) in Guangdong, southern China. METHODS: Individuals infected with URFs with similar genetic characteristics were recruited during routine surveillance of pretreatment drug resistance. Near full-length genomes (NFLGs) were amplified with two overlapping fragments using a serial dilution nested PCR approach after reverse transcription. We used SimPlot and IQ-TREE softwares to conduct recombination analyses and phylogenetic inferences. Time-scaled maximum clade credibility (MCC) phylogenetic trees were reconstructed using BEAST software to estimate evolutionary origins. Genotypic drug resistance mutations were interpreted via the Stanford HIV Database, and coreceptor usage was predicted using geno2pheno coreceptor 2.5 and the HIVcoPRED tool. RESULTS: Four NFLG sequences were obtained and identified as a novel CRF209_cpx, generated by recombination among CRF01_AE, CRF07_BC and subtype B. Phylogenetic analyses revealed that all the parental segments clustered with lineages prevalent among MSM in China. Bayesian evolutionary analysis estimated that the most recent common ancestor (tMRCA) of CRF209_cpx to have evolved between 2011 and 2013. The fifth strain was identified as a URF recombined from nascent CRF209_cpx and B. No transmitted drug resistance mutation was detected in these five sequences. The four CRF209_cpx sequences primarily utilized the CXCR4 coreceptor, while the URF exhibited R5/X4 dual tropism. CONCLUSIONS: The emergence of the complex CRF209_cpx and novel URF of CRF209_cpx/B highlights the active HIV-1 epidemic within the MSM population in Guangdong, underscoring the necessity for enhanced molecular surveillance and precise public health intervention in this key population.

HIV-1↗
PubMed1999

Secondary vaccination with vaccinia virus recombinants: role of residual virulence of recombinants and immunogenicity of extrinsic antigens.

ICR mice were immunized intraperitoneally with two doses (10(6) PFU per dose) of vaccinia virus (VV) recombinants of variable virulence expressing either the strongly immunogenic glycoprotein E (gE) of varicella zoster virus (VZV) or weakly immunogenic hepatitis B virus (HBV) preS2-S (S) antigen. Recombinants expressing gE were able to elicit primary and secondary anti-gE antibody irrespective of their residual virulence; after the second dose they did so even in the presence of VV antibody resulting from primary vaccination dose or under other conditions limiting VV replication. As for the S-recombinants, pronounced anti-S antibody development was only observed in mice which had received the more virulent recombinant virus as the first dose. A repeated dose of S-recombinants was unable to elicit a secondary anti-S antibody response. The present findings do not support the assumption that the poor immunogenicity of some extrinsic antigens could be overcome by administering repeated doses of the particular VV recombinant.

Aging↗
PubMed2001

An efficient homologous recombination vector pTV(I) contains a hot spot for increased recombinant protein expression in Chinese hamster ovary cells.

We employed reverse genetics to clone a 5.0 kb genomic DNA hot spot HIRPE (hot spot for increased recombinant protein expression) flanking the plasmid integration site from a recombinant Chinese hamster ovary (CHO) cell line. DNA sequence analysis of the 5.0 kb fragment revealed that HIRPE is enriched for repetitive elements, Alu-like sequences and matrix-associated regions that are known to be linked with transcriptionally active regions in a number of mammalian systems. The construction of a homologous recombination vector, pTV1, containing the 5.0 kb HIRPE genomic DNA, a recombinant gene human CTLA4-Ig, and the dhfr gene as a positive selection marker is described. It was observed that the pTV1 vector targeted the CTLA4Ig gene to a preferred locus in the CHO genome contributing to high recombinant gene expression in transfected CHO cells. Preliminary studies suggest that similar to the observation with the parental cell line, pTV1-generated transfectomas that were analyzed appear to harbor an inverted duplication of the genomic DNA at the plasmid integration site.

Abatacept↗
PubMed1982

P1 site-specific recombination: nucleotide sequence of the recombining sites.

Site-specific recombination between molecules of bacteriophage P1 DNA occurs at sites called loxP and requires the action of a protein that is the product of the P1 cre gene. Although recombination between two loxP sites is very efficient, recombination between loxP and a unique site in the bacterial chromosome (loxB) is inefficient and generates two hybrid lox sites called loxR and loxL. We present here the nucleotide sequences of all four lox sites. Analysis of these sequences indicates that (i) a region of extensive homology is not present at the loxP X loxB crossover point, in contrast to the 15-base pair common-core sequence in the bacteriophage lambda att sites, and (ii) the sites contain a region of dyad symmetry with 8- to 13-base pair inverted repeats separated by an 8- to 9-base pair sequence. The loxP X loxB crossover point falls in the sequence that separates the inverted repeats, and deletions that remove either the left or the right inverted repeat of loxP inactivate the site. These two observations are consistent with the conclusion that the region of dyad symmetry is important in los recombination. We have shown further that the loxP X loxP crossover point occurs in a 63-base pair sequence containing the loxP X loxB crossover point, suggesting that, despite the great difference in efficiencies of the two reactions, the crossover points may occur at the same place in both. Explanations for the different recombination properties of the various lox sites are discussed.

Base Sequence↗
PubMed1994

An implanted recombination hot spot stimulates recombination and enhances sister chromatid cohesion of heterologous YACs during yeast meiosis.

Heterologous yeast artificial chromosomes (YACs) do not recombine with each other and missegregate in 25% of meiosis I events. Recombination hot spots in the yeast Saccharomyces cerevisiae have previously been shown to be associated with sites of meiosis-induced double-strand breaks (DSBs). A 6-kb fragment containing a recombination hot spot/DSB site was implanted onto two heterologous human DNA YACs and was shown to cause the YACs to undergo meiotic recombination in 5-8% of tetrads. Reciprocal exchanges initiated and resolved within the 6-kb insert. Presence of the insert had no detectable effect on meiosis I nondisjunction. Surprisingly, the recombination hot spots acted in cis to significantly reduce precocious sister-chromatid segregation. This novel observation suggests that DSBs are instrumental in maintaining cohesion between sister chromatids in meiosis I. We propose that this previously unknown function of DSBs is mediated by the stimulation of sister-chromatid exchange and/or its intermediates.

Base Sequence↗
PubMed1992

Highly efficient generation of recombinant baculoviruses by enzymatically medicated site-specific in vitro recombination.

We have used the Cre-lox system of bacteriophage P1 to develop a highly efficient in vitrosystem for construction of recombinant baculoviruses. A positive visual selection has been included to make identification of recombinant viral progeny rapid and straightforward. We report recombination frequencies as high as 5 x 10(7) recombinants/micrograms starting plasmid DNA and under certain conditions, up to 50% of the viral progeny are recombinants. Genes inserted into the baculovirus genome can be readily recovered in a simple one step process and re-inserted after manipulation if required. We have confirmed the structure of recovered plasmids by diagnostic restriction endonuclease digestion and the structure of recombinant viral genomes by Southern analysis. Possible uses and the significance of the system are discussed and experiments currently being done to improve it are described.

Bacteriophages↗
PubMed1998

Genetic recombination in Bacillus subtilis 168: effects of recU and recS mutations on DNA repair and homologous recombination.

Bacillus subtilis recombination-deficient mutants were constructed by inserting a selectable marker (cat gene) into the yppB and ypbC coding regions. The yppB:cat and ypbC:cat null alleles rendered cells sensitive to DNA-damaging agents, impaired plasmid transformation (25- and 100-fold), and moderately affected chromosomal transformation when present in an otherwise Rec+ B. subtilis strain. The yppB gene complemented the defect of the recG40 strain. yppB and ypbC and their respective null alleles were termed "recU" and "recU1" (recU:cat) and "recS" and "recS1" (recS:cat), respectively. The recU and recS mutations were introduced into rec-deficient strains representative of the alpha (recF), beta (addA5 addB72), gamma (recH342), and epsilon (recG40) epistatic groups. The recU mutation did not modify the sensitivity of recH cells to DNA-damaging agents, but it did affect inter- and intramolecular recombination in recH cells. The recS mutation did not modify the sensitivity of addAB cells to DNA-damaging agents, and it marginally affected recF, recH, and recU cells. The recS mutation markedly reduced (about 250-fold) intermolecular recombination in recH cells, and there were reductions of 10- to 20-fold in recF, addAB, and recU cells. Intramolecular recombination was blocked in recS recF, recS addAB, and recS recU cells. RecU and RecS have no functional counterparts in Escherichia coli. Altogether, these data indicate that the recU and recS proteins are required for DNA repair and intramolecular recombination and that the recF (alpha epistatic group), addAB (beta), recH (gamma), recU (epsilon), and recS genes provide overlapping activities that compensate for the effects of single mutation. We tentatively placed recS within a new group, termed "zeta".

Bacillus subtilis↗
PubMed1999

The use of recombinant fusion proteases in the affinity purification of recombinant proteins.

In the affinity purification of recombinant fusion proteins, the rate-limiting step is usually the efficient proteolytic cleavage and removal of the affinity tail and the protease from the purified recombinant protein. We have developed a rapid, convenient, and efficient method of affinity purification that can overcome this limitation. In one example of the method, the protease 3C from a picornavirus (3Cpro), which cleaves specific sequences containing a minimum of 6-7 amino acids, has been expressed as a fusion with glutathione S-transferase. The resultant recombinant "fusion protease" cleaves fusion proteins bearing (from the amino-terminus) the same affinity tail as the fusion protease, a 3Cpro cleavage recognition site, and the recombinant protein of interest. The recombinant protein is purified in a single chromatographic step, which removes both the affinity tail and the fusion protease. The advantages over existing methods include much improved specificity of proteolytic cleavage, complete removal of the protease and the affinity tail in one step, and the option of adding any desired amount of fusion protease to ensure efficient cleavage. The potential flexibility of the method is shown by the use of various affinity tails and alternative fusion proteases.

3C Viral Proteases↗
PubMed1996

Immunoglobulin D(H) recombination signal sequence targeting: effect of D(H) coding and flanking regions and recombination partner.

V(D)J recombination is targeted by recombination signal sequences (RSS) located immediately adjacent to immune receptor gene segments. While the RSS flanking D(H) segments appear to be equivalent, they are not randomly utilized. During D(H) to J(H) rearrangement, the 3' D(H) RSS is virtually exclusively utilized, suggesting that the 3' D(H) RSS could simply be a better target for the recombinase. However, when we examined V(H) to D(H) (without J(H)) rearrangements, we found that the preference for D(H) RSS use changes, so that the 5' D(H) RSS are preferred. This suggests that the 3' D(H) RSS are not simply superior targets for the V(D)J recombinase, but instead that certain 12/23-bp spacer RSS combinations work better together to target recombination than do others. We have analyzed a series of artificial recombination substrates to delineate cis sequences that affect D(H) RSS selection. Our data suggest that coding sequences adjacent to the D(H) RSS, flanking sequences outside the D(H) gene segment itself, and recombination partner all affect D(H) RSS targeting.

Animals↗
PubMed2004

A conserved sequence in the mouse variable T cell receptor alpha recombination signal sequence 23-bp spacer can affect recombination.

Although the V-gene segments coding for the TCR alpha and delta chains are mixed together in the alpha delta locus and are recombined by the same processes, some gene segments (TRAV) are rearranged only with TCR Jalpha gene segments, some (TRDV) only with TCR Ddelta gene segments and some (TRADV) with both. To date, no molecular signal is known that can characterize these three different types of gene segments. Studying the recombination signal sequences (RSS) of all mouse TCR V-gene segments we observed that 80% of the TRAV contain a palindrome sequence (CTGCAG) or its related variant CTGTAG in their 23-bp spacer. Using gel-shift assays we show that these sequences are specifically recognized by some nuclear proteins that are expressed by fresh thymocytes, fresh lymphocytes and tumor cells. Recombination assays on plasmid substrates in a pre-B cell line showed that RSS containing the CTGCAG sequence can impair recombination. From the protein fractions containing the CTGCAG-binding activity, three proteins were identified: G3BP1 (a nucleic-acid-binding protein with a proposed helicase activity) and two proteins from the high-mobility group (HMG) family--HMGB2 and HMGB3. We hypothesize that these proteins can affect recombination at the TCR alpha delta locus.

Animals↗
PubMed1993

Recombinant technology as an alternative to chemical peptide synthesis: expression and characterization of HIV-1 Rev recombinant peptides.

Recombinant DNA technology has been widely used for the production of proteins in recent years. In this paper, we describe the expression and the purification of two specific peptides corresponding to parts of the human immunodeficiency virus Rev protein. The strategy of this method relies on the chemical synthesis of a pair of two complementary oligodeoxynucleotides corresponding to the coding region of the peptide of interest and the subsequent cloning into a prokaryotic expression vector. Transformation of Escherichia coli with these synthetic gene constructs yielded high production levels of recombinant protein in the bacteria. The recombinant protein was composed of two moieties, one corresponding to an "affinity handle" and the second corresponding to the peptide. Chemical cleavage of the fused protein followed by a combination of affinity chromatography and rp-HPLC led to rapid and convenient peptide purification. Peptide fused to the affinity handle as well as cleaved peptide were fully characterized by N-terminal microsequencing and mass spectrometry. The data presented demonstrate that although the major recombinant products had the expected amino acid composition, we detected unexpected processing such as alternative cleavage within the signal peptide, modified cysteines, and deamidations. These results emphasize the importance of the complete characterization of recombinant products by efficient analytical tools such as N-terminal microsequencing and mass spectrometry.

Amino Acid Sequence↗
PubMed1995

Allele-dependent recombination frequency: homology requirement in meiotic recombination at the hot spot in the mouse major histocompatibility complex.

Meiotic recombination break joints in the mouse major histocompatibility complex (MHC) are clustered within short segments known as hot spots. We systematically investigated the requirement for sequence homology between two chromosomes for recombination activity at the hot spot next to the Lmp2 gene. The results indicated that a high rate of recombination required a high degree of similarity of overall genome structure at the hot spot. In particular, the same copy number of repetitive sequences within the hot spot was essential for a high frequency of recombination, suggesting that recombination in mouse meiosis is more sensitive to heterozygous deletion or insertion of DNA than to mismatches of single-base substitutions.

Alleles↗
PubMed1998

A partial hprt gene duplication generated by non-homologous recombination in V79 Chinese hamster cells is eliminated by homologous recombination.

Here, the sequence in the hprt gene of the duplication mutant SPD8 originating from V79 Chinese hamster cells was determined. The duplication arose after non-homologous recombination between exon 6 and intron 7, resulting in an extra copy of the 3' portion of exon 6, of exon 7 and of flanking intron regions. Only a duplication of exon 7 is present in the mRNA, since the duplicated exon 6 lacks its 5' splice site and is removed during RNA processing. The findings in this study suggest that the non-homologous recombination mechanism which occurred here may have been initiated by endonucleases, rather than by a spontaneous double strand break. Subsequently, 14 spontaneous SPD8 revertants with a functional hprt gene were isolated and characterized using PCR and sequencing. The data revealed that although the SPD8 cell line arose by non-homologous recombination, it reverts spontaneously by homologous recombination. Interestingly, the downstream copy of exon 7 was restored by this process. This was indicated by the presence of a specific mutation, a T-to-G transversion, close to the breakpoint, a characteristic unique to the SPD8 clone. Our results suggest that the spontaneous reversion of this cell line by homologous recombination may involve an exchange, rather than a conversion mechanism.

Animals↗
PubMed1995

First experimental evidence of recombination in infectious bronchitis virus. Recombination in IBV.

A high frequency of recombination has been shown to occur during replication of the coronavirus mouse hepatitis virus (MHV) in vitro as well as in vivo. Although sequencing of field strains of coronavirus infectious bronchitis virus (IBV) has indicated that IBV strains also undergo recombination, there has been no experimental evidence to support this deduction. To investigate whether recombination occurs in IBV, embryonated eggs were coinfected with IBV-Beaudette and IBV-M41. Potential recombinants were detected by strain-specific polymerase chain reaction (PCR) amplifications, using oligonucleotides corresponding to regions in the 3' end of the genome. Sequencing of the PCR products confirmed that a number of recombinations had occurred between the two strains.

Animals↗
PubMed1989

A mutation of Streptomyces lividans which prevents intraplasmid recombination has no effect on chromosomal recombination.

A mutation (rec-46) of Streptomyces lividans, previously shown to prevent (or greatly diminish) homologous and illegitimate intraplasmid recombination, was shown to have no effect on generalised chromosomal recombination occurring in matings or in protoplast fusions, nor to affect homologous recombination between a recombinant plasmid and the host chromosome. By comparison with Escherichia coli mutants defective in various aspects of recombination, the rec-46 mutation is similar to those in recF, recJ, recO and topA.

Chromosome Mapping↗