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PubMed1981

Transposon Tn3 encodes a site-specific recombination system: identification of essential sequences, genes, and actual site of recombination.

The bacterial transposon Tn3 encodes a site-specific recombination system. The recombination requires the product of tnpR, a gene previously identified as a repressor of the transposase. This recombination is site specific and takes place somewhere within the sequence C-G-A-A-A-T-A-T-T-A-T-A-A-A-T-T-A-T-C but requires at least one additional sequence outside this. The phenotype of mutations in this recombination system suggests that transposition proceeds by a mechanism in which cointegrates are intermediates.

Base Sequence↗

PubMed1987

Growth of human hemopoietic colonies in response to recombinant gibbon interleukin 3: comparison with human recombinant granulocyte and granulocyte-macrophage colony-stimulating factor.

Supernatants of COS-1 cells transfected with gibbon cDNA encoding interleukin 3 (IL-3) with homology to sequences for human IL-3 were tested for ability to promote growth of various human hemopoietic progenitors. The effect of these supernatants as a source of recombinant IL-3 was compared to that of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) as well as to that of medium conditioned by phytohemagglutinin-stimulated leukocytes. The frequency of multilineage colonies, erythroid bursts, and megakaryocyte colonies in cultures containing the COS-1 cell supernatant was equivalent to the frequency observed in the controls and significantly higher than found in cultures plated with recombinant GM-CSF. G-CSF did not support the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. In contrast, growth of granulocyte-macrophage colonies was best supported with GM-CSF, while recombinant IL-3 yielded colonies at lower or at best equivalent frequency. The simultaneous addition of higher concentrations of GM-CSF to cultures containing IL-3 in optimal amounts did not enhance the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. However, the frequency of such colonies and bursts increased with GM-CSF when cultures were plated with suboptimal concentrations of IL-3. Growth of colonies within the granulocyte-macrophage lineage is optimally supported by GM-CSF and does not increase with further addition of IL-3.

Animals↗

PubMed2004

Mapping of a functional recombination motif that defines isotype specificity for mu-->gamma3 switch recombination implicates NF-kappaB p50 as the isotype-specific switching factor.

Ig class switch recombination (CSR) requires expression of activation-induced deaminase (AID) and production of germline transcripts to target S regions for recombination. However, the mechanism of CSR remains unclear. Here we show that an extrachromosomal S plasmid assay is AID dependent and that a single consensus repeat is both necessary and sufficient for isotype-specific CSR. Transfected switch substrates specific for mu-->gamma3 and mu-->gamma1 are stimulated to switch with lipopolysaccharide (LPS) alone or LPS and interleukin-4, respectively. An Sgamma3/Sgamma1 substrate containing only three Sgamma3-associated nucleotides reconstituted LPS responsiveness and permitted mapping of a functional recombination motif specific for mu-->gamma3 CSR. This functional recombination motif colocalized with a binding site for NF-kappaB p50, and p50 binding to this site was previously established. We show a p50 requirement for plasmid-based mu-->gamma3 CSR using p50-deficient B cells. Switch junctions from p50-deficient B cells showed decreased lengths of microhomology between Smu and Sgamma3 relative to wild-type cells, indicating a function for p50 in the mechanics of CSR. We note a striking parallel between the affects of p50 and Msh2 deficiency on Smu/Sgamma3 junctions. The data suggest that p50 may be the isotype-specific factor in mu-->gamma3 CSR and epistatic with Msh2.

Animals↗

PubMed2002

Distribution of recombination crossovers and the origin of haplotype blocks: the interplay of population history, recombination, and mutation.

Recent studies suggest that haplotypes are arranged into discrete blocklike structures throughout the human genome. Here, we present an alternative haplotype block definition that assumes no recombination within each block but allows for recombination between blocks, and we use it to study the combined effects of demographic history and various population genetic parameters on haplotype block characteristics. Through extensive coalescent simulations and analysis of published haplotype data on chromosome 21, we find that (1) the combined effects of population demographic history, recombination, and mutation dictate haplotype block characteristics and (2) haplotype blocks can arise in the absence of recombination hot spots. Finally, we provide practical guidelines for designing and interpreting studies investigating haplotype block structure.

Algorithms↗

PubMed2004

Recombination Analysis Tool (RAT): a program for the high-throughput detection of recombination.

MOTIVATION: Recombination can be a prevailing drive in shaping genome evolution. RAT (Recombination Analysis Tool) is a Java-based tool for investigating recombination events in any number of aligned sequences (protein or DNA) of any length (short viral sequences to full genomes). It is an uncomplicated and intuitive application and allows the user to view only the regions of sequence alignments they are interested in. RESULTS: RAT was applied to viral sequences. Its utility was demonstrated through the detection of a known recombinant of HIV and a detailed analysis of Noroviruses, the most common cause of viral gastroenteritis in humans. AVAILABILITY: RAT, along with a user's guide, is freely available from http://jic-bioinfo.bbsrc.ac.uk/bioinformatics-research/staff/graham_etherington/RAT.htm.

DNA, Viral↗

PubMed2005

IRILmap: linkage map distance correction for intermated recombinant inbred lines/advanced recombinant inbred strains.

Intermated Recombinant Inbred Lines (IRILs) in plants, or Advanced Recombinant Inbred Strains in animals, are constructed by carrying out generations of intermating between F2 individuals before starting recurrent inbreeding generations by selfing or sib-mating. IRILs are powerful for high-resolution genetic mapping because they have undergone more recombination than usual Recombinant Inbred Lines (RILs). However, there is no mapping software able to generate actual centiMorgan distances from the segregation data obtained with IRILs. IRILmap software converts genetic distances computed with any linkage mapping program designed for RILs, so that IRIL-derived data can be used to get actual centiMorgan distances, directly comparable to F2, backcross or RIL-derived maps.

Algorithms↗

PubMed2003

Regulation of V(D)J recombination by nucleosome positioning at recombination signal sequences.

A key component in the regulation of V(D)J recombination is control of the accessibility of RAG proteins to recombination signal sequences (RSS). Nucleosomes are known to inhibit this accessibility. We show here that the signal sequence itself represses accessibility by causing nucleosome positioning over the RSS. This positioning is mediated, in vitro and in vivo, by the conserved nonamer of the RSS. Consistent with this strong positioning, nucleosomes at RSSs are resistant to remodelling by nucleosome sliding. In vivo we find that consensus RSSs are preferentially protected, whereas those that lack a consensus nonamer, including some cryptic RSSs, fail to position nucleosomes. Decreased protection of these non-consensus RSSs correlates with their increased use in recombination assays. We therefore suggest that nucleosome positioning by RSSs provides a previously unanticipated level of protection and regulation of V(D)J recombination.

3T3 Cells↗

PubMed1996

Segregation and recombination of Solanum brevidens synteny groups in progeny of somatic hybrids with S. tuberosum: intragenomic equals or exceeds intergenomic recombination.

The Solanum brevidens genome (2n = 2x = 24) was examined with randomly applied polymorphic DNA (RAPD) markers in a second backcross population derived from a S. brevidens + S. tuberosum somatic hybrid. RAPD markers cosegregated into 12 different S. brevidens synteny groups. Most synteny groups were nonrecombinant. However, nearly 40% of the S. brevidens synteny groups detected in this population were recombinant deletions that carried at least one, but not all, synteny group-specific RAPD markers. All S. brevidens synteny groups (except chromosome 5) were involved in recombination, and recombination occurred within most intervals between markers. About 20% of the recombinant S. brevidens synteny groups involved a single synteny group-specific marker. The inheritance of some single-marker representatives was followed in four BC3 families. At least nine changes in S. brevidens synteny groups had occurred during backcrossing. Six of the nine changes involved translocation of S. brevidens markers between nonhomologous S. brevidens chromosomes, and three S. brevidens markers may have been introgressed into the potato genome.

Genome, Plant↗

PubMed1999

CYS3, a hotspot of meiotic recombination in Saccharomyces cerevisiae. Effects of heterozygosity and mismatch repair functions on gene conversion and recombination intermediates.

We have examined meiotic recombination at the CYS3 locus. Genetic analysis indicates that CYS3 is a hotspot of meiotic gene conversion, with a putative 5'-3' polarity gradient of conversion frequencies. This gradient is relieved in the presence of msh2 and pms1 mutations, indicating an involvement of mismatch repair functions in meiotic recombination. To investigate the role of mismatch repair proteins in meiotic recombination, we performed a physical analysis of meiotic DNA in wild-type and msh2 pms1 strains in the presence or absence of allelic differences at CYS3. Neither the mutations in CYS3 nor the absence of mismatch repair functions affects the frequency and distribution of nearby recombination-initiating DNA double-strand breaks (DSBs). Processing of DSBs is also similar in msh2 pms1 and wild-type strains. We conclude that mismatch repair functions do not control the distribution of meiotic gene conversion events at the initiating steps. In the MSH2 PMS1 background, strains heteroallelic for frameshift mutations in CYS3 exhibit a frequency of gene conversion greater than that observed for either marker alone. Physical analysis revealed no modification in the formation of DSBs, suggesting that this marker effect results from subsequent processing events that are not yet understood.

Alleles↗

PubMed2000

Recombination of chromosomes 3A(m) and 5A(m) of Triticum monococcum with homeologous chromosomes 3A and 5A of wheat: the distribution of recombination across chromosomes.

Recombination of chromosomes 3A(m) and 5A(m) of Triticum monococcum with closely homeologous chromosomes 3A and 5A of T. aestivum was compared with recombination across corresponding homologous chromosome pairs. Differentiation between the homeologues impacted recombination in the proximal regions of the long arms the most and in the distal regions of the long arms the least. It is concluded that this variation principally reflects allocation of multiple crossovers across an arm and positive crossover interference across chromosome arms. Recombination rates between homeologous chromosomes 5A(m) and 5A differed in the opposite sexes.

Recombination, Genetic↗

PubMed1980

Region-specific recombination in phage T4. II. Structure of the recombinants.

In this paper, we present results of crosses designed to elucidate the structure of recombinants in the tail-fiber region of bacteriophage T4, in which a glucosylation-dependent recombinations mechanism is operative, and the cause of the "special" recombination in glycosylated crosses is discussed. We present evidence that, when phage are nonglycosylated, recombination in the tail-fiber region proceeds via long heteroduplex overlaps. Mismatched bases within such regions (in nonglycosylated phage) are repaired efficiently (as contrasted to those of glucosylated phage), but asymmetrically; that is, there may be an equal probability of resolving the mismatch to mutant or wild type.

Capsid↗

PubMed2001

Control of directionality in integrase-mediated recombination: examination of recombination directionality factors (RDFs) including Xis and Cox proteins.

Similarity between the DNA substrates and products of integrase-mediated site-specific recombination reactions results in a single recombinase enzyme being able to catalyze both the integration and excision reactions. The control of directionality in these reactions is achieved through a class of small accessory factors that favor one reaction while interfering with the other. These proteins, which we will refer to collectively as recombination directionality factors (RDFs), play architectural roles in reactions catalyzed by their cognate recombinases and have been identified in conjunction with both tyrosine and serine integrases. Previously identified RDFs are typically small, basic and have diverse amino acid sequences. A subset of RDFs, the cox genes, also function as transcriptional regulators. We present here a compilation of all the known RDF proteins as well as those identified through database mining that we predict to be involved in conferring recombination directionality. Analysis of this group of proteins shows that they can be grouped into distinct sub-groups based on their sequence similarities and that they are likely to have arisen from several independent evolutionary lineages. This compilation will prove useful in recognizing new proteins that confer directionality upon site-specific recombination reactions encoded by plasmids, transposons, phages and prophages.

Amino Acid Sequence↗

PubMed2001

An efficient refolding method for the preparation of recombinant human prethrombin-2 and characterization of the recombinant-derived alpha-thrombin.

Human recombinant prethrombin-2 was produced in Escherichia coli. The expressed prethrombin-2 formed intracellular inclusion bodies from which the protein was refolded by a simple one-step dilution process in buffer consisting of 50 mM Tris-HCl, containing 20 mM CaCl(2), 500 mM NaCl, 1 mM EDTA, 600 mM arginine, 1 mM cysteine, 0.1 mM cystine, 10% (v/v) glycerol, and 0.2% (w/v) Brij-58 at pH 8.5. After refolding, prethrombin-2 was purified by hirudin-based COOH-terminal peptide affinity chromatography, and then activated with Echis carinatus snake venom prothrombin activator (ecarin). The activated protein, alpha-thrombin, was then tested for several activities including activity toward chromogenic substrate, release of fibrinopeptide A from fibrinogen, activation of protein C, and thrombin-activatable fibrinolysis inhibitor, reactivity with antithrombin, clotting activity, and platelet aggregation. The kinetic data showed no differences in activity between our recombinant alpha-thrombin and plasma-derived alpha-thrombin. The yield of refolded recombinant human prethrombin-2 was about 4-7% of the starting amount of solubilized protein. In addition, the final yield of purified refolded protein was 0.5-1%, and about 1 mg of recombinant prethrombin-2 could be isolated from 1 liter of E. coli cell culture.

Animals↗

PubMed1996

Differentiation between proteolytic activation and autocatalytic conversion of human prothrombin. Activation of recombinant human prothrombin and recombinant D419N-prothrombin by snake venoms from Echis carinatus and Oxyuranus scutellatus.

Recombinant human prothrombin (r-prothrombin) and recombinant mutant prothrombin with active site Asp419 substituted by Asn (D419N-prothrombin) were expressed in recombinant CHO cells, isolated and purified from the fermentation supernatant. The r-Prothrombin and D419N-prothrombin were digested by both Echis carinatus venom and Oxyuranus scutellatus venom. Prior to, during and after activation, generation of thrombin activity and the proteolytic degradation of the prothrombin polypeptide chain were analysed. Owing to the recombinant preparation and inactivity of D419N-prothrombin and its activation products, the proteolytic action of E.carinatus and O.scutellatus venoms could be studied without addition of thrombin inhibitor, without interference from autocatalytic digestion of prothrombin and in the absence of any other blood coagulation protease. The comparison between the activation of r-prothrombin and D419N-prothrombin by snake venoms permitted differentiation between proteolytic activation and autocatalytic conversion of prothrombin. Incubation of D419N-prothrombin with E.carinatus venom resulted in the generation of stable D419N-meizothrombin by hydrolysis of the peptide bond Arg320-Ile321. By contrast, O.scutellatus venom exhibited activity towards peptide bonds Arg320-Ile321 and Arg271-Thr272 and lower activity towards peptide bond Arg155-Ser156, thus converting D419-prothrombin into D419N-thrombin and also liberating Fragment-1, Fragment-2 and Fragment-1/2 activation peptide. Activation of r-prothrombin by E.carinatus and O.scutellatus venoms demonstrated the autocatalytic potential of prothrombin-derived molecules and indicated that meizothrombin hydrolysed the cleavage between Fragment-2 and thrombin A-chain in the meizothrombin molecule, but not in prothrombin, preferentially at position Arg284-Thr285. By contrast, both meizothrombin and thrombin exhibited no detectable activity towards peptide bond Arg320-Ile321 between thrombin A- and B-chain, although this site exhibits the optimum sequence for thrombin cleavage.

Amino Acid Sequence↗

PubMed1992

Control of human coagulation by recombinant serine proteases. Blood clotting is activated by recombinant factor XII deleted of five regulatory domains.

The availability of engineered serine proteases allows one to study the activation, substrate specificity and regulation of human coagulation and fibrinolytic activities. Human coagulation factor XII is composed of the protease catalytic region at the C-terminus, a hinge proline-rich region and regulatory domains at the N-terminus. From cDNA clones coding for factor XII, two DNA molecules were constructed, one being full length and the other being deleted of exons coding for the regulatory domains. Engineered factor-XII cDNA species were inserted by a homologous recombination technique into vaccinia viruses, which were used to infect the human hepatoma cell line HepG2. Two recombinant proteins were prepared from the culture media and identified by their antigenic properties and electrophoretic mobilities. The recombinant protein of larger size was identified as the full-length factor XII of 80 kDa and its specific activities and activation patterns, determined both by the coagulation and the amidolytic assays, are very similar to these of native human factor XII. The recombinant protein of smaller size was identified as a 319-amino-acid-deleted factor-XII protein of 32 kDa, containing only the entire protease region and part of the proline-rich hinge. This protein was expected to be the 'minimal' portion of factor XII able to sustain protease but unable to recognize substrates and surfaces necessary to activate the contact phase of coagulation. However, this 'minimal' factor-XII protein displays a marked protease activity and, although lacking five regulatory domains of factor XII, is bound and activated by negative charges and promotes coagulation with high efficiency.

Base Sequence↗

PubMed1999

Reconstitution of active recombinant Shiga toxin (Stx)1 from recombinant Stx1-A and Stx1-B subunits independently produced by E. coli clones.

Escherichia coli clones expressing recombinant Shiga toxin (Stx)1-A and recombinant Stx1-B subunits, were established. Culture supernatants of these clones were examined for inhibitory activity on in vitro protein synthesis using luciferase as a reporter enzyme. Culture supernatant of the clone expressing Stx1-A, but not Stx1-B, showed the inhibitory activity. Neither recombinant Stx1-A nor Stx1-B showed Vero cell cytotoxicity. For reconstitution of biologically active toxin, the culture supernatants of the Stx1-A clone and the Stx1-B clone were mixed. The reconstituted recombinant Stx1 showed both Vero cell cytotoxicity and inhibition of in vitro protein synthesis.

Animals↗

PubMed1988

Recombinant interleukin-1 alpha and recombinant tumor necrosis factor alpha synergize in vivo to induce early endotoxin tolerance and associated hematopoietic changes.

Endotoxin, the lipopolysaccharide (LPS) derived from gram-negative bacteria, invokes a wide range of responses in susceptible hosts. It is known that virtually all responses to LPS are mediated by the action of macrophage-derived cytokines (such as interleukin-1 [IL-1], tumor necrosis factor [TNF], and others) which are produced principally by macrophages and maximally within several hours of LPS administration. One manifestation of LPS administration which is not well understood is the phenomenon of "early endotoxin tolerance." In response to a single sublethal injection of LPS, experimental animals become refractory to challenge with a homologous or heterologous LPS preparation 3 to 4 days later. Animals rendered tolerant exhibit mitigated toxicity and a reduced capacity to produce circulating cytokines (i.e., colony-stimulating factor or interferon) in response to the challenge LPS injection. Previous studies have also shown that this state of transient, acquired hyporesponsiveness to LPS is accompanied by a marked increase in the size of cells in the bone marrow which are enriched in numbers of macrophage progenitors. In this study, we examined the capacity of recombinant IL-1 or recombinant TNF or both to induce early endotoxin tolerance and its associated hematopoietic changes. Neither cytokine alone was able to mimic LPS for induction of tolerance. Combined administration of recombinant IL-1 and recombinant TNF doses which were not toxic when administered individually led to synergistic toxicity (as assessed by death or weight loss). However, within a nontoxic range, the two cytokines synergized to induce a significant reduction in the capacity to produce colony-stimulating factor in response to LPS, as well as the characteristic increase in bone marrow cell size and macrophage progenitors shown previously to be associated with LPS-induced tolerance.

Animals↗

PubMed1967

Molecular recombination in T4 bacteriophage deoxyribonucleic acid. I. Tertiary structure of early replicative and recombining deoxyribonucleic acid.

A replicative hybrid resulting from the infection of heavy (substituted with 5-bromodeoxyuridine) bacteria with light (not substituted with 5-bromodeoxyuridine) radioactive bacteriophage was isolated from a CsCl density gradient. Sedimentation studies indicate that 60% of the deoxyribonucleic acid (DNA) behaves as if it were in units more than four times as large as an intact reference molecule. Under the electron microscope, hybrid molecules appeared tangled, showed puffs and loops, occupied a small area, and often had a total length twice that of mature phage. This indicates that sucrose gradient sedimentation is not applicable as a method for estimating the relative molecular size of replicative forms of DNA. After denaturation, the separated strands of hybrid were of the same size as those of reference DNA. CsCl density gradient analysis revealed no terminal covalent addition of new material to the old parental strand. The possibility of a continuous growth of the DNA molecule, either on a single-stranded level or as a double helical structure, is disproved. When chloramphenicol (CM) was added at critical times after infection, DNA synthesis continued at a constant rate. The parental label soon assumed and retained a hybrid density, despite concomitant synthesis of DNA, throughout the rest of the period of incubation in CM. The hybrid moiety, however, actively participated in replication and exchanged its partner strand for a new one; this was demonstrated by changing the density label during incubation in CM. A new enzyme synthesized shortly after infection introduced single-stranded "nicks" into the parental DNA. Since nicking can be inhibited by chloramphenicol, the responsible enzyme is not of host origin. The time of the appearance of this enzyme coincided with the onset of molecular recombination. Another enzyme, which mediates the repair of the continuity of the polynucleotide chain after recombination, appeared after recombination. If selectively inhibited by chloramphenicol, recombinant molecules remained unrepaired, and, upon denaturation, the parental fragment was liberated in pure form.

Bromodeoxyuridine↗