Frequent Recombination Events Research Articles

Interspecific recombination between two ruminant alphaherpesviruses, bovine herpesviruses 1 and 5.

Homologous recombination between different species of alphaherpesviruses has been described between herpes simplex viruses 1 and 2 but has not yet been observed between other alphaherpesviruses. In the present study we chose to assess to what extent in vitro recombination can occur between members of a well-defined group of closely related viruses such as ruminant alphaherpesviruses. At 24 h after infection of epithelial bovine kidney cells with a double-deleted mutant of bovine herpesvirus 1 (BoHV-1) (containing green fluorescent protein and red fluorescent protein genes) and different ruminant alphaherpesviruses, four types of progeny viruses were detected and distinguished according to their phenotype. Frequent recombination events between identical or different strains of BoHV-1 were observed (up to 30%), whereas only two BoHV-1/BoHV-5 recombinants were identified, and no recombinants between BoHV-1 and less closely related caprine and cervine herpesviruses were detected. Restriction analysis of the genomes of the two BoHV-1/BoHV-5 recombinants showed different genetic backgrounds. One possessed a restriction pattern close to BoHV-1, whereas the other one was close to BoHV-5. This exhaustive analysis of each combination of coinfection in a unique situation of five closely related alphaherpesviruses revealed the importance of a high degree of genetic relatedness and similar parental virus growth kinetics for successful interspecific recombination.

An Arabidopsis homologue of bacterial RecA that complements an E. coli recA deletion is targeted to plant mitochondria.

Homologous recombination results in the exchange and rearrangement of DNA, and thus generates genetic variation in living organisms. RecA is known to function in all bacteria as the central enzyme catalyzing strand transfer and has functional homologues in eukaryotes. Most of our knowledge of homologous recombination in eukaryotes is limited to processes in the nucleus. The mitochondrial genomes of higher plants contain repeated sequences that are known to undergo frequent rearrangements and recombination events. However, very little is known about the proteins involved or the biochemical mechanisms of DNA recombination in plant mitochondria. We provide here the first report of an Arabidopsis thaliana homologue of Escherichia coli RecA that is targeted to mitochondria. The mt recA gene has a putative mitochondrial presequence identified from the A. thaliana genome database. This nuclear gene encodes a predicted product that shows highest sequence homology to chloroplast RecA and RecA proteins from proteobacteria. When fused to the GFP coding sequence, the predicted presequence was able to target the fusion protein to isolated mitochondria but not to chloroplasts. The mitochondrion-specific localization of the mt recA gene product was confirmed by Western analysis using polyclonal antibodies raised against a synthetic peptide from a unique region of the mature mtRecA. The Arabidopsis mt recA gene partially complemented a recA deletion in E. coli, enhancing survival after exposure to DNA-damaging agents. These results suggest a possible role for mt recA in homologous recombination and/or repair in Arabidopsis mitochondria.

VO1, a temperate bacteriophage of the type 19A multiresistant epidemic 8249 strain of Streptococcus pneumoniae: analysis of variability of lytic and putative C5 methyltransferase genes.

A temperate bacteriophage (VO1) has been isolated from the Streptococcus pneumoniae type 19F multiresistant epidemic 8249 strain (South African strain). Structural analysis of the specific integration site, protein composition, restriction patterns, and molecular dissection of the lytic system of this phage revealed high sequence similarity with MM1, a temperate phage from the Spain23F-1 strain of pneumococcus, another multiresistant epidemic clone. The different pneumococcal strains sequenced so far exhibit an identical and single attB located in the same site of the genome. Remarkably, the LytA amidase coded by VO1 showed clear differences with that of the host bacterium in contrast with the situation previously documented for bacterial- and phage-coded amidases of pneumococcus. In addition, a new gene (orfmet) putatively coding for a C5 methyltransferase has been identified. A noticeable variability affecting the presence (or absence) of this supernumerary gene(s) in the same region of the genomes of three otherwise highly similar phages (i.e., VO1, MM1, and HB-3) suggests frequent recombinational events leading to introduce variability in this genome region. The peculiarities of genes like lytA and orfmet in VO1 provide interesting insights on mechanisms of horizontal transfer and lysogenic state co-evolution.

Var gene diversity in Plasmodium falciparum is generated by frequent recombination events

Var gene diversity in Plasmodium falciparum is generated by frequent recombination events

Frequent homologous recombination events between molecules of one RNA component in a multipartite RNA virus.

Brome mosaic bromovirus (BMV), a tripartite plus-sense RNA virus, has been used as a model system to study homologous RNA recombination among molecules of the same RNA component. Pairs of BMV RNA3 variants carrying marker mutations at different locations were coinoculated on a local lesion host, and the progeny RNA3 in a large number of lesions was analyzed. The majority of doubly infected lesions accumulated the RNA3 recombinants. The distribution of the recombinant types was relatively even, indicating that both RNA3 counterparts could serve as donor or as acceptor molecules. The frequency of crossovers between one pair of RNA3 variants, which possessed closely located markers, was similar to that of another pair of RNA3 variants with more distant markers, suggesting the existence of an internal recombination hot spot. The majority of crossovers were precise, but some recombinants had minor sequence modifications, possibly marking the sites of imprecise homologous crossovers. Our results suggest discontinuous RNA replication, with the replicase changing among the homologous RNA templates and generating RNA diversity. This approach can be easily extended to other RNA viruses for identification of homologous recombination hot spots.

DNA rearrangements at the extremities of the Streptomyces ambofaciens linear chromosome: Evidence for developmental control

In Streptomyces, a genomic instability results from frequent recombination events which occur at the ends of the linear chromosomal DNA. These events are believed to be responsible for the variability observed in these regions among Streptomyces species and strains. In order to identify functions able to control this type of genome plasticity, mutators as well as mutants produced at different stages of development have been characterized in S. ambofaciens. Their characterization suggests the existence of a relationship between genomic instability and colony development.

Sequence analysis of Stx2-converting phage VT2-Sa shows a great divergence in early regulation and replication regions.

In enterohemorrhagic Escherichia coli, Shiga toxin is produced by lysogenic prophages. We have isolated the prophage VT2-Sa that is responsible for production of Shiga toxin type 2 protein, and determined the complete nucleotide sequence of this phage DNA. The entire DNA sequence consisted of 60,942 bp, exhibiting marked similarity to the 933W phage genome. However, several differences were observed in the immunity and replication regions, where cI, cII, cIII, N, cro, O, and P genes were present: Predicted amino acid sequences of N, cI, cro, O and P in the VT2-Sa genome did not show significant similarity to the counterparts of the 933W genome; however its cI showed higher similarity to lambda. Furthermore, O and P closely resembled those of phage HK022. These observations suggest that the various degrees of homology observed in the immunity and replication regions of VT2-Sa could have resulted from frequent recombination events among the lambdoid phages, and that these regions play a key role as a functional unit for phage propagation in competition with other lambdoid phages.

Study of the Structure of Replicative Intermediates of HSV-1 DNA by Pulsed-Field Gel Electrophoresis

DNA from HSV-1-infected cells was separated by pulsed-field gel electrophoresis into two virus-specific bands: one that migrated as the linear monomer genome (152 kb) and another that remained at the origin of the gel. The latter band contained the replicating HSV-1 DNA, as determined by pulse-labeling with [ 3H]thymidine. To investigate the structure of this "gel origin" DNA, we constructed a HSV-1 KOS mutant bearing a unique Pac I restriction site (HSV-1 PAC1DTK). Partial digestion of gel origin PAC1DTK DNA at late times postinfection (24-48 hr) demonstrated the presence of linear concatemers on pulsed-field gel electrophoresis. Within each concatemer, the long (L) regions of adjacent monomer genomes were found in the two possible orientations. In addition, shorter-than-unit-size fragments that corresponded in size to the left end fragments of the viral genome were detected with the U L region in the two possible orientations. At early times postinfection (8-12 hr), digestion with PacI released only a trace of linear fragments, and most of the gel origin DNA did not migrate on pulsed-field gel electrophoresis. Multiple cuts with EcoRI (a restriction enzyme that cuts the HSV-1 KOS genome 12 times) were necessary to release linear fragments that migrated from the origin of the gel. These results indicate that replicative intermediates of HSV-1 DNA are linked in a large network that needs to be unraveled before packaging takes place. This network may be composed of linear molecules linked together by frequent recombination events or of products of a mode of replication other then simple rolling circle (e.g., theta replication).

The primary structure of the rat guanylyl cyclase A/atrial natriuretic peptide receptor gene

We have isolated and characterized three genomic clones and a genomic fragment amplified by the polymerase chain reaction that contain the rat guanylyl cyclase-A (GC-A)/atrial natriuretic peptide receptor gene. The gene spans about 17.5 kilobases and includes 22 exons and 21 introns. All of the exon-intron junction sequences coincide with the GT/AG consensus. GC-A consists of at least the following four distinguishable domains: extracellular ligand binding, transmembrane, kinase-like, and cyclase catalytic. Exon 7 encodes the putative transmembrane domain. The kinase-like and catalytic domains are encoded by exons 8-15 and 16-22, respectively. The 5' end of the transcript, estimated by primer extension and S1 mapping, is 370 nucleotides upstream of the methionine initiation codon. The initiator sequence (-3 to +5) of CACACTCC has two mismatches when compared with a consensus initiator sequence of CTCANTCT. The 5'-flanking region contains three potential Sp1-binding sites and an inverted CCAAT box, but no apparent TATA box. Three different and short interspersed, repetitive sequences are found within intervening sequences and within the 5'- and 3'-flanking regions of the gene (five rat identifier, two rat type 2 Alu equivalent, and seven Alu-like sequences). They fall between the four major domains suggestive that these may be sites for frequent recombination events. This first reported structure of a gene for a member of this new enzyme/receptor family should facilitate the search for new family members, as well as allow studies to progress on the mechanisms by which the gene is regulated.

The divergence between two oncogenic Herpesvirus saimiri strains in a genomic region related to the transforming phenotype

Herpesvirus saimiri strains can be divided into at least three subgroups (A, B, C) based on sequence divergence at the left end of viral unique sequence DNA. Strains of subgroups A and C are highly oncogenic and readily transform simian T-lymphocytes in vitro to interleukin-2 independent growth, while subgroup B strains do not. A left terminal reading frame of a H. saimiri subgroup A strain was shown previously to correlate with the oncogenic phenotype and in vitro transforming potential; the deduced polypeptide was termed STP-A. Furthermore, this same region contains an open reading frame (ORF) for dihydrofolate reductase (DHFR) and genes for five virus-specific U RNAs (HSURs). We now show by sequence analysis of the corresponding region in a subgroup C strain that DHFR and HSUR genes are present in both virus subgroups; however, no sequence homologous to the STP-A reading frame was found in this subgroup C virus. At a position and orientation similar to STP-A, two ORFs were found for peptides sharing a putative transmembrane domain. One of them encodes a peptide with collagen-like repetitions. In addition to the lack of similarity to STP-A, these two reading frames also did not show any similarity to known oncogenes. The organization of sequences at the left junction of unique L- and repetitive H-DNA of H. saimiri suggests frequent recombinational events, possibly accelerating the uptake of foreign genes by the virus.

Specific allelic variation among linked HLA class II genes.

Locus-specific oligonucleotides have been used as probes to detect polymorphic alleles for the HLA genes DQ beta, DQ alpha, DX alpha, and DO beta. These genes lie between the HLA DR and DP genes on chromosome 6, a region frequently implicated in intra-HLA recombination. In order to distinguish among these highly homologous HLA class II genes, we have identified sequences in the coding regions that are locus specific and have synthesized short oligonucleotide probes corresponding to these regions. We have used these probes in a gel hybridization procedure to analyze restriction-enzyme-digested genomic DNA and to assign polymorphic bands to a particular locus. Taq I-digested DNA hybridized with a DQ-beta-specific probe detects a single band per haplotype. The size of this band corresponds precisely to the expressed DQ beta gene and distinguishes among the serologically defined DQ alleles, providing a rapid method for genomic DQ typing. Similar analysis with an oligonucleotide probe specific for DX alpha, the nonexpressed alpha gene in the DQ subregion, detects 2 DX alleles, and hybridization with an oligonucleotide specific for the newly described DO beta gene detects 2 alleles at DO beta. Heterogeneity in linkage patterns among DQ, DX, and DO genes suggests that frequent recombinational events at multiple intergenic sites contributed to the generation of present-day haplotypes. One such recombinational event was identified directly in a family with serologically HLA-identical siblings, in which genomic analysis indicated a parental recombination event mapping between DR and DX, which correlated with unexpected alloreactivity.

Genetic recombination within the human T-cell receptor alpha-chain gene complex.

Genetic analyses of the human T-cell receptor (TCR) alpha-chain genes indicate that recombination events may occur frequently within this gene complex. Examination of the inheritance of restriction fragment length polymorphisms (RFLP) detected by using probes for constant or variable region gene segments made it possible to assign TCR alpha haplotypes to the 16 parents and 43 offspring of eight families studied. A total of six RFLP, three for the constant region and three for variable region gene segments, were examined in the present studies. Most enzyme and probe combinations tested revealed no polymorphism and those finally selected for the study showed limited polymorphism in that only two or, in one case, three allelic forms of the gene were seen. In spite of limited variability at this level, extensive heterogeneity was observed for the combinations of markers present in haplotypes, suggesting that frequent recombination events have occurred. Most strikingly, multiple combinations of RFLP occurring in close proximity of the TCR alpha constant region gene were observed in this study. A high recombination frequency for the TCR alpha gene complex is further supported by the observation that two children, one in each of two families, inherited recombinant TCR alpha haplotypes.

Homologous recombination between hepadnaviral genomes following in Vivo DNA transfection: Implications for studies of viral infectivity

The cloned DNA of duck hepatitis B virus (DHBV) can initiate a productive infection of susceptible ducklings when presented by direct intrahepatic injection. We have examined the effects of the structure of the incoming DNA upon the outcome of this in vivo transfection. Plasmid-linked DHBV genomes of greater than unit length regularly give rise to infectious virus irrespective of the location of the plasmid insert on the transcriptional map. When mutant DHBV genomes are coinjected with subgenomic viral DNA fragments spanning the mutation, wild-type recombinants arise. These results indicate that, as in transfection of cultured cells, in vivo transfection of hepatocytes is regularly followed by homologous recombination involving incoming DNA molecules. These frequent recombination events will complicate efforts to use in vivo transfection for certain genetic analyses of hepadnaviruses.

Japanese wild mouse, Mus musculus molossinus, has duplicated immunoglobulin gamma 2a genes.

The immunoglobulin heavy chain loci of the mouse contain eight tightly linked genes that encode the constant region (C) of the immunoglobulin heavy chains (H). As no recombination occurred within the immunoglobulin CH gene loci among more than 3,000 crosses of mice1–3, the mouse CH genes were believed to be inherited as a set, designated the Igh haplotype. Recent molecular cloning experiments4–9 have demonstrated that the organization of the mouse Igh loci of BALB/c (Igha haplotype) is 5′-Cμ-(4.5 kilobases [kb])-Cδ-(55kb)-Cγ3-(34kb)-Cγ1-(21 kb)-Cγ2b-(15 kb)-Cγ2a-(14 kb)-Cɛ-(12 kb)-Cα-3′. The structural analyses of four Cγ subclass genes have shown that they are essentially identical in terms of lengths and locations of each of the structural and intervening sequences6,10–12. Comparison of the nucleotide sequences of the Cγ1, Cγ2b and Cγ2a genes has shown that limited portions of the Cγ gene are conserved between compared pairs of the Cγ genes12. The results imply that the nucleotide sequences of the Cγ gene segments have been exchanged by recombination among related clustered genes. Similar studies on the Cγ2a genes of different haplotypes, Igha and Ighb, have also suggested that these two types of Cγ2a gene have undergone recombinational exchange of a part of the gene sequence13. These results imply, in contrast to the previous belief, that frequent recombinational events have taken place within the Igh loci and that their nucleotide sequences have been rearranged during evolution. Here we present direct evidence that a Japanese wild mouse, Mus musculus molossinus, contains duplicated Cγ2a genes while the copy numbers of the neighbouring CH genes, that is, the Cγ1, Cγ2b and Cɛ genes, remain constant. The results indicate that the duplicated Cγ2a genes of M. m. molossinus arose from unequal crossing-over between homologous chromosomes.

Purification and characterization of a murine tumor cell surface glycoprotein of 75,000 daltons that is related to the major envelope glycoprotein of murine leukemia virus

An antigen which competes with murine C-type viral glycoprotein in an interspecies radioimmunocompetition assay has been purified from the surface of EL-4 tumor cells. The EL-4 tumor cell is virus particle negative. The amount of p30 was extremely low and there was no detectable p12 or p15, confirming that the tumor cell was not expressing a murine oncornavirus. In homologous competition radioimmunoassays, the tumor cell was negative for Rauscher and AKR gp71, but in an interspecies assay employing 125I-Rauscher gp71 and anti-feline leukemia virus serum, there was a reactive antigen. The gp71-like antigen was on the surface of the tumor cell since, by immunofluorescence and immunoelectronmicroscopy, the EL-4 cell could be labeled with antiserum against Rauscher virus gp71. The gp71 cross-reactive antigen was purified by lithium diiodosalycilate extraction, DEAF; chromatography, and lentil lectin affinity chromatography. It is a glycoprotein of about 75,000 daltons which could be precipitated by various broadly reactive antisera to murine gp71s and antisera to murine virus. Based on radioimmunocompetition assays, the purified gp75 was not related to AKR or Rauscher gp71, but did compete in an assay using BALB/2 xenotropic gp71 and anti-C57/L virus serum. It also competed in an assay using 125I-Moloney virus gp71 and anti-Moloney virus serum, but not in a more type-specific assay assay 125I-Moloney virus gp71 and anti-Moloney gp71 serum. Tryptic peptide maps of iodinated proteins were prepared and the cell surface antigen was compared with AKR gp71, Moloney gp71, Rauscher gp71, and xenotropic BALB/2 and NZB gp71s. The EL-4 gp75 was not identical with or very similar structurally to any of the viral gp71s. The differences in the structures of the various viral gp71s shown here and also in other laboratories are consistent with the idea that the env genes of murine viruses which code for gp71s are sites of frequent recombinational events. Recombination between different endogenous viral sequences or between viral and host allelic genomes could have resulted in many immunologically related proteins on viruses, cell surfaces and, in sera of mice, which are immunologically related, about 70,000-dalton molecular weight, but have divergent structure.

Sacro-iliac changes and urinary infection in patients with spinal cord injuries.

The mouse multimember family of <i>Qa-2</i> oligomorphic class I MHC genes is continuously undergoing duplications and deletions that alter the number of the two “prototype” Qa-2 sequences, <i>Q8</i> and <i>Q9</i>. The frequent recombination events within the Q region lead to strain-specific modulation of the cumulative Qa-2 expression levels. Q9 protects C57BL/6 hosts from multiple disparate tumors and functions as a major CTL restriction element for shared tumor-associated Ags. We have now analyzed functional and structural properties of Q8, a class I MHC that differs significantly from Q9 in the peptide-binding, CTL-interacting α₁ and α₂ regions. Unexpectedly, we find that the extracellular domains of Q8 and Q9 act similarly during primary and secondary rejection of tumors, are recognized by cross-reactive antitumor CTL, have overlapping peptide-binding motifs, and are both assembled via the transporter associated with the Ag processing pathway. These findings suggest that shared Ag-presenting functions of the “odd” and “even” <i>Qa-2</i> loci may contribute to the selective pressures shaping the haplotype-dependent quantitative variation of Qa-2 protein expression.