Single Recombination Event Research Articles

Identification of adenovirus 7h heterogeneity in the E3 region.

Adenovirus genotype 7h was previously reported to be originated from a recombination event between adenovirus genotypes 7p and 3p. Based on those findings, further characterization of other adenovirus 7h strains become important to determine whether all adenovirus 7h strains arose from a single recombinational event. To explore such a possibility, 160 clinical isolates were studied after developing a PCR assay using a primer set designed to amplify the region corresponding to E3-7,7 Kd of adenovirus ADV 7p and E3-9 Kd of adenovirus 3p. The assay was able to differentiate most of the subgenus B strains from adeno 7h with the genotype 3d. The study of several adenovirus 7h clinical isolates revealed the existence of three variants of adeno 7h. One of the variants, 7h3, shows a high degree of similarity with gene E3-9 Kd of ADV 3p, but lacks the corresponding AUG codon. Our results suggest that more than one recombination event may explain the detection of three different types of adenovirus 7h. The genotype variants of adeno 7h were detected in different years, indicating that the recombination events took place independently from each other. The study of the recombination region may allow further understanding of the function of several viral polypeptides in the immune response, and understanding the mechanism involved in virulence associated to adenovirus 7h.

Typeability of Tn1546-like elements in vancomycin-resistant enterococci using long-range PCRs and specific analysis of polymorphic regions.

Molecular subtyping of the VanA-type resistance element Tn1546 in an international collection of 81 genomically diverse vancomycin-resistant enterococci (VRE) from human, animal, and environmental reservoirs was evaluated by restriction analysis of long-range PCR amplicons (PCR-RFLP), single gene PCRs, Southern blot analysis of genomic digests, and partial DNA sequencing. A dominant Tn1546-RFLP in accordance with Enterococcus faecium BM4147 was detected in 43 of the 49 long-range PCR positive strains from ecologically diverse sources in several European countries and the US. Tn1546-like elements from the 32 (40%) long-range PCR negative strains were typed into 17 different groups by single-gene PCRs and Southern blot analysis of the ORF1, ORF2, vanS-vanH, vanX-vanY, and vanZ regions. All these isolates showed deletions in the ORF1 and/or vanZ primer binding regions explaining the failure of long-range PCR amplification. Enlarged vanS-vanH or vanX-vanY fragments were detected in 7 (22%) and 16 (50%) of the long-range PCR negative strains, respectively. The enlarged vanS-vanH regions of five clinical isolates from the US (n = 2), Ireland (n = 2), and Norway (n = 1) contained identical IS1251-like insertions indicating intercontinental spread of the vanA gene cluster. Intergenic vanS-vanH IS1251 insertions have so far not been reported in European studies. Structural rearrangements of Tn1546-like elements may represent single recombination events that can serve as fingerprints in the molecular examination of vanA gene cluster evolution and transmission. The optimal strategy for such analysis has yet to be determined. Two alternative long-range PCRs with subsequent RFLP analysis were successfully used to type the majority of vanA gene clusters in an ecologically and geographically heterogeneous VRE strain collection, but failed to detect and type a group of variant Tn1546-like elements truncated in the left-end ORF1/ORF2 region. Further subtyping of such variants should specifically target the polymorphic vanS-vanH and vanX-vanY regions.

The spontaneous coat color mutant white nose (wn) maps to murine chromosome 15.

The spontaneous coat color mutant white nose (wn) maps to murine chromosome 15.

The STR polymorphisms in intron 8 may provide information about the molecular evolution of RH haplotypes.

We identified simple-sequence repeat polymorphisms in intron 8 of the RHD and RHCE genes, both of which contained the 5-bp repeat unit (AAAAT)n. We analyzed the polymorphisms of this short tandem repeat (STR) in 104 Japanese RhD-positive and 124 RhD-negative (87 RHD gene negative and 37 nonfunctional RHD gene positive) donors by the polymerase chain reaction (PCR) and subsequent typing by electrophoresis and silver staining. We found five alleles (10, 11, 12, 13, and 14 repeats) in the RHD gene and four (7, 8, 9, and 10 repeats) in the RHCE gene. The Rh phenotypes were closely associated with polymorphisms of the STR. The Ce allele had 12 repeats in the RHD gene and 9 repeats in the RHCE gene at high frequency. The cE allele frequently had 10-12 repeats in the RHD gene and 10 repeats in the RHCE gene. The 10 repeats in the RHCE gene were identified exclusively in the 87 RHD gene-negative donors and 9 repeats were identified only in those with the RhC antigen. These results indicate that both haplotypes of dce and dcE arose from single RHD gene deletion and recombination events, respectively. In the 37 RhD-negative donors with a nonfunctional RHD gene, 12 repeats in the RHD gene and 9 repeats in the RHCE gene were frequently observed. Thus, the RhD-negative with a nonfunctional RHD gene combination might have arisen from the DCe haplotype via a mutation that abolished RHD gene expression. These findings suggest that the STR polymorphisms might shed light upon the molecular evolution of RH haplotypes.

Mapping of intra-locus duplications and introgressed DNA: aids to map-based cloning of genes from complex genomes illustrated by physical analysis of the Rx locus in tetraploid potato

We describe here novel approaches to high-resolution mapping of repeated and introgressed DNA which may prove generally useful in map-based cloning from complex genomes. These approaches were developed in order to clone the Rx locus in potato. First, we prepared a BAC library from a tetraploid plant carrying Rx in the duplex condition (Rx, Rx, rx, rx). BAC clones were then isolated with close markers on either side of Rx. However these clones did not extend across Rx: in the cloned DNA the closest markers to Rx were separated by single recombination events on either side of Rx. To bridge the gap we exploited the finding that the BAC clones on the right side of Rx contained resistance-gene homologues. Anticipating that there would be duplicated copies of these resistance gene homologues in the vicinity of Rx, we used low-stringency PCR conditions to identify additional markers. One of these markers was completely linked to Rx in our mapping population and was used to isolate a BAC (BAC77) that had not been previously identified by screening with Rx-flanking markers. Based on two criteria it was concluded that BAC77 spans Rx. There was a chromosomal recombination in one plant of our mapping population that separated the BAC77 right end from Rx. On the other side of Rx it was found that the BAC77 left end was outside the region of DNA carrying Rx that had been introgressed into potato cv Amaryl from a Solanum tuberosum ssp. andigena accession CPC1673.

Phylogenetic evidence for recombination in dengue virus.

A split decomposition analysis of dengue (DEN) virus gene sequences revealed extensive networked evolution, indicative of recombination, among DEN-1 strains but not within serotypes DEN-2, DEN-3, or DEN-4. Within DEN-1, two viruses sampled from South America in the last 10 years were identified as recombinants. To map the breakpoints and test their statistical support, we developed a novel maximum likelihood method. In both recombinants, the breakpoints were found to be in similar positions, within the fusion peptide of the envelope protein, demonstrating that a single recombination event occurred prior to the divergence of these two strains. This is the first report of recombination in natural populations of dengue virus.

Homologous expression of soluble methane monooxygenase genes in Methylosinus trichosporium OB3b.

An homologous expression system has been developed for soluble methane monooxygenase (sMMO) genes from Methylosinus trichosporium OB3b. sMMO-minus mutants were previously obtained after marker-exchange mutagenesis, by the insertion of a kanamycin-resistance cassette into the mmoX gene of the sMMO operon. Complementation of the sMMO-minus genotype was achieved by conjugation with broad-host-range plasmids containing the native promoter and sMMO operon from Ms. trichosporium OB3b (pVK100Sc and pHM2). In wild-type methanotrophs, copper ions present in the growth medium at concentrations greater than 0.25 microM inhibit transcription of sMMO genes. The stable maintenance of pVK100Sc resulted in transconjugant methanotrophs with a decreased sensitivity to copper, since expression of sMMO occurred at copper sulphate concentrations of 7.5 microM. sMMO activity was only detected in soluble extracts after the addition of purified sMMO reductase component, which is inhibited by copper ions in vitro. This phenomenon could have arisen due to the increased number of sMMO gene copies (derived from pVK100Sc) in the cell. Transconjugants obtained from conjugations with pHM2 expressed sMMO at copper concentrations of 0-2.5 microM only and sMMO activity was not restored by the addition of purified reductase component at copper concentrations higher than 2.5 microM. Southern hybridization showed that the plasmid had integrated into the chromosome, probably by a single homologous recombination event. This is the first report of homologous sMMO expression in a methanotroph with enzyme activities that are comparable to the activity reported in wild-type strains. This expression system will be useful for site-directed mutagenesis of active-site residues of sMMO from Ms. trichosporium OB3b.

Fine-Scale Mapping of a Novel Dementia Gene, PLOSL, by Linkage Disequilibrium

Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL; MIM 221770) is a rare hereditary cause of presenile dementia with autosomal recessive inheritance. Its unique feature is the cystic bone lesions that accompany the dementia. About 160 cases have been reported to date, mostly in Finland and Japan. The etiology and pathogenesis of PLOSL are unknown. We recently assigned the locus for PLOSL in the Finnish population to chromosome 19q13.1 (P. Pekkarinenet al.,1998,Am. J. Hum. Genet.62, 362–272). In the present study, we restrict the critical region for PLOSL to 153 kb by linkage-disequilibrium mapping. First, three new microsatellite markers were revealed in the PLOSL critical region. These and three other markers spanning the critical region were analyzed in Finnish PLOSL families. Strong linkage disequilibrium (multipointPvalue < 10−47) was detected between the markers and PLOSL, and for two markers, D19S1176 and D19S610, all the PLOSL chromosomes shared identical 171- and 218-bp alleles, respectively. Haplotype analysis revealed five different haplotypes in the Finnish PLOSL chromosomes. But all of them shared the region between markers D19S1175 and D19S608 that could be traced to one ancestor haplotype by single recombination events, thus defining the critical region as 153 kb. Multipoint association analysis also assigned the most likely location of the PLOSL locus within this interval to the immediate vicinity of marker D19S610. A promising positional candidate for PLOSL, an amyloid precursor-like protein, was studied by sequencing, but no mutations were detected. These results lay the basis for the cloning of this novel dementia gene and for diagnostics in the Finnish population using haplotype analysis.

A novel subfamily of LINE-derived elements in mice.

Hybrid sequences have been described previously that consist of a 5' region homologous to ORF2 of LINEs and a 3' end that shares homology with a sequence located in the first intron of Cepsilon immunoglobulin. The present investigation has revealed 14 new sequences from seven murine species, that show high homology to those observed earlier. Database search has found several new homologous hybrid sequences including one located in the mouse T-cell receptor (Tcra) locus. Several interesting features of this sequence include identical 15-bp flanking short direct repeats as well as poly-A signal and A-rich sequence at the 3' end. We have classified this set of sequences as LINE-derived elements (LDEs), which constitute a newly observed subfamily. Comparative analysis of these sequences suggests that a single recombination event was responsible for the production of an LDE progenitor. The phylogenetic tree shows a number of elements that pre-existed in the common ancestor of murine species and displays different evolutionary rates. The time of LDE origin is estimated at approximately 10-15 MYA.

Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function.

A recA deletion mutant of Mycobacterium smegmatis has been isolated by homologous recombination using a sacB counterselection strategy. Deletion of the recA gene from the chromosome was demonstrated by Southern hybridizations and by polymerase chain reaction (PCR). Western analysis using anti-RecA antibodies confirmed that the RecA protein was not made by the mutant strain. The recA deletion strain exhibited enhanced sensitivity to UV irradiation and failed to undergo homologous recombination. The results obtained from the recombination assays suggest that in wild-type M. smegmatis the majority of colonies arise from single cross-over homologous recombination events with only a very minor contribution from random integrations. The deficiencies in UV survival and recombination were complemented by introduction of the cloned M. smegmatis recA gene. Overexpression of RecA was found to be toxic in the absence of recX, which is found downstream of and co-transcribed with recA and is thus also affected by the deletion of recA. The M. smegmatis recA deletion strain was also complemented by the M. tuberculosis recA gene with or without its intein; most importantly, the frequency of double cross-over homologous recombination events was identical regardless of whether the M. tuberculosis recA gene contained or lacked the intein. Thus, the low frequency of homologous recombination observed in M. tuberculosis is not due to the presence of an intein-coding sequence in its recA gene per se.

Biometry and DNA fingerprinting detect limited genetic differentiation among populations of the apomictic blackberry Rubus nessensis (Rosaceae)

Clonal plant species have in many recent studies exhibited unexpectedly high levels of genetic differentiation. This seems to hold true also for several apomictic (agamospermous) species in spite of their potential ability to spread single genotypes over large areas through seed dispersal. By contrast, previous DNA fingerprint studies have demonstrated apomictic Rubus species to be extremely homogeneous. The present study was undertaken to estimate the extent and distribution of intraspecific genetic variation in R. nessensis. Populations differed significantly for many biometrical characters but this variation was evenly distributed, with few significant differences between single pairs of populations. Patterns indicating ecoclinal variation, which could be adaptive in origin, were not found. Populations also clustered close together in a two-dimensional CVA plot, with only one population being somewhat further removed. DNA analysis of later collected material from this population as well as from three others yielded identical DNA fingerprints with each of two different multi-locus DNA probes. Taken together with previous DNA fingerprint studies, these results indicate that a major part of the R. nessensis plants in Northern Europe have been derived from a single recombinational event, possibly making this genet the most widespread land-living organism in the world. The encountered variation in biometrical characters is probably instead caused by e.g. minor somatic mutations. Rubus species have relatively large, bird-dispersed seeds that probably travel further than the small, wind-dispersed seeds found in most previously investigated apomicts (e.g. in Asteraceae). The potential for single genotypes to disperse over large areas should therefore be greater in Rubus than in many other apomictic species groups.

A complex major histocompatibility complex D locus variant generated by an unusual recombination mechanism in mice.

A spontaneous variant of the mouse class I major histocompatibility complex D(b) gene, designated D(bm28), is characterized. This mutation consists of a cluster of nucleotide substitutions in exon 3 that resembles the product of a classical gene conversion event in that the substituted nucleotides appear to be templated. However, D(bm28) is distinctive, because no single donor gene containing the nucleotide sequence of the mutation exists in the genome of the parent strain. The mutation is consistent with the expected result of an interaction of two donor genes at the target locus during a single recombination event. While no known genetic mechanism gives rise to this class of mutation, we have established that 10 percent of spontaneous class I mutations in the mouse major histocompatibility complex have this complex phenotype. This process occurs at the D locus and the K locus. The significance of this kind of genetic interaction may extend beyond the major histocompatibility complex and have importance in shaping other multigene families.

Molecular mapping of the potato virus Y resistance gene Rysto in potato

Ry sto is a dominant gene which confers resistance to potato virus Y (PVY) in potato. We have used bulked segregant analysis of an F1 tetraploid potato population to identify three AFLP markers linked to and on either side of Ry sto . The tomato homologue of one of these AFLP markers was assigned to linkage group XI by analysis of an F2 mapping population of tomato, suggesting that Ry sto is also on chromosome XI of the potato genome. This map position was confirmed by the demonstration that Ry sto was linked to markers which had been previously mapped to chromosome XI of the potato genome. Four additional AFLP markers were identified that were closely linked to Ry sto in a population of 360 segregating progeny of a potato cross between a resistant (Ry sto ) and a susceptible parent. Two of these markers were on either side of Ry sto , separated by only a single recombination event. The other two markers co-segregated with Ry sto .

Prediction of multiple hypermutable codons in the human PAH gene: codon 280 contains recurrent mutations in Quebec and other populations.

The predicted mutability profile (MUTPRED) of the phenylalanine hydroxylase (PAH) gene shows that the 48 CpG sites (template and atemplate strands) are either empty of known mutations (7 sites), harbour "PKU" alleles involving CpG doublets (16 sites), or contain mutations that do not involve a C-->T or G-->A substitution in the doublet. These hypermutable sites harbour 32 different mutations in association with at least 66 different haplotypes and hyperphenylalaninemia. The E280K mutation in exon 7 of the PAH gene is a cause of phenylketonuria. It occurs on four different haplotypes in Europeans and on haplotypes 1 and 2 in Quebec. Whereas a single recombination event could explain the two haplotype associations in Quebec, the mutation does involve a CpG dinucleotide. By analyzing multiallelic markers 5' (STR) and 3' (VNTR) to the E280K allele on 12 mutant and 30 normal chromosomes, we conclude that recurrent mutation is the likely origin of E280K in Quebec. The PAH mutation database shows that the allele accounts for 1.5% of PKU chromosomes worldwide.

An investigation into the role of phenotypic plasticity in evolution

Phenotypic plasticity can modify evolutionary pathways and accelerate the course of evolution. This was brought out in a quantitative model by Hinton & Nowlan (1987, Complex Systems 1,497-502). The present work confirms and extends their results. We consider a population of genetically haploid individuals of fixed size. Genotypes are represented by one-dimensional arrays (strings) of genes. Each gene can be in one of three allelic states, designated 1, 0 and X.1 and 0 stand for fixedstates, that is for states with predetermined effects on the phenotype. Xstands for a plasticstate: the phenotypic effect of an Xcan be equivalent to that of a 1 or a 0, the actual choice being realized by a process of random coin-tossing. Our model, in contrast to that of Hinton and Nowlan, assumes a relatively smooth dependence of fitness on distance from a pre-assigned target genotype. From the fitness values, the number of individuals reaching reproductive maturity is determined. Reproduction involves random mating and a single recombinational event, with one of the two progeny genotypes becoming, in turn, a possible parental genotype for the next generation. We find that it is because of the special assumptions in the Hinton and Nowlan model that phenotypic plasticity invariably accelerates evolution. The relationship is not as straightforward with realistic fitness schemes. Instead, the general result is that plasticity, up to a certain optimal level, slows down the rate of evolutionary change but improves the level of adaptation finally reached.

Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination.

An integron is a genetic unit that includes the determinants of the components of a site-specific recombination system capable of capturing and mobilizing genes that are contained in mobile elements called gene cassettes. An integron also provides a promoter for expression of the cassette genes, and integrons thus act both as natural cloning systems and as expression vectors. The essential components of an integron are an int gene encoding a site-specific recombinase belonging to the integrase family, an adjacent site, attI, that is recognized by the integrase and is the receptor site for the cassettes, and a promoter suitably oriented for expression of the cassette-encoded genes. The cassettes are mobile elements that include a gene (most commonly an antibiotic-resistance gene) and an integrase-specific recombination site that is a member of a family of sites known as 59-base elements. Cassettes can exist either free in a circularized form or integrated at the attI site, and only when integrated is a cassette formally part of an integron. A single site-specific recombination event involving the integron-associated attI site and a cassette-associated 59-base element leads to insertion of a free circular cassette into a recipient integron. Multiple cassette insertions can occur, and integrons containing several cassettes have been found in the wild. The integrase also catalyses excisive recombination events that can lead to loss of cassettes from an itegron and generate free circular cassettes. Due to their ability to acquire new genes, integrons have a clear role in the evolution of the genomes of the plasmids and transposons that contain them.

Plasmid evolution by acquisition of mobile gene cassettes: plasmid pIE723 contains the aadB gene cassette precisely inserted at a secondary site in the IncQ plasmid RSF1010

Gene cassettes are mobile DNA elements which contain a specific recombination site, a 59-base element, recognized by the site-specific recombination system of integrons. Gene cassettes are normally found inserted at a unique site in an integron, downstream of a promoter which directs transcription of the cassette-associated genes. However, insertion of a gene cassette into a secondary site in a plasmid which does not contain an integron is also formally possible. Sequence analysis of the aadB gene in pIE723, a plasmid closely related to the IncQ plasmid RSF1010, revealed the presence of the complete aadB cassette inserted at a secondary site downstream of a known RSF1010 promoter. The site of insertion of the aadB cassette in RSF1010 conformed to the consensus for secondary sites recognized by the integron integrase (Int), and it is likely that the cassette was inserted via a single Int-mediated recombination event between the 59-base element of a free, circular aadB cassette and a secondary site in RSF1010. The cassette-associated recombination site was inactivated by the insertion, and Int-mediated excision of the aadB cassette from this non-specific location was not detectable, indicating that the cassette is stably inserted. The movement of gene cassettes to secondary sites is likely to play an important role in the acquisition of new genes by bacterial and plasmid genomes.

Restoration of half the normal dystrophin sequence in a double‐deletion Duchenne muscular dystrophy family

Two male cousins with Duchenne muscular dystrophy were found to have different maternal dystrophin gene haplotypes and different deletion mutations. One propositus showed two noncontiguous deletions--one in the 5', proximal deletional hotspot region, and the other in the 3', more distal deletional hotspot region. The second propositus showed only the 5' deletion. Using multiple fluorescent exon dosage and fluorescent multiplex CA repeat linkage analyses, we show that the mother of each propositus carries both deletions on the same grandmaternal X chromosome. This paradox is explained by a single recombinational event between the 2 deleted regions of one of the carrier's dystrophin genes, giving rise to a son with a partially "repaired" gene retaining only the 5' deletion.

Evidence for origin, by recurrent mutation, of the phenylalanine hydroxylase R408W mutation on two haplotypes in European and Quebec populations.

The R408W mutation in the phenylalanine hydroxylase gene (PAH) of phenylketonuria patients occurs on haplotypes 2.3 and 1.8 in Europeans. The mutation involves a CpG dinucleotide; nonetheless, a single recombination event might also explain the two haplotype associations. By analysis of an STR in the PAH gene 5' to the 408 codon and of the VNTR system in the 3' UTR, we identified unique features of the haplotype 1.8 chromosome harbouring the R408W mutation which are not accounted for by recombination. We conclude that recurrent mutation is the origin of R408W on different PAH haplotypes in Europeans.

Restoration of a normal level of immunoglobulin production in a hybridoma cell line following modification of the chromosomal immunoglobulin μ gene by gene replacement

In the present investigation, we have measured the levels of IgM mRNA and protein in recombinant cell lines in which the chromosomal immunoglobulin μ gene has been modified by gene replacement (gene conversion or double reciprocal recombination) or vector integration (single reciprocal recombination) events. Our studies reveal that chromosomal immunoglobulin μ genes modified by gene replacement are expressed at wild-type levels whereas those modified by vector integration have lower levels of immunoglobulin μ gene expression. These results suggest that gene replacement may a preferred method for the construction of hybridoma and myeloma cell lines producing optimized immunoglobulins and for studies of immunoglobulin gene function.