A clustering of RNA recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus

Coronaviruses undergo RNA recombination at a very high frequency. To understand the mechanism of recombination in murine coronavirus, we have performed RNA sequencing of viral genomic RNA to determine the precise sites of recombination in a series of recombinants which have crossovers within the gene encoding the peplomer protein. We found that all of the recombination sites are clustered within a region of 278 nucleotides in the 5'-half of the gene. This region in which all of the crossovers occurred represents a small fraction of the distance between the two selection markers used for the isolation of these recombinant viruses. This result suggests that this region may be a preferred site for RNA recombination. The crossover sites are located within and immediately adjacent to a hypervariable area of the gene. This area has undergone deletions of varying sizes in several virus strains which have been passaged either in vivo or in vitro. These results suggest that a similar RNA structure may be involved in the occurrence of both recombination and deletion events.     

Studies on the recombination between RNA genomes of poliovirus: the primary structure and nonrandom distribution of crossover regions in the genomes of intertypic poliovirus recombinants

A series of intertypic (type 3/type 1) poliovirus recombinants was obtained whose crossover sites were expected to be located in the middle of the viral genome, between the loci encoding type-specific antigenic properties, on the 5' side, and an altered sensitivity to guanidine, on the 3' side. The primary structures of the crossover regions in the genomes of these recombinants were determined by the primer extension method. The length of the crossover sites (the uninterrupted sequences shared by the recombinant and both parental genomes that are flanked, in the recombinant RNAs, by two heterotypic segments) varied between 2 and 32 nucleotides, but the majority of the sites were 5 nucleotides long or shorter. The crossover sites were nonrandomly distributed over the presumably available genome region: only a single such site was found within the gene for polypeptide 2A, whereas an apparent clustering of the crossover sites was encountered in other genomic segments. When the crossover sites were superimposed on a model of the secondary structure of the relevant region of the viral RNA molecule, a pattern consistent with the previously proposed mechanism of poliovirus recombination (L.I. Romanova, V.M. Blinov, E.A. Tolskaya, E.G. Viktorova, M.S. Kolesnikova, E.I. Guseva, and V.I. Agol (1986) Virology 155, 202-213) was observed. It is suggested that the nonrandom distribution of the crossover sites in the genomes of intertypic poliovirus recombinants was due to two factors: the existence of preferred sites for recombination, and selection against recombinants with a lowered level of viability.     

Comparisons of defective HTLV-I proviruses predict the mode of origin and coding potential of internally deleted genomes.

Cell lines infected with a variety of HTLV-I isolates were examined for the presence of defective proviruses that contain deletions spanning the gag, pol, and env genes. Internally deleted proviruses were identified by Southern blotting and by PCR amplification with 5' and 3' primers complementary to gag and tax sequences, respectively. PCR products representing eight defective proviruses from seven different cell lines were subsequently cloned and sequenced. The objectives of this study were twofold: first, we sought to determine whether nucleotide sequences surrounding sites of deletion shared common features that might reveal the mechanisms by which the defective genomes originated. Second, we asked whether deleted proviruses encode Gag fusion proteins with related C-terminal residues derived from open reading frames in the pX region. While most of the defective proviruses had incurred a single, large deletion, two of them displayed a more complex pattern of multiple rearrangements. Alignments of bases flanking the 5' and 3' deletion endpoints within each provirus showed tracts of sequence identity consistent with a mechanism involving aberrant intramolecular strand-transfer events during replication. We suggest that the amount or activity of HTLV-I polymerase in virions may contribute both to the poor infectivity of the virus and to the high deletion frequency. Two of the eight proviruses that were examined encoded a gag gene joined to an extended open reading frame; the other six had very short open reading frames (one to six amino acids) derived from pX or env regions joined to gag that showed no apparent amino acid sequence similarity.     

Termini of human chromosomes display elevated rates of mitotic recombination

The strand-specific in situ hybridization technique of CO-FISH was used to probe telomeres of human mitotic cells in order to determine the spontaneous frequency of crossover. This approach allowed the detection of recombinational crossovers occurring anywhere along the length of individual chromosomes, including reciprocal events taking place between sister chromatids. Although the process of sister chromatid exchange (SCE) is the most prominent type of recombination in somatic mammalian cells, our results show that SCEs accounted for less than a third of the recombinational events revealed by CO-FISH. It is concluded that chromosomal regions near the termini of chromosome arms undergo extraordinarily high rates of spontaneous recombination, producing terminal crossovers whose small size precludes detection by standard cytogenetic methods. That similar results were observed for transformed epithelial cells, as well as primary fibroblasts, suggests that the phenomenon is a common characteristic of human cells. These findings are noteworthy because, although telomeric and subtelomeric DNA is known to be preferentially involved in certain types of recombination, the tips of somatic mammalian chromosomes have not previously been identified as preferred sites for crossover. Implications of these results are discussed in terms of limitations imposed on CO-FISH for its proposed use in directional hybridization mapping.     

Mechanism of DI RNA formation in tombusviruses: dissecting the requirement for primer extension by the tombusvirus RNA dependent RNA polymerase in vitro

Tombusviruses, which are positive-strand RNA viruses of plants, frequently generate defective interfering (DI) RNAs that consist of three to four noncontiguous segments of the parental RNA. Replicase jumping was postulated to cause multiple deletions leading to the de novo formation of DI RNAs in planta. This model was tested using a partially purified RNA-dependent RNA polymerase (RdRp) preparation from tombusvirus-infected plants in vitro. The tombusvirus RdRp was capable of primer extension without the need for sequence complementarity between the primer and the acceptor template in vitro, although the most efficient primer extension was obtained with primers forming a 5-bp duplex with the acceptor region. Primers forming 14- to 20-bp duplexes with the acceptor region were used less efficiently by the tombusvirus RdRp in vitro. In addition, primers with 3' noncomplementary nucleotides were also extended by the tombusvirus RdRp, albeit with a reduced efficiency. The preference of the tombusvirus RdRp for short base-paired primers in vitro is consistent with the lack of extended sequence similarities at the junction sites in the de novo generated tombusvirus-associated DI RNAs. The in vitro experiments also revealed that the acceptor region plays a significant role in primer extension. Comparison of tombusvirus-derived, heterologous and artificial acceptor regions revealed that the conserved regions present in DI RNAs are the best acceptor regions when they are available in the minus-strand orientation. These data suggest that recombination/deletion events may be more frequent at some regions, rather than occurring randomly throughout the parental genome. In addition, these findings support a model that predicts a higher frequency of replicase jumping, i.e., recombination/deletion events, during plus-strand synthesis than during minus-strand synthesis.     

Molecular determinants of HIV-1 intersubtype recombination potential

Sequence differences in the dimerization initiation signal (DIS) affect the rate of recombination between subtype B and subtype C HIV-1. To test the hypothesis that DIS sequences can be used to predict intersubtype recombination potentials, we measured the recombination rate between CRF01_A/E (AE) and B, which contain mismatches in the DIS, and between AE and C, which have an identical DIS. Compared with the intrasubtype recombination rate, the recombination rate between AE and subtype B virus was 9-fold lower, and the rate between AE and subtype C virus was 2-fold lower. Thus, DIS sequences can be used to predict the recombination potential between HIV-1 subtypes. Further analyses revealed that the 2-fold lower recombination rate between AE and C viruses can be restored to the intrasubtype recombination rate by matching a part of the LTR and a portion of the viral genome. Therefore, the lower intersubtype recombination rate between AE and C is not caused by a given region but is a cumulative effect by more than one region.     

Mapping recombination sites in the HLA class II region.

Studies of linkage disequilibrium across the HLA class II region have been useful in predicting where recombination is most likely to occur. Strong associations exist within the 100kb region from DQB1 to DRB1, suggesting a low frequency of recombination in this region. Conversely, a lack of association between alleles of TAP1 and TAP2 (about 15kb) has been observed, suggesting that recombination occurs here with relative frequency. Analysis of familial-derived recombinant chromosomes is likely to enrich the current knowledge of recombination across the class II region which has been derived from disequilibrium studies of known class II haplotypes. Families containing recombinant chromosomes within the 700kb interval between the DPB1 and DRB1 genes have been identified for such purposes. Using single-strand conformation polymorphism analysis, 122 novel polymorphic markers distributed throughout the class II region were identified and used to delineate the site of crossover events in 26 class II recombinant chromosomes. The three regions where recombination occurred most frequently are as follows: the 45kb interval between DN-A and RING3 in 10 cases, the 40kb interval between DQB3 and DQB1 in 6 cases, and an 8.8kb segment of the TAP2 gene in 3 cases. The final seven recombinant chromosomes require further resolution, but all of these potentially fall within one of the three intervals mentioned. Two of the three TAP2 recombinant chromosomes have been mapped to overlapping 139bp and 850bp segments of intron 2. Associations between polymorphic markers immediately flanking each of the 3 defined regions using data from unrelated individuals will be compared with the familial recombination data observed in these regions.     

Site-specific recombination by Gin of bacteriophage Mu: inversions and deletions

A 3000-bp invertible segment in the DNA of bacteriophage Mu determines the host range of the phage. The inversion is catalyzed by the phage-coded protein Gin; the recombination sites are short inverted repeats. Gin protein is only made in low amounts by Mu. To further investigate the Gin-mediated recombination reaction a Gin overproducing strain was constructed. The gin gene was cloned on a plasmid behind the PL-promotor of phage lambda. This results in a 100-fold higher inversion frequency of a Mu gin phage as compared to the situation when Gin is expressed from its own promoter. A test system was developed suitable for the detection of Gin action in vivo and in vitro: the lacZ gene of E. coli was cloned within the invertible region in such a way that it is only expressed when the region is in one specific orientation. Thus inversions can be detected or selected as a switch from Lac- to Lac+. This system was used to determine the inversion frequency under different experimental conditions. The ability of Gin to catalyze deletions was investigated by inverting in vitro one of the two recombination sites using restriction enzymes and genetically marking the DNA between those sites. Deletions do occur, although at a lower frequency than inversions.     

Factors influencing recombination frequency and distribution in a human meiotic crossover hotspot

Little is known about the factors that influence the frequency and distribution of meiotic recombination events within human crossover hotspots. We now describe the detailed analysis of sperm recombination in the NID1 hotspot. Like the neighbouring MS32 hotspot, the NID1 hotspot is associated with a minisatellite, suggesting that hotspots predispose DNA to tandem repetition. Unlike MS32, crossover resolution breakpoints in NID1 avoid the minisatellite, producing a cold spot within the hotspot. This avoidance may be related to the palindromic nature of the minisatellite interfering with the generation and/or processing of recombination intermediates. The NID1 hotspot also contains a single nucleotide polymorphism (SNP) close to the centre, which appears to directly influence the frequency of crossover initiation. Quantitative gene conversion assays show that this SNP affects the frequency of gene conversion and crossover to a very similar extent, providing evidence that conversions and crossovers are triggered by the same recombination initiating events. The recombination-suppressing allele is over-transmitted to recombinant progeny, and provides the most dramatic example to date of recombination-mediated meiotic drive, of a magnitude sufficient to virtually guarantee that the recombination suppressor will eventually replace the more active allele in human populations.     

Intrachromosomal recombination after targeted monocopy integration in Penicillium chrysogenum: stabilization of the direct repeats to prevent loss of the inserted gene

Monocopy systems obtained by targeted integration at the pyrG locus of P. chrysogenum led to the formation of unstable direct repeats in the genome. A previously isolated pyrG mutant was sequenced and the mutation was found to be located at nucleotide position 665 of the pyrG gene. A different pyrG mutation was introduced in vitro at the BamHI site of this gene. Recombination products arising from monocopy systems using the bleomycin/phleomycin resistance gene (ble) as a model were studied to elucidate the intrachromosomal recombination mechanisms. Experimental results showed that both gene conversion and deletion events occurred spontaneously at the integration site. Gene conversion products were obtained at a frequency of one in 3.4x10(4) viable transformant spores. When gene conversion occurred, the inserted exogenous gene was retained and was flanked by rearranged direct repeats of the pyrG gene, each containing at least one pyrG mutation. Deletion events resulted in the loss at high frequency of the inserted exogenous gene. Genetic stabilization of a monocopy system was obtained when both pyrG repeats (formed at the targeted integration site) contained at least one identical mutation, since in this case further recombinations can be easily counter-selected.     

Cycles of chromosome instability are associated with a fragile site and are increased by defects in DNA replication and checkpoint controls in yeast

We report here that a normal budding yeast chromosome (ChrVII) can undergo remarkable cycles of chromosome instability. The events associated with cycles of instability caused a distinctive "sectoring" of colonies on selective agar plates. We found that instability initiated at any of several sites on ChrVII, and was sharply increased by the disruption of DNA replication or by defects in checkpoint controls. We studied in detail the cycles of instability associated with one particular chromosomal site (the "403 site"). This site contained multiple tRNA genes known to stall replication forks, and when deleted, the overall frequency of sectoring was reduced. Instability of the 403 site involved multiple nonallelic recombination events that led to the formation of a monocentric translocation. This translocation remained unstable, frequently undergoing either loss or recombination events linked to the translocation junction. These results suggest a model in which instability initiates at specific chromosomal sites that stall replication forks. Forks not stabilized by checkpoint proteins break and undergo multiple rounds of nonallelic recombination to form translocations. Some translocations remain unstable because they join two "incompatible" chromosomal regions. Cycles of instability of this normal yeast chromosome may be relevant to chromosome instability of mammalian fragile sites and of chromosomes in cancer cells.     

Predominance of a rare type of HIV-1 in Estonia

An earlier study has indicated that a complex recombinant HIV-1 strain dominates the epidemic in Estonia. The objective of this study was to further investigate the molecular epidemiology and genetic structure of HIV-1 in Estonia. Most of the investigated individuals became infected after August 2000 when HIV-1 started to spread rapidly among Estonian intravenous drug users (IDUs). Two viral DNA regions, gag/pol and gp41, were sequenced and subtyped from peripheral blood mononuclear cells or plasma from 141 individuals. Phylogenetic analysis in the gp41 region revealed that the most frequent type of the virus among IDUs was a circulating recombinant form, CRF06_cpx, whereas a few samples showed highest sequence similarity to a subtype A strain circulating in Ukraine and Russia. Likewise, in the gag/pol region, most of the samples were classified as CRF06_cpx, with a few classified as subtype A. In this region, however, 16% of the sequences turned out to be mosaic unique recombinant forms consisting of CRF06_cpx and subtype A. At least 9 mosaic forms were identified, each with distinct patterns of multiple crossover. To characterize Estonian CRF06_cpx as well as recombinant isolates in more detail, 4 near-full-length HIV-1 genomes were sequenced.     

High resolution analysis of haplotype diversity and meiotic crossover in the human TAP2 recombination hotspot

Little is known about the nature of recombination hotspots in the human genome and the relationship between crossover activity and patterns of linkage disequilibrium. We have therefore used both haplotype analysis and direct detection of crossovers in sperm to characterize a putative recombination hotspot in the TAP2 gene within the class II region of the MHC. Haplotype diversity provided evidence for a localized hotspot within intron 2 of this gene. Sperm DNA typing using allele-specific PCR primers to selectively amplify recombinant TAP2 molecules revealed a highly localized meiotic crossover hotspot approximately 1.2 kb long, unusually abundant in sequence polymorphisms and flanked by DNA much less active in recombination. Sperm crossover appeared to be fully reciprocal, and almost all crossover products were simple, involving a single exchange between adjacent heterozygous markers. This hotspot appears to be much more active in female than male meiosis. No primary sequence similarities could be found between any of the very few well defined crossover hotspots in the human genome, all of which show recombinationally active domains 1-2 kb long. Direct comparison of recombination frequency and haplotype diversity in TAP2 showed that linkage disequilibrium measures were a poor predictor of crossover frequency in this region, with non-recombining markers sometimes in free association and with examples of pairs of markers spanning the recombination hotspot showing substantial or even absolute linkage disequilibrium.     

Random nature of coronavirus RNA recombination in the absence of selection pressure.

RNA-RNA recombination is thought to occur preferentially at certain selected sites and in only a few RNA viruses; the mechanism for these restrictions is unknown. In this paper we report the development of a recombination assay for coronavirus, using polymerase chain reaction, in the absence of selection pressure. Our results showed that within a 1-kb region of the peplomer gene, RNA recombination occurred at almost every potential crossover site. Thus, coronavirus RNA recombination appears to be more random than previously realized. However, after serial passages of the recombinant viruses in tissue culture, the recombination sites among the progeny viruses became clustered in the region which contains the previously reported "hot spot" for coronavirus recombination. These results suggest that RNA recombination is common and random in nature, but only certain recombinants can be selected. Thus, the presence of recombinational "hot spots" for coronavirus or other RNA viruses most likely resulted from selection of certain recombinant viruses and not restriction on the occurrence of RNA recombination. The failure to detect recombinants in other RNA viruses may therefore be due to unfavorable properties of recombinant viruses. This approach can be used to detect recombinants in these viruses.     

The basis for the mechanistic bias for deletional over inversional V(D)J recombination.

V(D)J recombination between recognition sites in the genome is characterized by certain biases. At some loci, proximal sites undergo recombination substantially more frequently than distal ones. The joining of DH/JH is an example of this. Because the DH element bears signal sequences on each side, inversion would be expected as often as deletion in DH/JH recombination. However, the markedly favored outcome is deletion, entailing utilization of the closer recombination site. One model proposed to explain these biases is the tracking model in which the recombinase tracks from one site to the other. Here, we have directly tested for various types of tracking in V(D)J recombination and have found no indication that it occurs. In addition, we have created DH-JH minilocus substrates for analysis of the basis for the preference for deletion. We find that we can reproduce the deletional bias for the system. Moreover, by flipping the orientation of the D segment, we can reverse the bias such that the frequency of inversions can exceed the number of deletions. These results indicate (1) that there is no intrinsic topological preference in this reaction, and (2) that the sequence of the signal and coding ends determines the bias.     

Double crossover in the human Xp/Yp pseudoautosomal region and its bearing on interference

Most models on crossover and crossover interference have assumedthat the Intensity of interference depends inversely on thephysical distance separating the respective intervals. The possibility,however, also exists that Interference depends on genetic ratherthan on physical distance. As the human pseudoautosomal region(PAR) on Xp/Yp is physically small, yet genetically a hot spotof recombination In male meiosls, studies on the existence ofmultiple crossover events may be particularly useful In addressingthe question of interference. Our results demonstrate for thefirst time a double crossover during male meiosls In the humanPAR on Xp/Yp. The occurrence of a single obligatory recombinationevent in this region can, therefore, no longer be taken as adogma. However, double crossover events seem to remain exceptionaland, thus, the model originally suggested by Burgoyne remainsglobally correct. As both recombination events can be localizedwithin a narrow range of physical distance, theories describinginterference due to stertc hindrance may have to be reconsidered.This finding may, therefore, have general implications for ourunderstanding of the mechanism of positive and negative crossoverInterference In mammalian genomes and may be interesting especiallyfor linkage mapping where double recombinations In small intervalstend to be considered as genotyplng errors.     

Activation-induced cytidine deaminase induces DNA break repair events more frequently in the Ig switch region than other sites in the mammalian genome

Activation-induced cytidine deaminase (AID) produces DNA breaks in immunoglobulin genes during antibody diversification. Double-stranded breaks (DSB) in the switch region mediate class switch recombination, and contribute to gene conversion and somatic hypermutation in the variable regions. However, the relative extent to which AID induces DSB in these regions or between these and other actively expressed sequences is unknown. Here, we exploited an enhancer-trap plasmid that identifies DSB in actively expressed loci to investigate the frequency and position of AID-induced vector integration events in mouse hybridoma cells. Compared to control cells, wild-type AID stimulates plasmid integration into the genome by as much as 29-fold. Southern and digestion-circularization PCR analysis revealed non-uniformity in the integration sites, with biases of 30- and 116-fold for the immunoglobulin kappa light chain and mu heavy chain genes, respectively. Further, within the immunoglobulin mu gene, 73% of vector integrations map to the mu switch region, an enhancement of five- and 12-fold compared to the adjacent heavy chain variable and mu gene constant regions, respectively. Thus, among potential highly transcribed genes in mouse hybridoma cells, the immunoglobulin heavy and light chain genes are important AID targets, with the immunoglobulin mu switch region being preferred compared to other genomic sites.