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PubMed2001

Fibroblast growth factor-induced gene expression and cartilage pattern formation in chick limb bud recombinants.

To clarify the roles of fibroblast growth factors (FGF) in limb cartilage pattern formation, the effects of various FGF on recombinant limbs that were composed of dissociated and reaggregated mesoderm and ectodermal jackets were examined. Fibroblast growth factor-soaked beads were inserted just under the apical ectodermal ridge (AER) of recombinant limbs and the recombinant limbs were grafted and allowed to develop. Control recombinant limbs without FGF beads formed one or two cartilage elements. Recombinants with FGF-4 beads formed up to five cartilage elements, which were aligned along the anteroposterior (AP) axis. Each cartilage element showed digit-like segmentation. In contrast, recombinants with FGF-2 beads showed formation of multiple thick and unsegmented cartilage rods, which elongated inside and outside the AP plane from the distal end of the recombinants. Recombinants with FGF-8 beads formed a truncated cartilage pattern and recombinants with FGF-10 beads formed a cartilage pattern similar to that of the control recombinants. The expression of the Fgf-8, Msx-1 and Hoxa-13 genes in the developing recombinant limbs were examined. FGF-4 induced extension of the length of the Fgf-8-positive epidermis, or AER, along the AP axis 5 days after grafting, at which time the digits are specified. FGF-2 induced expansion of the Msx-1-positive area, first in the proximal direction and then along the dorsoventral axis. The functions of these FGF in recombinant and normal limb patterning are discussed in this paper.

Animals↗
PubMed2003

Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants.

Homologous recombination can be used to generate recombinants on episomes or directly on the Escherichia coli chromosome with PCR products or synthetic single-stranded DNA (ssDNA) oligonucleotides (oligos). Such recombination is possible because bacteriophage lambda-encoded functions, called Red, efficiently recombine linear DNA with homologies as short as 20-70 bases. This technology, termed recombineering, provides ways to modify genes and segments of the chromosome as well as to study homologous recombination mechanisms. The Red Beta function, which binds and anneals ssDNA to complementary ssDNA, is able to recombine 70-base oligos with the chromosome. In E. coli, methyl-directed mismatch repair (MMR) can affect these ssDNA recombination events by eliminating the recombinant allele and restoring the original sequence. In so doing, MMR can reduce the apparent recombination frequency by >100-fold. In the absence of MMR, Red-mediated oligo recombination can incorporate a single base change into the chromosome in an unprecedented 25% of cells surviving electroporation. Our results show that Beta is the only bacteriophage function required for this level of recombination and suggest that Beta directs the ssDNA to the replication fork as it passes the target sequence.

Base Sequence↗
PubMed2002

Long CTG.CAG repeat sequences markedly stimulate intramolecular recombination.

Previous studies have shown that homologous recombination is a powerful mechanism for generation of massive instabilities of the myotonic dystrophy CTG.CAG sequences. However, the frequency of recombination between the CTG.CAG tracts has not been studied. Here we performed a systematic study on the frequency of recombination between these sequences using a genetic assay based on an intramolecular plasmid system in Escherichia coli. The rate of intramolecular recombination between long CTG.CAG tracts oriented as direct repeats was extraordinarily high; recombinants were found with a frequency exceeding 12%. Recombination occurred in both RecA(+) and RecA(-) cells but was approximately 2-11 times higher in the recombination proficient strain. Long CTG.CAG tracts recombined approximately 10 times more efficiently than non-repeating control sequences of similar length. The recombination frequency was 60-fold higher for a pair of (CTG.CAG)(165) tracts compared with a pair of (CTG.CAG)(17) sequences. The CTG.CAG sequences in orientation II (CTG repeats present on a lagging strand template) recombine approximately 2-4 times more efficiently than tracts of identical length in the opposite orientation relative to the origin of replication. This orientation effect implies the involvement of DNA replication in the intramolecular recombination between CTG.CAG sequences. Thus, long CTG.CAG tracts are hot spots for genetic recombination.

Escherichia coli↗
PubMed1986

Meiosis can induce recombination in rad52 mutants of Saccharomyces cerevisiae.

The RAD52 and RAD50 genes have previously been shown to be required for normal meiotic recombination and for various types of recombination occurring in mitotic cells. Recent evidence suggests that rad52 mutants might be defective in an intermediate recombination step; we therefore examined recombination during meiosis in several rad52 mutants at several different loci and in genetic backgrounds that yield efficient sporulation and synchronous meiosis. Similar to previous reports, spores from rad52 diploids are inviable and meiotic recombination is greatly reduced by rad52 mutations. However, intragenic recombinants were detected when cells were plated on selective media during meiosis; rad52 mutants experience induction of recombination between homologues under these special conditions. The frequencies of recombination at four loci were considerably greater than the mitotic controls; however, they were still at least 20 times lower than corresponding Rad+ strains. The prototrophs induced by meiosis in rad52 mutants were not typical meiotic recombinants because incubation in nutrient-rich medium before plating to selective medium resulted in the complete loss of recombinants. We propose that previously observed single-strand breaks that accumulate in rad52 mutants may be associated with recombinational intermediates that are resolved when cells are returned to selective mitotic media and that the meiosis-induced recombination in rad52 cells does not involve double-strand breaks.

DNA Transposable Elements↗
PubMed1996

The efficiency of meiotic recombination between dispersed sequences in Saccharomyces cerevisiae depends upon their chromosomal location.

To examine constrains imposed on meiotic recombination by homologue pairing, we measured the frequency of recombination between mutant alleles of the ARG4 gene contained in pBR322-based inserts. Inserts were located at identical loci on homologues (allelic recombination) or at different loci on either homologous or heterologous chromosomes (ectopic recombination). Ectopic recombination between interstitially located inserts on heterologous chromosomes had an efficiency of 6-12% compared to allelic recombination. By contrast, ectopic recombination between interstitial inserts located on homologues had relative efficiencies of 47-99%. These findings suggest that when meiotic ectopic recombination occurs, homologous chromosomes are already colocalized. The efficiency of ectopic recombination between inserts on homologues decreased as the physical distance between insert sites was increased. This result is consistent with the suggestion that during meiotic recombination, homologues are not only close to each other, but also are aligned end to end. Finally, the efficiency of ectopic recombination between inserts near telomeres (within 16 kb) was significantly greater than that observed with inserts > 50 kb from the nearest telomere. Thus, at the time of recombination, there may be a special relationship between the ends of chromosomes not shared with interstitial regions.

Argininosuccinate Lyase↗
PubMed2002

Forty-one near full-length HIV-1 sequences from Kenya reveal an epidemic of subtype A and A-containing recombinants.

OBJECTIVE: To further define the genetic diversity of HIV-1 in Kenya using approaches that clearly distinguish subtypes from inter-subtype recombinants. DESIGN: Near full genome sequencing and analysis were used, including sensitive new tools for detection and mapping of recombinants. METHODS: Purified peripheral blood mononuclear cell DNA from 41 HIV-1 positive blood donations collected from six hospitals across southern Kenya was used to amplify near full-length genomes by nested PCR. These were sequenced on an ABI 3100 automated sequencer and analyzed phylogenetically. RESULTS: Among 41 near full-length genomes, 25 were non-recombinant (61%) and 16 were recombinant (39%). Of the 25 pure subtypes, 23 were subtype A, one was subtype C and one was subtype D. Most recombinants consisted of subtype A and either subtype C or subtype D; a few contained A2, a recently identified sub-subtype. Two A2/D recombinants had identical breakpoints and may represent a circulating recombinant form. A third A2/D recombinant had the same structure as a previously described Korean isolate, and these may constitute a second A2-containing circulating recombinant form. CONCLUSIONS: In Kenya, 93% of HIV-1 genomes were subtype A or A-containing recombinant strains. Almost 40% of all strains were recombinant. Vaccine candidates tested in Kenya should be based on subtype A strains, but the methods used for evaluation of breakthrough infections during future vaccine trials should be capable of identifying non-A subtypes, the A2 sub-subtype, and recombinants.

Disease Outbreaks↗
PubMed2004

Geographically and temporally distant natural recombinant isolates of Plum pox virus (PPV) are genetically very similar and form a unique PPV subgroup.

Natural recombinant Plum pox virus (PPV) isolates were detected in Albania, Bulgaria, Czech Republic, Germany, Hungary and Slovakia. Despite different geographical origins and dates of isolation, all the recombinant isolates were closely related at the molecular level and shared the same recombination breakpoint as well as a typical signature in their N-terminal coat protein sequence, suggesting a common origin. Biological assays with four recombinant isolates demonstrated their capacity to be aphid-transmitted to various Prunus hosts. One of these isolates had a threonine-to-isoleucine mutation in the conserved PTK motif of its HC-Pro and showed a drastically decreased, although not abolished, aphid transmissibility. The complete genome sequence of one of the recombinant isolates, BOR-3, was determined, as well as some partial sequences in the HC-Pro and P3 genes for additional natural recombinant isolates. Analysis of the phylogenetic relationships between the recombinant isolates and other sequenced PPV isolates confirmed that the recombinant isolates form a phylogenetically homogeneous lineage. In addition, this analysis revealed an ancient recombination event between the PPV-D and M subgroups, with a recombination breakpoint located in the P3 gene. Taken together, these results indicate that recombinant isolates represent an evolutionarily successful, homogeneous group of isolates with a common history and unique founding recombination event. The name PPV-Rec is proposed for this coherent ensemble of isolates.

Animals↗
PubMed2000

Perspective: sex, recombination, and the efficacy of selection--was Weismann right?

The idea that sex functions to provide variation for natural selection to act upon was first advocated by August Weismann and it has dominated much discussion on the evolution of sex and recombination since then. The goal of this paper is to further extend this hypothesis and to assess its place in a larger body of theory on the evolution of sex and recombination. A simple generic model is developed to show how fitness variation and covariation interact with selection for recombination and illustrate some important implications of the hypothesis: (1) the advantage of sex and recombination can accrue both to reproductively isolated populations and to modifiers segregating within populations, but the former will be much larger than the latter; (2) forces of degradation that are correlated across loci within an individual can reduce or reverse selection for increased recombination; and (3) crossing-over (which can occur at different places in different meioses) will create more variability than having multiple chromosomes and so will have more influence on the efficacy of selection. Several long-term selection experiments support Weismann's hypothesis, including those showing a greater response to selection in populations with higher rates of recombination and higher rates of recombination evolving as a correlated response to selection for some other character. Weismann's hypothesis is also consistent with the sporadic distribution of obligate asexuality, which indicates that clones have a higher rate of extinction than sexuals. Weismann's hypothesis is then discussed in light of other patterns in the distribution of sexuality versus asexuality. To account for variation in the frequency of obligate asexuality in different taxa, a simple model is developed in which this frequency is a function of three parameters: the rate of clonal origin, the initial fitness of clones when they arise, and the rate at which that fitness declines over time. Variation in all three parameters is likely to be important in explaining the distribution of obligate asexuality. Facultative asexuality also exists, and for this to be stable it seems there must be ecological differences between the sexual and asexual propagules as well as genetic differences. Finally, the timing of sex in cyclical parthenogens is most likely set to minimize the opportunity costs of sex. None of these patterns contradict Weismann's hypothesis, but they do show that many additional principles unrelated to the function of sex are required to fully explain its distribution. Weismann's hypothesis is also consistent with what we know about the mechanics and molecular genetics of recombination, in particular the tendency for chromatids to recombine with a homolog rather than a sister chromatid at meiosis, which is opposite to what they do during mitosis. However, molecular genetic studies have shown that cis-acting sites at which recombination is initiated are lost by gene conversion as a result, a factor that can be expected to affect many fine details in the evolution of recombination. In summary, although Weismann's hypothesis must be considered the leading candidate for the function of sex and recombination, nevertheless, many additional principles are needed to fully account for their evolution.

Biological Evolution↗
PubMed2001

Selection for recombination in small populations.

The reasons that sex and recombination are so widespread remain elusive. One popular hypothesis is that sex and recombination promote adaptation to a changing environment. The strongest evidence that increased recombination may evolve because recombination promotes adaptation comes from artificially selected populations. Recombination rates have been found to increase as a correlated response to selection on traits unrelated to recombination in several artificial selection experiments and in a comparison of domesticated and nondomesticated mammals. There are, however, several alternative explanations for the increase in recombination in such populations, including two different evolutionary explanations. The first is that the form of selection is epistatic, generating linkage disequilibria among selected loci, which can indirectly favor modifier alleles that increase recombination. The second is that random genetic drift in selected populations tends to generate disequilibria such that beneficial alleles are often found in different individuals; modifier alleles that increase recombination can bring together such favorable alleles and thus may be found in individuals with greater fitness. In this paper, we compare the evolutionary forces acting on recombination in finite populations subject to strong selection. To our surprise, we found that drift accounted for the majority of selection for increased recombination observed in simulations of small to moderately large populations, suggesting that, unless selected populations are large, epistasis plays a secondary role in the evolution of recombination.

Animals↗
PubMed2005

Rep helicase suppresses short-homology-dependent illegitimate recombination in Escherichia coli.

To study roles of Rep helicase in short-homology-dependent illegitimate recombination, we examined the effect of a rep mutation on illegitimate recombination and found that the frequency of spontaneous illegitimate recombination is enhanced by the rep mutation. In addition, illegitimate recombination was synergistically enhanced by the rep mutation and UV irradiation, showing that Rep helicase plays a role in suppression of spontaneous as well as UV-induced illegitimate recombination. The defect in RecQ helicase also has a synergistic effect on the increased illegitimate recombination in the rep mutant. It was also found that the illegitimate recombination induced by the rep mutation is independent of the RecA function with or without UV irradiation. Nucleotide sequence analyses of the recombination junctions showed that the illegitimate recombination induced by the rep mutation mostly takes place between short homologous sequences. Based on the fact that the defect of Rep helicase induces replication arrest during replication, resulting in the formation of DNA double-strand breaks, we propose a model for illegitimate recombination, in which double-strand breaks caused by defect of Rep helicase promotes illegitimate recombination via short-homology-dependent-end-joining. In addition, the mechanism of synergistic action between the rep mutation and UV irradiation on illegitimate recombination is discussed.

Adenosine Triphosphatases↗
PubMed2005

Effects of recJ, recQ, and recFOR mutations on recombination in nuclease-deficient recB recD double mutants of Escherichia coli.

The two main recombination pathways in Escherichia coli (RecBCD and RecF) have different recombination machineries that act independently in the initiation of recombination. Three essential enzymatic activities are required for early recombinational processing of double-stranded DNA ends and breaks: a helicase, a 5'-->3' exonuclease, and loading of RecA protein onto single-stranded DNA tails. The RecBCD enzyme performs all of these activities, whereas the recombination machinery of the RecF pathway consists of RecQ (helicase), RecJ (5'-->3' exonuclease), and RecFOR (RecA-single-stranded DNA filament formation). The recombination pathway operating in recB (nuclease-deficient) mutants is a hybrid because it includes elements of both the RecBCD and RecF recombination machineries. In this study, genetic analysis of recombination in a recB (nuclease-deficient) recD double mutant was performed. We show that conjugational recombination and DNA repair after UV and gamma irradiation in this mutant are highly dependent on recJ, partially dependent on recFOR, and independent of recQ. These results suggest that the recombination pathway operating in a nuclease-deficient recB recD double mutant is also a hybrid. We propose that the helicase and RecA loading activities belong to the RecBCD recombination machinery, while the RecJ-mediated 5'-->3' exonuclease is an element of the RecF recombination machinery.

Adenosine Triphosphatases↗
PubMed1983

Quantitation of a simian virus 40 nonhomologous recombination pathway.

We describe an infectious-center in situ plaque hybridization procedure which quantitates simian virus 40 (SV40) nonhomologous recombination in terms of the number of recombinant-producing cells in the DNA transfected cell population. Using this assay to measure the efficiency of recombination with SV40 DNA in permissive monkey BSC-1 cells, we found that: (i) over a range of DNA concentrations, polyomavirus DNA (which is partially homologous to SV40 DNA) cannot be distinguished from nonhomologous phi X174 RF1 DNA with respect to its ability to recombine with SV40 DNA; (ii) at defined DNA concentrations, polyomavirus and phi X174 RF1 DNA compete with each other for recombination with SV40 DNA; (iii) virtually all segments of the phi X174 genome recombine, apparently at random, with SV40 DNA; (iv) the frequency of recombinant-producing cells, among the successfully transfected (virion-producing) cells, depends upon the input SV40 DNA concentration in the transfection solution; and (v) replication-defective SV40 mutant DNAs compete with wild-type SV40 DNA for recombination with phi X174 RF1 DNA. From these observations, we conclude that the efficiency of recombination with SV40, in the system under study, is unaffected by nucleotide sequence homology and that a limiting stage in the recombination pathway occurs before SV40 DNA replication. Comparison of the dependency of recombination on initial SV40 DNA concentration with the dependency on initial phi X174 RF1 DNA concentration indicates that SV40 DNA sequences are a controlling factor in the nonhomologous recombination pathway.

Animals↗
PubMed1988

High levels of genetic recombination among cotransfected plasmid DNAs in poxvirus-infected mammalian cells.

The frequency of recombination between transfected plasmid DNAs was measured by using cultured cells infected with a variety of poxviruses. Plasmid derivatives of pBR322 containing XhoI linker insertion mutations in the tetracycline gene were used to assess recombination frequencies in rabbit cells infected with the leporipoxviruses Shope fibroma virus and myxoma virus and the orthopoxvirus vaccinia virus. Recombination frequencies were calculated by Southern blotting, which detects novel plasmid restriction fragments generated by genetic recombination, and by a plasmid rescue procedure in which the reconstruction of an intact tetracycline gene in the transfected rabbit cell was monitored by transformation back into Escherichia coli. The highest recombination frequencies were measured in cells infected with Shope fibroma virus and myxoma virus, and a minimum recombination frequency of at least one recombination event per 7 kilobases was calculated within 24 h posttransfection under these conditions. The deduced recombination frequency in vaccinia virus-infected cells was at least fivefold lower and was not detectable in mock-infected cells, suggesting that the induced recombination activity detected by these methods was under viral control. The results of kinetic studies, analysis with methylation-sensitive restriction enzymes, and the use of phosphonoacetic acid, a specific inhibitor of poxvirus DNA polymerase, indicated that recombination between transfecting DNAs occurred concomitantly with DNA replication but that the two processes could be partially uncoupled. We conclude that the dramatic expansion of recombination activities in the cytoplasm of poxvirus-infected cells is virus specific and offers a good model system with which to analyze the mechanism of recombination in a eucaryotic environment.

Animals↗
PubMed1993

Epstein-Barr virus recombinants from overlapping cosmid fragments.

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, Viral↗
PubMed1998

Characterization of wild-type adeno-associated virus type 2-like particles generated during recombinant viral vector production and strategies for their elimination.

The pSub201-pAAV/Ad plasmid cotransfection system was developed to eliminate homologous recombination which leads to generation of the wild-type (wt) adeno-associated virus type 2 (AAV) during recombinant vector production. The extent of contamination with wt AAV has been documented to range between 0.01 and 10%. However, the precise mechanism of generation of the contaminating wt AAV remains unclear. To characterize the wt AAV genomes, recombinant viral stocks were used to infect human 293 cells in the presence of adenovirus. Southern blot analyses of viral replicative DNA intermediates revealed that the contaminating AAV genomes were not authentic wt but rather wt AAV-like sequences derived from recombination between (i) AAV inverted terminal repeats (ITRs) in the recombinant plasmid and (ii) AAV sequences in the helper plasmid. Replicative AAV DNA fragments, isolated following amplification through four successive rounds of amplification in adenovirus-infected 293 cells, were molecularly cloned and subjected to nucleotide sequencing to identify the recombinant junctions. Following sequence analyses of 31 different ends of AAV-like genomes derived from two different recombinant vector stocks, we observed that all recombination events involved 10 nucleotides in the AAV D sequence distal to viral hairpin structures. We have recently documented that the first 10 nucleotides in the D sequence proximal to the AAV hairpin structures are essential for successful replication and encapsidation of the viral genome (X.-S. Wang et al., J. Virol. 71:3077-3082, 1997), and it was noteworthy that in each recombinant junction sequenced, the same 10 nucleotides were retained. We also observed that adenovirus ITRs in the helper plasmid were involved in illegitimate recombination with AAV ITRs, deletions of which significantly reduced the extent of wt AAV-like particles. Furthermore, the combined use of recombinant AAV plasmids lacking the distal 10 nucleotides in the D sequence and helper plasmids lacking the adenovirus ITRs led to complete elimination of replication-competent wt AAV-like particles in recombinant vector stocks. These strategies should be useful in producing clinical-grade AAV vectors suitable for human gene therapy.

Base Sequence↗
PubMed2002

Human immunodeficiency virus type 1 recombination: rate, fidelity, and putative hot spots.

Previously, we reported that human immunodeficiency virus type 1 (HIV-1) recombines approximately two to three times per genome per replication cycle, an extremely high rate of recombination given the relatively small genome size of HIV-1. However, a recombination hot spot involving sequence of nonretroviral origin was identified in the vector system utilized, raising the possibility that this hot spot skewed the rate of recombination, and the rate of recombination observed was an overestimation. To address this issue, an HIV-1-derived vector system was used to examine the rate of recombination between autologous HIV-1 sequences after restricting replication to a single cycle in the absence of this hot spot. Viral DNA and RNA were analyzed by a combination of the heteroduplex tracking assay, restriction enzyme analysis, DNA sequencing, and reverse transcription-PCR. The results indicate that HIV-1 undergoes recombination at a minimum rate of 2.8 crossovers per genome per cycle. Again, this is a very high rate given the small size of the HIV-1 genome. The results also suggested that there might be local hot spots of recombination at different locations throughout the genome since 13 of the 33 strand transfers identified by DNA sequencing shared the same site of recombination with one or two other clones. Furthermore, identification of crossover segments also allowed examination of mutations at the point of recombination, since it has been predicted from some studies of cell-free systems that mutations may occur with a frequency of 30 to 50% at crossover junctions. However, DNA sequence analysis of crossover junctions indicated that homologous recombination during viral replication was not particularly mutagenic, indicating that there are other factors or conditions not yet reproduced in cell-free systems which contribute to fidelity during retroviral recombination.

Cell Line↗
PubMed2003

High rates of human immunodeficiency virus type 1 recombination: near-random segregation of markers one kilobase apart in one round of viral replication.

One of the genetic consequences of packaging two copies of full-length viral RNA into a single retroviral virion is frequent recombination during reverse transcription. Many of the currently circulating strains of human immunodeficiency virus type 1 (HIV-1) are recombinants. Recombination can also accelerate the generation of multidrug-resistant HIV-1 and therefore presents challenges to effective antiviral therapy. In this study, we determined that HIV-1 recombination rates with markers 1.0, 1.3, and 1.9 kb apart were 42.4, 50.4, and 47.4% in one round of viral replication. Because the predicted recombination rate of two unlinked markers is 50%, we conclude that markers 1 kb apart segregated in a manner similar to that for two unlinked markers in one round of retroviral replication. These recombination rates are exceedingly high even among retroviruses. Recombination rates of markers separated by 1 kb are 4 and 4.7% in one round of spleen necrosis virus and murine leukemia virus replication, respectively. Therefore, HIV-1 recombination can be 10-fold higher than that of other retroviruses. Recombination can be observed only in the proviruses derived from heterozygous virions that contain two genotypically different RNAs. The high rates of HIV-1 recombination observed in our studies also indicate that heterozygous virions are formed efficiently during HIV-1 replication and most HIV-1 virions are capable of undergoing recombination. Our results demonstrate that recombination is an effective mechanism to break the genetic linkage between neighboring sequences, thereby reassorting the HIV-1 genome and increasing the diversity in the viral population.

Cells, Cultured↗
PubMed1993

Homologous and illegitimate recombination in developing Xenopus oocytes and eggs.

Exogenous DNA is efficiently recombined when injected into the nuclei of Xenopus laevis oocytes. This reaction proceeds by a homologous resection-annealing mechanism which depends on the activity of a 5'-->3' exonuclease. Two possible functions for this recombination activity have been proposed: it may be a remnant of an early process in oogenesis, such as meiotic recombination or amplification of genes coding for rRNA, or it may reflect materials stored for embryogenesis. To test these hypotheses, recombination capabilities were examined with oocytes at various developmental stages. Late-stage oocytes performed only homologous recombination, whereas the smallest oocytes ligated the restriction ends of the injected DNA but supported no homologous recombination. This transition from ligation to recombination activity was also seen in nuclear extracts from these same stages. Exonuclease activity was measured in the nuclear extracts and found to be low in early stages and then to increase in parallel with recombination capacity in later stages. The accumulation of exonuclease and recombination activities during oogenesis suggests that they are stored for embryogenesis and are not present for oocyte-specific functions. Eggs were also tested and found to catalyze homologous recombination, ligation, and illegitimate recombination. Retention of homologous recombination in eggs is consistent with an embryonic function for the resection-annealing mechanism. The observation of all three reactions in eggs suggests that multiple pathways are available for the repair of double-strand breaks during the extremely rapid cleavage stages after fertilization.

Animals↗