Extensive homologous chloroplast DNA recombination in the pt14 Nicotiana somatic hybrid

Summary In a previous study, six recombination sites have been confirmed in the chloroplast DNA (cpDNA) of pt14, a somatic hybrid of Nicotiana tabacum and Nicotiana plumbaginifolia. In the present study, physical mapping revealed six recombination sites in the 11.4-kb SalI fragment alone, only one of which has been previously identified. This fragment is located in the large unique region. We assume, therefore, that the pt14 cpDNA is a fine mosaic of the parental genomes with a recombination site about every 2 kb. A 748-bp region that comprised the intergenic region between ORF73 and ORF74B, and 460 bp of the petD intron have been sequenced. Parent-specific sequences in the pt14 DNA defined the regions within which recombination took place. The exact site of recombination events could not be determined because the parental sequences were identical between the polymorphic markers, and these sequences have been preserved in the pt14 line.     

Next generation synthetic vectors for transformation of the plastid genome of higher plants

Plastid transformation vectors are E. coli plasmids carrying a plastid marker gene for selection, adjacent cloning sites and flanking plastid DNA to target insertions in the plastid genome by homologous recombination. We report here on a family of next generation plastid vectors carrying synthetic DNA vector arms targeting insertions in the rbcL-accD intergenic region of the tobacco (Nicotiana tabacum) plastid genome. The pSS22 plasmid carries only synthetic vector arms from which the undesirable restriction sites have been removed by point mutations. The pSS24 vector carries a c-Myc tagged spectinomycin resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. The synthetic vectors will enable direct manipulation of passenger genes in the transformation vector targeting insertions in the rbcL-accD intergenic region that contains many commonly used restriction sites. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Extensive homologous recombination in classical swine fever virus: A re-evaluation of homologous recombination events in the strain AF407339

In this short report, the genome-wide homologous recombination events were re-evaluated for classical swine fever virus (CSFV) strain {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339. We challenged a previous study which suggested only one recombination event in {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339 based on 25 CSFV genomes. Through our re-analysis on the 25 genomes in the previous study and the 41 genomes used in the present study, we argued that there should be possibly at least two clear recombination events happening in {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339 through genome-wide scanning. The reasons for identifying only one recombination event in the previous study might be due to the limited number of available CSFV genome sequences at that time and the limited usage of detection methods. In contrast, as identified by most detection methods using all available CSFV genome sequences, two major recombination events were found at the starting and ending zones of the genome {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339, respectively. The first one has two parents {"type":"entrez-nucleotide","attrs":{"text":"AF333000","term_id":"13383931","term_text":"AF333000"}}AF333000 (minor) and {"type":"entrez-nucleotide","attrs":{"text":"AY554397","term_id":"49860681","term_text":"AY554397"}}AY554397 (major) with beginning and ending breakpoints located at 19 and 607 nt of the genome respectively. The second one has two parents {"type":"entrez-nucleotide","attrs":{"text":"AF531433","term_id":"22347661","term_text":"AF531433"}}AF531433 (minor) and {"type":"entrez-nucleotide","attrs":{"text":"GQ902941","term_id":"298113017","term_text":"GQ902941"}}GQ902941 (major) with beginning and ending breakpoints at 8397 and 11,078 nt of the genome respectively. Phylogenetic incongruence analysis using neighbor-joining algorithm with 1000 bootstrapping replicates further supported the existence of these two recombination events. In addition, we also identified additional 18 recombination events on the available CSFV strains. Some of them may be trivial and can be ignored. In conclusion, CSFV might have relatively high frequency of homologous recombination events. Genome-wide scanning of identifying recombination events should utilize multiple detection methods so as to reduce the risk of misidentification.     

Induction of homologous recombination in Saccharomyces cerevisiae

Summary We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, of the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of, homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.     

Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer

Expression of genes in plant chloroplasts provides an opportunity for enhanced production of target proteins. We report the introduction and expression of a fusion DPT protein containing immunoprotective exotoxin epitopes of Corynebacterium diphtheriae, Bordetella pertussis, and Clostridium tetani in tobacco chloroplasts. Using biolistic-mediated transformation, a plant-optimized synthetic DPT gene was successfully transferred to tobacco plastomes. Putative transplastomic T0 plants were identified by PCR, and Southern blot analysis confirmed homoplasmy in T1 progeny. ELISA assays demonstrated that the DPT protein retained antigenicity of the three components of the fusion protein. The highest level of expression in these transplastomic plants reached 0.8% of total soluble protein. To assess whether the functional recombinant protein expressed in tobacco plants would induce specific antibodies in test animals, a mice feeding experiment was conducted. For mice orally immunized with freeze-dried transplastomic leaves, production of IgG and IgA antibodies specific to each toxin were detected in serum and mucosal tissues. R. E. Soria-Guerra and A. G. Alpuche-Solís contributed equally to this work.     

Targeting human Rad51 by specific DNA aptamers induces inhibition of homologous recombination

Human Rad51 (HsRad51), a key element of the homologous recombination repair pathway, is related to the resistance of cancer cells to chemo- and radio-therapies. This protein is thus a good target for the development of anti-cancer treatments. We have searched for new inhibitors directed against HsRad51 using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach. We have selected three aptamers displaying strong effects on strand exchange activity. Analysis by circular dichroism shows that they are highly structured DNA molecules. Our results also show that they affect the first step of the strand exchange reaction by promoting the dissociation of DNA from the ATP/HsRad51/DNA complex. Moreover, these inhibitors bind only weakly to RecA, a prokaryotic ortholog of HsRad51. Both the specificity and the efficiency of their inhibition of recombinase activity offer an analytical tool based on molecular recognition and the prospect of developing new therapeutic agents.     

Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells

Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5′- and 3′-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis.     

VP8* antigen produced in tobacco transplastomic plants confers protection against bovine rotavirus infection in a suckling mouse model

Group A rotavirus is a major leading cause of diarrhea in mammalian species worldwide. In Argentina, bovine rotavirus (BRV) is the main cause of neonatal diarrhea in calves. VP4, one of the outermost capsid proteins, is involved in various virus functions. Rotavirus infectivity requires proteolytic cleavage of VP4, giving an N-terminal non-glycosilated sialic acid-recognizing domain (VP8*), and a C-terminal fragment (VP5*) that remains associated with the virion. VP8* subunit is the major determinant of the viral infectivity and one of the neutralizing antigens.In this work, the C486 BRV VP8* protein was produced in tobacco chloroplasts. Transplastomic plants were obtained and characterized by Southern blot, northern blot and western blot. VP8* was highly stable in the transplastomic leaves, and formed insoluble aggregates that were partially solubilized by sonication. The recombinant protein yield was 600 μg/g of fresh tissue (FT). Both the soluble and insoluble fractions of the VP8* plant extracts were able to induce a strong immune response in female mice as measured by ELISA and virus neutralization test. Most important, suckling mice born to immunized dams were protected against oral challenge with virulent rotavirus. Results presented here contribute to demonstrate the feasibility of using antigens expressed in transplastomic plants for the development of subunit vaccines.     

Nucleases in homologous recombination as targets for cancer therapy

Genomic DNA is constantly challenged from endogenous as well as exogenous sources. The DNA damage response (DDR) mechanism has evolved to combat these challenges and ensure genomic integrity. In this review, we will focus on repair of DNA double-strand breaks (DSB) by homologous recombination and the role of several nucleases and other recombination factors as suitable targets for cancer therapy. Their inactivation as well as overexpression have been shown to sensitize cancer cells by increasing toxicity to DNA-damaging agents and radiation or to be responsible for resistance of cancer cells. These factors can also be used in targeted cancer therapy by taking advantage of specific genetic abnormalities of cancer cells that are not present in normal cells and that result in cancer cell lethality.     

High fidelity extrachromosomal recombination and gene targeting in plants

The precision of extrachromosomal homologous recombination and gene targeting in plant cells was investigated. Recombination was directed to introns of selectable marker genes where potential changes could persist without affecting the function and therefore the selectability of the genes. Approximately 9 kb of crossover regions was rescued and sequenced. Changes were detected at a frequency below one point mutation per 1000 bp, indicating that extrachromosomal recombination and gene targeting both appear to occur with high fidelity.     

RadB acts in homologous recombination in the archaeon Haloferax volcanii,consistent with a role as recombination mediator

Homologous recombination plays a central role in the repair of double-strand DNA breaks, the restart of stalled replication forks and the generation of genetic diversity. Regulation of recombination is essential since defects can lead to genome instability and chromosomal rearrangements. Strand exchange is a key step of recombination – it is catalysed by RecA in bacteria, Rad51/Dmc1 in eukaryotes and RadA in archaea. RadB, a paralogue of RadA, is present in many archaeal species. RadB has previously been proposed to function as a recombination mediator, assisting in RadA-mediated strand exchange. In this study, we use the archaeon Haloferax volcanii to provide evidence to support this hypothesis. We show that RadB is required for efficient recombination and survival following treatment with DNA-damaging agents, and we identify two point mutations in radA that suppress the ΔradB phenotype. Analysis of these point mutations leads us to propose that the role of RadB is to act as a recombination mediator, which it does by inducing a conformational change in RadA and thereby promoting its polymerisation on DNA.     

Role of PCNA and TLS polymerases in D-loop extension during homologous recombination in humans

Homologous recombination (HR) is essential for maintaining genomic integrity, which is challenged by a wide variety of potentially lethal DNA lesions. Regardless of the damage type, recombination is known to proceed by RAD51-mediated D-loop formation, followed by DNA repair synthesis. Nevertheless, the participating polymerases and extension mechanism are not well characterized. Here, we present a reconstitution of this step using purified human proteins. In addition to Pol δ, TLS polymerases, including Pol η and Pol κ, also can extend D-loops. In vivo characterization reveals that Pol η and Pol κ are involved in redundant pathways for HR. In addition, the presence of PCNA on the D-loop regulates the length of the extension tracks by recruiting various polymerases and might present a regulatory point for the various recombination outcomes.     

Construction of the first shuttle vectors for gene cloning and homologous recombination in Mycoplasma agalactiae

Mycoplasma agalactiae is a worldwide ruminant pathogen that causes significant economic losses by inflicting contagious agalactia in sheep and goats. The development of efficient control strategies requires a better understanding of the mycoplasma factors that promote successful infection. However, lack of genetic tools has been a major impediment in studying the pathogenic mechanisms of M. agalactiae. This study describes the identification and cloning of the M. agalactiae origin of replication (oriC) in order to construct the first shuttle vectors for targeted gene disruption, gene complementation and expression studies. Additionally, this report provides the first evidence of the occurrence of homologous recombination and the functionality of heterologous tetM determinant in this pathogen.     

Single-stranded oligonucleotide-mediated gene repair in mammalian cells has a mechanism distinct from homologous recombination repair

Single-stranded DNA oligonucleotide (SSO)-mediated gene repair has great potentials for gene therapy and functional genomic studies. However, its underlying mechanism remains unclear. Previous studies from other groups have suggested that DNA damage response via the ATM/ATR pathway may be involved in this process. In this study, we measured the effect of two ATM/ATR inhibitors caffeine and pentoxifylline on the correction efficiency in SSO-mediated gene repair. We also checked their effect on double-stranded break (DSB)-induced homologous recombination repair (HRR) as a control, which is well known to be dependent on the ATM/ATR pathway. We found these inhibitors could completely inhibit DSB-induced HRR, but could only partially inhibit SSO-mediated process, indicating SSO-mediated gene repair is not dependent on the ATM/ATR pathway. Furthermore, we found that thymidine treatment promotes SSO-mediated gene repair, but inhibits DSB-induced HRR. Collectively, our results demonstrate that SSO-mediated and DSB-induced gene repairs have distinct mechanisms.     

Gene replacement by homologous recombination in plants

After the elucidation of the sequence of the yeast genome a major effort was started to elucidate the biological function of all open reading frames of this organisms by targeted gene replacement via homologous recombination. The establishment of the complete sequence of the genome of Arabidopsis thaliana would principally allow a similar approach. However, over the past dozen years all attempts to establish an efficient gene targeting technique in flowering plants were in the end not successful. In contrast, in Physcomitrella patens an efficient gene targeting procedure has been set up, making the moss a valuable model system for plant molecular biologists. But also for flowering plants recently several new approaches – some of them based on the availability of the genomic sequence of Arabidopsis – were initiated that might finally result on the set up of a general applicable technique. Beside the production of hyper-recombinogenic plants either via expression or suppression of specific gene functions or via undirected mutagenesis, the application of chimeric oligonucleotides might result in major progress.     

High-throughput cloning of human liver complete open reading frames using homologous recombination in Escherichia coli

In this article, we describe a high-throughput cloning method, seamless enzyme-free cloning (SEFC), which allows one-step assembly of DNA fragments in vivo via homologous recombination in Escherichia coli. In the method, the desired open reading frame (ORF) is amplified by use of ORF-specific primers with flanking sequences identical to the two ends of a linearized vector. The polymerase chain reaction (PCR) product and the linearized vector are then cotransformed into E. coli cells, where the ORF is incorporated into the vector in vivo. SEFC is a simple, reliable, and inexpensive method of cloning in which PCR fragments are fused into expression vectors without unwanted amino acids or extra in vitro manipulations apart from the single PCR amplification step. Using this method, we successfully cloned human liver complete ORFs into the yeast AD and DB vectors and generated a clone resource of 4964 AD-ORFs and 4676 DB-ORFs in 3 months. This approach will be useful for daily DNA cloning and for creating proteome-scale clone resources.     

Mechanisms for recombination between stably integrated vector sequences in CHO cells

Possible mechanisms for homologous recombination in CHO cells have been investigated using a stably integrated vector, pIII-14gpt. The vector contains 2 inactive neo gene fragments in tandem arrangement. Functional neo gene activity can be restored by recombination between homologous regions in the 2 fragments. Cells in which this event has taken place become resistant to the antibiotic G418. Possible mechanisms for neo gene reactivation in this system are unequal exchange between chromatids, intra-chromatidal deletion and gene conversion.

DNA from a total of 74 G418-resistant cell clones have been isolated, and analyzed on Southern blots using neo-specific probes. Rearrangements of neo-specific restriction fragments were found to have occurred in all cell clones. In 50% of the revertants, these rearrangements can be explained by a deletion which brings the complementary regions in the 2 neo gene gragments together.

One single revertant (1.3%) shows a possible gene conversion event. The other isolated revertants (about 48%) contain more complex rearrangements. These results indicate that the predominating recombination mechanism for reactivation of the neo gene in this system is either a deletion within a chromatid or an unequal exchange between sister chromatids.     

Involvement of the inverted repeat of the yeast 2-micron plasmid in Flp site-specific and RAD52-dependent homologous recombination

Site-specific recombination within the Saccharomyces cerevisiae 2-micron DNA plasmid is catalyzed by the Flp recombinase at specific Flp Recognition Target (FRT) sites, which lie near the center of two precise 599-bp Inverted Repeats (IRs). However, the role of IR DNA sequences other than the FRT itself for the function of the Flp reaction in vivo is not known. In the present work we report that recombination efficiency differs depending on whether the FRT or the entire IR serves as the substrate for Flp. We also provide evidence for the involvement of the IR in RAD52-dependent homologous recombination. In contrast, the catalysis of site-specific recombination between two FRTs does not require the function of RAD52. The efficiency of Flp site-specific recombination between two IRs cloned in the same orientation is about one hundred times higher than that obtained when only the two FRTs are present. Moreover, we demonstrate that a single IR can activate RAD52-dependent homologous recombination between two flanking DNA regions, providing new insights into the role of the IR as a substrate for recombination and a new experimental tool with which to study the molecular mechanism of homologous recombination. Received: 14 June 1999 / Accepted: 3 November 1999     

Overexpression of cyclin E does not influence homologous recombination in Chinese hamster cells

Overexpressed cyclin E in tumours is a prognosticator for poor patient outcome. Cells that overexpress cyclin E have been shown to be impaired in S-phase progression and exhibit genetic instability that may drive this subset of cancers. However, the origin for genetic instability caused by cyclin E overexpression is unknown. Homologous recombination plays an important role in S-phase progression and is also regulated by the same proteins that regulate cyclin E-associated kinase activity, i.e., p53 and p21. To test the hypothesis that overexpressed cyclin E causes genetic instability through homologous recombination, we investigated the effect of cyclin E overexpression on homologous recombination in the hprt gene in a Chinese hamster cell line. Although cyclin E overexpression shortened the G1 phase in the cell cycle as expected, we could see no change in neither spontaneous nor etoposide-induced recombination. Also, overexpression of cyclin E did not affect the repair of DNA double-strand breaks and failed to potentiate the cytotoxic effects of etoposide. Our data suggest that genetic instability caused by overexpression of cyclin E is not mediated by aberrant homologous recombination.     

Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology

Summary To determine the minimal DNA sequence homology required for recombination in Bacillus subtilis, we developed a system capable of distinguishing between homologous and illegitimate recombination events during plasmid integration into the chromosome. In this system the recombination frequencies were measured between is pE194 derivatives carrying segments of the chromosomal -gluconase gene (bglS) of various lengths and the bacterial chromosome, using selection for erythromycin resistance at the non-permissive temperature. Homologous recombination events, resulting in disruption of the bglS gene, were easily detected by a colorimetric assay for -gluconase activity. A linear dependence of recombination frequency on homology length was observed over an interval of 77 bp. It was found that approximately 70 bp of homology is required for detectable homologous recombination. Homologous recombination was not detected when only 25 by of homology between plasmid and chromosome were provided. The data indicate that homology requirements for recombination in B. subtilis differ from those in Escherichia coli.