Low Recombination Frequency Research Articles

DNA oligonucleotide-assisted genetic manipulation increases transformation and homologous recombination efficiencies: Evidence from gene targeting of Dictyostelium discoideum

Artificial gene alteration by homologous recombination in living cells, termed gene targeting, presents fundamental and considerable knowledge of in vivo gene function. In principle, this method can possibly be applied to any type of genes and transformable cells. However, its success is limited due to a low frequency of homologous recombination between endogenous targeted gene and exogenous transgene. Here, we describe a general gene-targeting method in which co-transformation of DNA oligonucleotides (oligomers) could significantly increase the homologous recombination frequency and transformation efficiency. The oligomers were simply designed such that they were identical to both the ends of the homologous flanking regions of the targeting construct. Using this strategy, both targeted alleles of diploid cells were simultaneously replaced in a single transformation procedure. Thus, the simplicity and versatility of this method applicable to any type of cell may increase the application of gene targeting.

Distribution of mating types and genetic diversity induced by sexual recombination in Setosphaeria turcica in northern China

Mature ascocarps and ascospores in the heterothallic ascomycete fungus, Setosphaeria turcica, were successfully produced in Sach’s medium with barley culm as the mating stimulator after four weeks’ coincubation of two opposite mating type isolates at 25°C in darkness. A single isolate could not produce ascospores or ascocarps. The ascocarps were produced on the exposed surface and embedded parts of barley culm or in the upper layer of the medium. The asci linked themselves to ascocarp with their short handles and assembled at the bottom of the ascocarp. Many asci had four to six colorless mature ascospores with one to six septa. But asci with eight ascospores were also found. Using isolate 9914 and isolate 9961 as standard testers for mating types (MAT1 and MAT2), respectively, 94 isolates of S. turcica collected from northern China in 1999, 2003, and 2004 were grouped into three mating types: MAT1 (53 isolates), MAT2 (31 isolates) and MAT12 (10 isolates). The MAT12 isolates, which were first found in China, were compatible with not only MAT1 isolates but also MAT2 isolates. No MAT12 isolates were found in 1999, but 2 MAT12 isolates and 8 MAT12 isolates were found in 2001 and 2003, respectively. The geographic distribution of different mating types was unequal among locations. Generally the frequency of MAT1 was significantly higher than that of MAT2 and MAT12. The unequal distribution of mating types suggested a low frequency of genetic recombination. The pathogenicity of different mating type isolates was tested on the susceptible corn inbred B37 and the results revealed that the disease latency period, disease incidence, lesion area and conidia production were not significantly different among the three mating type groups. However, the pathogenicity of the progeny isolates of isolate 99-12 (MAT2, race 1) and isolate 99-15 (MAT1, race 0) was significantly different from the parent isolates, isolate 99-12 and isolate 99-15, suggesting that sexual recombination could cause significantly virulence variation in S. turcica. Random amplification of polymorphic DNA (RAPD) analysis also revealed high genotype diversity among the progeny isolates, indicating that the sexual recombination could also produce significant genetic variation in the fungal pathogen.

Analysis of the variability of CMV strains in the RL11D domain of the RL11 multigene family

Human cytomegalovirus (CMV) contains one of the largest genomes within the herpesvirus family and includes 12 multigene families. One of these is the RL11 family, whose members encode a characteristic domain, called RL11D. In the present study, the sequence variability of RL11D within the UL1, UL4, UL6, UL7, and UL10 genes of the RL11 family was investigated. For this purpose, these genes were analyzed in 70 clinical isolates obtained from urine, bronchoalveolar lavage, and blood of different patients. Substantial genetic variability among the clinical isolates was observed in all five genes analyzed. Based on phylogenetic analysis of variations in RL11D, the clinical isolates could be classified into three genotypes for UL1, 7, and 10 and, four genotypes for UL4 and 6. Further analysis showed statistically significant linkages between the following pairs of genes: UL6/UL7, UL4/UL7, UL1/UL4, and UL4/UL6. The data show that CMV strains exhibit a high interstrain variability in the RL11D domain of various RL11 family genes. Sequence variations, however, can be clearly grouped into a limited number of distinct genotypes. The genetic linkages found probably reflect a low frequency of recombination between genes that are arranged in close proximity on the viral genome.

Molecular identification of Pm12-carrying introgression lines in wheat using genomic and EST-SSR markers

Pm12, transferred from Aegilops speltoides (2n = 2x = 14, genome SS) to wheat, confers effective resistance to powdery mildew worldwide. By applying bulked segregant analysis in a BC3F2 segregating population consisting of 305 plants, 18 wheat genomic and EST-SSR markers linked to the resistance gene were identified. Pm12 was located in the 6SS portion of the T6BS-6SS.6SL translocation chromosome based on the physical bin positions of the genomic and EST-SSR markers in the Chinese Spring group six deletion stocks and their linkage relationship to the resistance gene. Twenty eight recombinants among 305 F2 plants indicated a low frequency of recombination between the alien chromosome segment and wheat chromosome 6B. Since recombination events occurred on both sides of Pm12, the materials generated provide opportunities for further reduction of alien chromatin by intercrossing selected individuals and using markers to select the required plants.

The Rate, Not the Spectrum, of Base Pair Substitutions Changes at a GC-Content Transition in the Human NF1 Gene Region: Implications for the Evolution of the Mammalian Genome Structure

The human genome is composed of long stretches of DNA with distinct GC contents, called isochores or GC-content domains. A boundary between two GC-content domains in the human NF1 gene region is also a boundary between domains of early- and late-replicating sequences and of regions with high and low recombination frequencies. The perfect conservation of the GC-content distribution in this region between human and mouse demonstrates that GC-content stabilizing forces must act regionally on a fine scale at this locus. To further elucidate the nature of these forces, we report here on the spectrum of human SNPs and base pair substitutions between human and chimpanzee. The results show that the mutation rate changes exactly at the GC-content transition zone from low values in the GC-poor sequences to high values in GC-rich ones. The GC content of the GC-poor sequences can be explained by a bias in favor of GC > AT mutations, whereas the GC content of the GC-rich segment may result from a fixation bias in favor of AT > GC substitutions. This fixation bias may be explained by direct selection by the GC content or by biased gene conversion.

Constraints to Genetic Exchange Support Gene Coadaptation in a Tripartite RNA Virus

Genetic exchange by recombination, or reassortment of genomic segments, has been shown to be an important process in RNA virus evolution, resulting often in important phenotypic changes affecting host range and virulence. However, data from numerous systems indicate that reassortant or recombinant genotypes could be selected against in virus populations and suggest that there is coadaptation among viral genes. Little is known about the factors affecting the frequency of reassortants and recombinants along the virus life cycle. We have explored this issue by estimating the frequency of reassortant and recombinant genotypes in experimental populations of Cucumber mosaic virus derived from mixed infections with four different pairs of isolates that differed in about 12% of their nucleotide sequence. Genetic composition of progeny populations were analyzed at various steps of the virus life cycle during host colonization: infection of leaf cells, cell-to-cell movement within the inoculated leaf, encapsidation of progeny genomes, and systemic movement to upper noninoculated leaves. Results indicated that reassortant frequencies do not correspond to random expectations and that selection operates against reassortant genotypes. The intensity of selection, estimated through the use of log-linear models, increased as host colonization progressed. No recombinant was detected in any progeny. Hence, results showed the existence of constraints to genetic exchange linked to various steps of the virus life cycle, so that genotypes with heterologous gene combinations were less fit and disappeared from the population. These results contribute to explain the low frequency of recombinants and reassortants in natural populations of many viruses, in spite of high rates of genetic exchange. More generally, the present work supports the hypothesis of coadaptation of gene complexes within the viral genomes.

Mapping of the juxtacentromeric heterochromatin-euchromatin frontier of human chromosome 21

Euchromatin and heterochromatin are functional compartments of the genome. However, little is known about the structure and the precise location of the heterochromatin-euchromatin boundaries in higher eukaryotes. Constitutive heterochromatin in centromeric regions is associated with (1) specific histone methylation patterns, (2) high levels of DNA methylation, (3) low recombination frequency, and (4) the repression of transcription. All of this contrasts with the permissive structure of euchromatin found along chromosome arms. On the sequence level, the transition between these two domains consists most often of patchworks of segmental duplications. We present here a comprehensive analysis of gene expression, DNA methylation in CpG islands, distribution of histone isoforms, and recombination activity for the juxtacentromeric (or pericentromeric) region of the long arm of human chromosome 21. We demonstrate that most HapMap data are reliable within this region. We show that high linkage disequilibrium between pairs of SNPs extends 719-737 kb from the centromeric alpha-satellite. In the same region we find a peak of histone isoforms H3K9Me3 and H3K27Me (715-822 kb distal to the alpha-satellite). In normal somatic cells, CpG islands proximal to this peak are highly methylated, whereas distal CpG islands are not or very little methylated. This methylation profile undergoes dramatic changes in cancer cells and during spermatogenesis. As a consequence, transcription from heterochromatic genes is activated in the testis, and aberrant gene activation can occur during neoplastic transformation. Our data indicate that the frontier between the juxtacentromeric heterochromatic domain and euchromatic domain of the long arm of chromosome 21 is marked by a heterochromatic peak located approximately 750 kb distal to the alpha-satellite.

SNP identification, linkage disequilibrium, and haplotype analysis for a 200-kb genomic region in a Korean population

Understanding patterns of linkage disequilibrium (LD) across genomes may facilitate association mapping studies to localize genetic variants influencing complex diseases, a recognition that led to the International Haplotype Mapping Project (HapMap). Divergent patterns of haplotype frequency and LD across global populations require that the HapMap database be supplemented with haplotype and LD data from additional populations. We conducted a pilot study of the LD and haplotype structure of a genomic region in a Korean population. A total of 165 SNPs were identified in a 200-kb region of 22q13.2 by direct sequencing. Unphased genotype data were generated for 76 SNPs in 90 unrelated Korean individuals. LD, haplotype diversity, and recombination rates were assessed in this region and compared with the HapMap database. The pattern of LD and haplotype frequencies of Korean samples showed a high degree of similarity with Japanese data. There was a strong correlation between high LD and low recombination frequency in this region. We found considerable similarities in local LD patterns between three Asian populations (Han Chinese, Japanese, and Korean) and the CEPH population. Haplotype frequencies were, however, significantly different between them. Our results should further the understanding of distinctive Korean genomic features and assist in designing appropriate association studies.

A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors

The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.

Identification of low frequency knockout mutants in Dictyostelium discoideum created by single or double homologous recombination

Generation and characterization of knockout clones is a widely used approach to evaluate the specific function of a gene product in Dictyostelium discoideum. The mutant clones are generally obtained by double homologous recombination in the target gene. A frequent limitation to obtaining mutants is the low frequency of homologous recombination. Here we present an easy method to identify rare mutants, based on PCR analysis of pools of clones. This method also allows the isolation of functional knockout mutants created by a single homologous recombination event, which can be more frequent than a double recombination event.

An isochore transition zone in the NF1 gene region is a conserved landmark of chromosome structure and function

The mammalian genome is organized as a mosaic of isochores, stretches of DNA with a distinct sequence composition. Isochores form the basis of the chromosomal banding pattern, which is tightly correlated with a number of structural and functional features. We have recently demonstrated that the transition from a GC-poor isochore to a GC-rich one in the NF1 gene region occurs within 5 kb and demarcates genomic regions with high and low recombination frequency. We now report that the same transition zone separates early replicating from late replicating chromatin on the molecular level. At the isochore transition the replication fork is stalled in mid-S phase and can be visualized by fiber-FISH techniques as a Y-shaped structure. The switch in GC content and in replication timing is conserved between human and mouse, emphasizing the importance of the transition zones as landmarks of chromosome organization and function.

Reduced chromosome association between the short arms of 5H homologues in Hordeum vulgare L. at metaphase I

Rod or ring bivalents at metaphase I (MI) reflect whether crossing-over has occurred on one or both pairs of chromosome arms, respectively, during meiotic prophase (pachytene). In cultivated barley (Hordeum vulgare) the highest frequencies of rod bivalents were found for satellite chromosomes (either 5H or 6H). To distinguish between chromosome 5H and 6H associations, a recombinant line derived from an H. vulgare × H. bulbosum hybrid with a hemizygous introgression from H. bulbosum on the short arm of chromosome 6H was probed with 45S rDNA and pSc119.2 using fluorescent in situ hybridization and compared with non-recombinant H. vulgare. The frequencies of rod bivalents, caused by failure of the short arms of 5H chromosomes (5HS) to associate during meiosis in the recombinant line, were similar to the frequencies of rod bivalents involving the satellite chromosomes in H. vulgare. Hence, rod bivalent formation in H. vulgare satellite chromosomes is probably based on reduced association of 5HS, corresponding to a low frequency of genetic recombination. 5HS is, therefore, more likely to show linkage disequilibrium than the other chromosome arms.

Rare creation of recombinant mtDNA haplotypes in mammalian tissues.

The problem of whether recombinant mtDNAs are created in mammalian cells has been controversial for many years. We show convincing evidence for the very rare creation of recombinant mtDNA haplotypes by isolating human somatic hybrid cells and by generating mice carrying two different mtDNA haplotypes. To avoid misinterpretation of PCR-jumping products as recombinants, we used purified mtDNAs for cloning and sequencing. The results showed that only three of 318 clones of mtDNA purified from mouse tissues corresponded to recombinant mtDNA haplotypes, whereas no recombinants were found in human somatic hybrid cells. Such an extremely low frequency of mtDNA recombination does not require any revision of important concepts on human evolution that are based on its absence. Considering the high concentration of reactive oxygen species around the mtDNA and its frequent strand breakage, recombinant clones would correspond to gene conversion products created by repair of nucleotide mismatches.

Highly efficient endogenous human gene correction using designed zinc-finger nucleases

Permanent modification of the human genome in vivo is impractical owing to the low frequency of homologous recombination in human cells, a fact that hampers biomedical research and progress towards safe and effective gene therapy. Here we report a general solution using two fundamental biological processes: DNA recognition by C2H2 zinc-finger proteins and homology-directed repair of DNA double-strand breaks. Zinc-finger proteins engineered to recognize a unique chromosomal site can be fused to a nuclease domain, and a double-strand break induced by the resulting zinc-finger nuclease can create specific sequence alterations by stimulating homologous recombination between the chromosome and an extrachromosomal DNA donor. We show that zinc-finger nucleases designed against an X-linked severe combined immune deficiency (SCID) mutation in the IL2Rgamma gene yielded more than 18% gene-modified human cells without selection. Remarkably, about 7% of the cells acquired the desired genetic modification on both X chromosomes, with cell genotype accurately reflected at the messenger RNA and protein levels. We observe comparably high frequencies in human T cells, raising the possibility of strategies based on zinc-finger nucleases for the treatment of disease.

Viral RNA-directed RNA Polymerases Use Diverse Mechanisms to Promote Recombination between RNA Molecules

An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Qbeta phage replicase and the poliovirus 3Dpol protein, to promote RNA recombination through a primer extension mechanism. The substrates of recombination were fragments of complementary strands of a Qbeta phage-derived RNA, such that if aligned at complementary 3'-termini and extended using one another as a template, they would produce replicable molecules detectable as RNA colonies grown in a Qbeta replicase-containing agarose. The results show that while 3Dpol efficiently extends the aligned fragments to produce the expected homologous recombinant sequences, only nonhomologous recombinants are generated by Qbeta replicase at a much lower yield and through a mechanism not involving the extension of RNA primers. It follows that the mechanisms of RNA recombination by poliovirus and Qbeta RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Qbeta replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Qbeta phage.

Efficient gene disruption in the koji-mold Aspergillus sojae using a novel variation of the positive-negative method.

When no phenotypic screen is available, gene disruption in the koji-mold Aspergillus sojae is a time-consuming process, owing to the low frequency of homologous recombination. To achieve efficient gene disruption in the koji-mold, we developed a novel positive-negative selection method to enrich for homologous recombinants. The pyrG gene from A. sojae was used as a positive selection marker for transformants, and the oliC31 gene of A. nidulans, which codes for a mutant form of subunit 9 of the F1FO-ATPase, was employed as a negative selection marker to facilitate elimination of non-homologous recombinants among the transformants. The positive-negative selection markers, in combination with a pyrG deletion strain as a host, enabled enrichment for homologous recombinants, and disruption of the genes niaD, areA and tannase was successfully demonstrated. In order to examine whether the positive-negative selection technique is effective for targeting any locus, even in the absence of information on gene function or phenotype, we attempted to disrupt the aflR gene of A. sojae, which codes for a putative transcription factor for the aflatoxin biosynthetic pathway, using the method. Despite the fact that this gene is not transcribed in A. sojae, aflR disruptants were efficiently obtained, suggesting that the method is indeed capable of targeting any locus, without additional ectopic integration, and is thus applicable for functional genomics studies in filamentous fungi, including A. sojae.

300. Targeted Gene Correction in Mouse Embryonic Stem Cells Using Helper-Dependent Adenoviral Vectors

Integration of a therapeutic gene into random sites on host chromosomes could potentially lead to serious problems such as cellular transformation and gene silencing. Gene targeting, the process of homologous recombination between exogenous and chromosomal DNA, would achieve safe, transcriptionally active and stable expression of an integrated gene. However, the low frequency of homologous recombination in mammalian cells prevents therapeutic application of this strategy. Recombinant adenoviral vectors (AdVs) might be an ideal vehicle for homologous recombination because of their highly efficient delivery of double-stranded DNA genomes into the nucleus of almost any cell type. We examined the ability of AdVs to correct an insertional mutation in exon 3 of the hypoxanthine phosphoribosyl transferase (Hprt) locus in male mouse embryonic stem (ES) cells. The cells were infected with four types of Hprt targeting AdVs (E1-deleted vectors with 1.7-kb and 6.7-kb wild-type Hprt sequences and helper-dependent (HD) AdVs with 6.7-kb and 18.6-kb wild-type Hprt sequences) at multiplicities of infection (MOI) of 10, 100 and 1000 genomes per cell. The ES cells that might have undergone homologous recombination and thus restored their HPRT activity were detected by HAT selection. The HD AdVs also encode the beta-geo marker gene, which flanks the Hprt sequence. Therefore, randomly integrated vectors could also be detected by G418 selection. The frequency of homologous recombination in cells infected with HD AdVs with an 18.6-kb homology was nearly 0.02%, an 800-fold increase compared to those cells infected by an E1-deleted vector with the 6.7-kb homology. No homologous recombination was detected with the E1-deleted vector with the 1.7-kb Hprt sequence. In contrast to the homologous recombination frequency, which showed an MOI-dependent increase, the random integration frequency reached maximum levels at an MOI of 100 and was similar between the HD AdVs. The ratio of random to targeted integration was 61% with the 18.6-kb HD AdV at an MOI of 1000. Treatment of the infected cells with trichostatin A, a histone deacetylase inhibitor, enhanced only the random integration frequency but had no effect on the frequency of targeted integration. The majority of HAT-resistant cell clones showed a pattern of faithful gene targeting. No randomly integrated vectors were observed in the cells with targeted vector integration, as analyzed by PCR. These findings suggest that adenovirus-mediated homologous recombination might be applicable for ex vivo gene therapy.

SCAR and CAPS mapping of CRb, a gene conferring resistance to Plasmodiophora brassicae in Chinese cabbage ( Brassica rapa ssp. pekinensis).

Clubroot disease, caused by Plasmodiophora brassicae Wor., is highly damaging for Chinese cabbage. The CR (clubroot resistant) Shinki DH (doubled haploid) line of Chinese cabbage carries a single dominant gene, CRb, which confers resistance to the P. brassicae races 2, 4, and 8. An F(2) population derived from a cross between the CR Shinki DH line and a susceptible line, 94SK, was used to map the CRb gene. Inoculation of F(3) families with SSI (single-spore isolate) resulted in a 1:2:1 segregation ratio. Use of the AFLP technique combined with bulked segregant analysis allowed five co-dominant AFLP markers, and four and seven dominant AFLP markers linked in coupling and repulsion, respectively, to be identified. Six of the 16 AFLP markers showing low frequencies of recombination with the CRb locus among 138 F(2) lines were cloned. A reliable conversion procedure allowed five AFLP markers to be successfully converted into CAPS and SCAR markers. An F(2) population (143 plants) was analyzed with these markers and a previously identified SCAR marker, and a genetic map around CRb covering a total distance of 6.75 cM was constructed. One dominant marker, TCR09, was located 0.78 cM from CRb. The remaining markers (TCR05, TCR01, TCR10, TCR08, and TCR03) were located on the other side of CRb, and the nearest of these was TCR05, at a distance of 1.92 cM.

Intra-specific and inter-specific recombination of tortricid-specific granuloviruses during co-infection in insect larvae.

Intra-specific recombination between two genotypes of the Cryptophlebia leucotreta granulovirus (CrleGV), namely CV3 and CV4, was studied by mixed infection experiments of larvae of C. leucotreta, followed by in vivo cloning and DNA restriction enzyme analyses of isolated progeny viruses. As a prerequisite for these studies a comparative restriction map for of CV3 and CV4 was constructed for eight restriction enzymes. The mixed infection experiments resulted in the isolation of the recombinant CrleGV CVR, which contained restriction sites typical for both parental viruses. Inter-specific recombination between two different granulovirus species, namely CrleGV CV3 and Cydia pomonella granulovirus (CpGV), was analogously investigated by mixed infections of larvae of C. leucotreta. A survey of more than 300 isolated CrleGV and CpGV clones did not reveal any recombinant, which indicated an extremely low recombination frequency in these experiments. By using a specific PCR approach, however, chimerical fragments from the highly conserved granulin gene sequence could be observed in DNA preparations of virus progeny. Cloning and sequencing indicated recombination between CrleGV and CpGV DNA. Our results suggest that recombination between granulovirus genotypes and granulovirus species result in eventually viable viruses and may contribute to the genetic diversity in this virus group.

Recombinant bovine herpesvirus-1 expressing p23 protein of Cryptosporidium parvum induces neutralizing antibodies in rabbits.

In order to develop a vaccine against cryptosporidiosis in cattle, we constructed a recombinant bovine herpesvirus-1 (BHV-1) expressing an immunodominant surface protein, p23, of Cryptosporidium parvum sporozoites. In the recombinant virus, the p23 gene under the control of a CAG promoter and a gene coding for an enhanced green fluorescent protein were integrated into the gG gene of BHV-1. Despite a low frequency of homologous recombination, cloning of the recombinants was easy because of the specific fluorescence of the plaques formed by recombinants. These plaques were among the plaques of the nonfluorescent parental virus. All clones selected for fluorescence also contained the p23 gene. In MDBK cells infected with the recombinant BHV-1, the antibody against the p23 protein recognized the p23 protein as an approximately 23-kDa specific band in Western blotting analysis. Rabbits immunized with the recombinant produced IgG against the p23 protein. It was also demonstrated that the sera of immunized rabbits reduced infection of C. parvum sporozoites in HCT-8 cells. The serum of an immunized rabbit reduced infection compared with the normal rabbit serum control. These results indicate that the recombinant BHV-1 induces neutralizing antibodies in rabbits.