Allelic Recombination Research Articles

Delineation of the roles of FadD22, FadD26 and FadD29 in the biosynthesis of phthiocerol dimycocerosates and related compounds in Mycobacterium tuberculosis

Phthiocerol and phthiodiolone dimycocerosates (DIMs) and phenolic glycolipids (PGLs) are complex lipids located at the cell surface of Mycobacterium tuberculosis that play a key role in the pathogenicity of tuberculosis. Most of the genes involved in the biosynthesis of these compounds are clustered on a region of the M. tuberculosis chromosome, the so‐called DIM + PGL locus. Among these genes, four ORFs encode FadD proteins, which activate and transfer biosynthetic intermediates onto various polyketide synthases that catalyze the formation of these lipids. In this study, we investigated the roles of FadD22, FadD26 and FadD29 in the biosynthesis of DIMs and related compounds. Biochemical characterization of the lipids produced by a spontaneous Mycobacterium bovis BCG mutant harboring a large deletion within fadD26 revealed that FadD26 is required for the production of DIMs but not of PGLs. Additionally, using allelic exchange recombination, we generated an unmarked M. tuberculosis mutant containing a deletion within fadD29. Biochemical analyses of this strain revealed that, like fadD22, this gene encodes a protein that is specifically involved in the biosynthesis of PGLs, indicating that both FadD22 and FadD29 are responsible for one particular reaction in the PGL biosynthetic pathway. These findings were also supported by in vitro enzymatic studies showing that these enzymes have different properties, FadD22 displaying a p‐hydroxybenzoyl‐AMP ligase activity, and FadD29 a fatty acyl‐AMP ligase activity. Altogether, these data allowed us to precisely define the functions fulfilled by the various FadD proteins encoded by the DIM + PGL cluster.

Delineation of the roles of FadD22, FadD26 and FadD29 in the biosynthesis of phthiocerol dimycocerosates and related compounds in Mycobacterium tuberculosis

Phthiocerol and phthiodiolone dimycocerosates (DIMs) and phenolic glycolipids (PGLs) are complex lipids located at the cell surface of Mycobacterium tuberculosis that play a key role in the pathogenicity of tuberculosis. Most of the genes involved in the biosynthesis of these compounds are clustered on a region of the M. tuberculosis chromosome, the so-called DIM + PGL locus. Among these genes, four ORFs encode FadD proteins, which activate and transfer biosynthetic intermediates onto various polyketide synthases that catalyze the formation of these lipids. In this study, we investigated the roles of FadD22, FadD26 and FadD29 in the biosynthesis of DIMs and related compounds. Biochemical characterization of the lipids produced by a spontaneous Mycobacterium bovis BCG mutant harboring a large deletion within fadD26 revealed that FadD26 is required for the production of DIMs but not of PGLs. Additionally, using allelic exchange recombination, we generated an unmarked M. tuberculosis mutant containing a deletion within fadD29. Biochemical analyses of this strain revealed that, like fadD22, this gene encodes a protein that is specifically involved in the biosynthesis of PGLs, indicating that both FadD22 and FadD29 are responsible for one particular reaction in the PGL biosynthetic pathway. These findings were also supported by in vitro enzymatic studies showing that these enzymes have different properties, FadD22 displaying a p-hydroxybenzoyl-AMP ligase activity, and FadD29 a fatty acyl-AMP ligase activity. Altogether, these data allowed us to precisely define the functions fulfilled by the various FadD proteins encoded by the DIM + PGL cluster.

Double Holliday junctions are intermediates of DNA break repair

Repair of DNA double-strand-breaks (DSBs) by homologous recombination is crucial for cell proliferation and tumor suppression. However, despite its importance, the molecular intermediates of mitotic DSB-repair remain undefined. The double Holliday Junction (dHJ), presupposed to be the central intermediate for more than 25 years1, has only been identified during meiotic recombination2. Moreover, evidence has accumulated for alternative, dHJ-independent mechanisms3–6, raising the possibility that dHJs are not formed during DSB-repair in mitotically cycling cells. Here we identify intermediates of DSB-repair using a budding yeast assay system designed to mimic physiological DSB repair. This system utilizes diploid cells and provides the possibility for allelic recombination either between sister-chromatids or between homologs, as well as direct comparison with meiotic recombination at the same locus. In mitotically cycling cells, we detect inter-homolog Joint Molecule (JM) intermediates whose size and strand-composition are identical to the canonical dHJ structures observed in meiosis2. However, in contrast to meiosis, JMs between sister chromatids form in preference to those between homologs. Moreover, JMs appear to represent a minor pathway of DSB repair in mitotic cells, being detected at ~10-fold lower levels (per DSB) than during meiotic recombination. Thus, although dHJs are identified as intermediates of DSB-promoted recombination in both mitotic and meiotic cells, their formation is distinctly regulated according to the specific dictates of the two cellular programs.

Mitotic inter-homologue junctions accumulate at damaged DNA replication forks in recQ mutants

Mitotic homologous recombination is utilised to repair DNA breaks using either sister chromatids or homologous chromosomes as templates. Because sister chromatids are identical, exchanges between sister chromatids have no consequences for the maintenance of genomic integrity unless they involve repetitive DNA sequences. Conversely, homologous chromosomes might differ in genetic content, and exchanges between homologues might lead to loss of heterozygosity and subsequent inactivation of functional genes. Genomic instability, caused by unscheduled recombination events between homologous chromosomes, is enhanced in the absence of RecQ DNA helicases, as observed in Bloom's cancer-prone syndrome. Here, we used two-dimensional gel electrophoresis to analyse budding yeast diploid cells that were modified to distinguish replication intermediates originating from each homologous chromosome. Therefore, these cells were suitable for analysing the formation of inter-homologue junctions. We found that Rad51-dependent DNA structures resembling inter-homologue junctions accumulate together with sister chromatid junctions at damaged DNA replication forks in recQ mutants, but not in the absence of Srs2 or Mph1 DNA recombination helicases. Inter-homologue joint molecules in recQ mutants are less abundant than sister chromatid junctions, but they accumulate with similar kinetics after origin firing under conditions of DNA damage. We propose that unscheduled accumulation of inter-homologue junctions during DNA replication might account for allelic recombination defects in recQ mutants.

Identification of Uncommon Recurrent Potocki-Lupski Syndrome-Associated Duplications and the Distribution of Rearrangement Types and Mechanisms in PTLS

Nonallelic homologous recombination (NAHR) can mediate recurrent rearrangements in the human genome and cause genomic disorders. Smith-Magenis syndrome (SMS) and Potocki-Lupski syndrome (PTLS) are genomic disorders associated with a 3.7 Mb deletion and its reciprocal duplication in 17p11.2, respectively. In addition to these common recurrent rearrangements, an uncommon recurrent 5 Mb SMS-associated deletion has been identified. However, its reciprocal duplication predicted by the NAHR mechanism had not been identified. Here we report the molecular assays on 74 subjects with PTLS-associated duplications, 35 of whom are newly investigated. By both oligonucleotide-based comparative genomic hybridization and recombination hot spot analyses, we identified two cases of the predicted 5 Mb uncommon recurrent PTLS-associated duplication. Interestingly, the crossovers occur in proximity to a recently delineated allelic homologous recombination (AHR) hot spot-associated sequence motif, further documenting the common hot spot features shared between NAHR and AHR. An additional eight subjects with nonrecurrent PTLS duplications were identified. The smallest region of overlap (SRO) for all of the 74 PTLS duplications examined is narrowed to a 125 kb interval containing only RAI1, a gene recently further implicated in autism. Sequence complexities consistent with DNA replication-based mechanisms were identified in four of eight (50%) newly identified nonrecurrent PTLS duplications. Our findings of the uncommon recurrent PTLS-associated duplication at a relative prevalence reflecting the de novo mutation rate and the distribution of 17p11.2 duplication types in PTLS reveal insights into both the contributions of new mutations and the different underlying mechanisms that generate genomic rearrangements causing genomic disorders.

Multilocus tetrasomic linkage analysis using hidden Markov chain model

The availability of reliable genetic linkage maps is crucial for functional and evolutionary genomic analyses. Established theory and methods of genetic linkage analysis have made map construction a routine exercise in diploids. However, many evolutionarily, ecologically, and/or agronomically important species are autopolyploids, with autotetraploidy being a typical example. These species undergo much more complicated chromosomal segregation and recombination at meiosis than diploids. In addition, there is evidence of polyploidy-induced and highly dynamic changes in the structure of the genome. These polysomic characteristics indicate the inappropriateness of the theory and methods of linkage analysis in diploids for use in these species and a gap in the theory and methodology of tetraploid map construction. This paper presents a theoretical model and statistical framework for multilocus linkage analysis in autotetraploids for use with dominant and/or codominant DNA molecular markers. The theory and methods incorporate the essential features of allele segregation and recombination under tetrasomic inheritance and the major challenges in statistical modeling and marker data analysis. We validated the method and explored its statistical properties by intensive simulation study and demonstrated its utility by analysis of AFLP and SSR marker data from an outbred autotetraploid potato population.

Identification of Zoonotic Genotypes of Giardia duodenalis

Giardia duodenalis, originally regarded as a commensal organism, is the etiologic agent of giardiasis, a gastrointestinal disease of humans and animals. Giardiasis causes major public and veterinary health concerns worldwide. Transmission is either direct, through the faecal-oral route, or indirect, through ingestion of contaminated water or food. Genetic characterization of G. duodenalis isolates has revealed the existence of seven groups (assemblages A to G) which differ in their host distribution. Assemblages A and B are found in humans and in many other mammals, but the role of animals in the epidemiology of human infection is still unclear, despite the fact that the zoonotic potential of Giardia was recognised by the WHO some 30 years ago. Here, we performed an extensive genetic characterization of 978 human and 1440 animal isolates, which together comprise 3886 sequences from 4 genetic loci. The data were assembled into a molecular epidemiological database developed by a European network of public and veterinary health Institutions. Genotyping was performed at different levels of resolution (single and multiple loci on the same dataset). The zoonotic potential of both assemblages A and B is evident when studied at the level of assemblages, sub-assemblages, and even at each single locus. However, when genotypes are defined using a multi-locus sequence typing scheme, only 2 multi-locus genotypes (MLG) of assemblage A and none of assemblage B appear to have a zoonotic potential. Surprisingly, mixtures of genotypes in individual isolates were repeatedly observed. Possible explanations are the uptake of genetically different Giardia cysts by a host, or subsequent infection of an already infected host, likely without overt symptoms, with a different Giardia species, which may cause disease. Other explanations for mixed genotypes, particularly for assemblage B, are substantial allelic sequence heterogeneity and/or genetic recombination. Although the zoonotic potential of G. duodenalis is evident, evidence on the contribution and frequency is (still) lacking. This newly developed molecular database has the potential to tackle intricate epidemiological questions concerning protozoan diseases.

InSaccharomyces cerevisiae, yKu and Subtelomeric Core X Sequences Repress Homologous Recombination Near Telomeres as Part of the Same Pathway

Unlike in meiosis where recombination near telomeres is repressed, subtelomeric regions appear to recombine with each other frequently in vegetative cells with no detrimental consequences. To test whether or not such recombination is prevented in the core of chromosomes for maintenance of genome stability, we measured allelic homologous recombination (HR) along chromosome arms and between different ectopic locations. We found that there is an increase of recombination at telomeres in wild-type cells compared with sequences at proximal subtelomeric and interstitial regions of the genome. We also screened for mutations that result in an increase in HR between a telomeric sequence and a more internal sequence, which normally exhibit very low rates of HR. YKU80 was hit most frequently in our screen, and we show that the yKu heterodimer specifically represses HR in the vicinity of telomeres. This repression of HR is not explained solely by the role of yKu in maintaining telomere length, silencing, or tethering to the nuclear periphery. Analysis of mutant strains harboring deleted core X sequences revealed a role for this subtelomeric element in preventing telomeric recombination. Furthermore, core X bestowed this protection as part of the same pathway as yKu. Our findings implicate a role for both yKu and core X in stabilizing the genome against recombination events involving telomeric sequences.

Cell Type–Specific Targeted Mutations of Kras and Pten Document Proliferation Arrest in Granulosa Cells versus Oncogenic Insult to Ovarian Surface Epithelial Cells

The small G-protein KRAS is crucial for mediating gonadotropin-induced events associated with ovulation. However, constitutive expression of KrasG12D in granulosa cells disrupted normal follicle development leading to the persistence of abnormal follicle-like structures containing nonmitotic cells. To determine what factors mediate this potent effect of KrasG12D, gene profiling analyses were done. We also analyzed KrasG12D;Cyp19-Cre and KrasG12;Pgr-Cre mutant mouse models that express Cre prior to or after the initiation of granulosa cell differentiation, respectively. KrasG12D induced cell cycle arrest in granulosa cells of the KrasG12D;Cyp19-Cre mice but not in the KrasG12D;Pgr-Cre mice, documenting the cell context-specific effect of KrasG12D. Expression of KrasG12D silenced the Kras gene, reduced cell cycle activator genes, and impaired the expression of granulosa cell and oocyte-specific genes. Conversely, levels of PTEN and phosphorylated p38 mitogen-activated protein kinase (MAPK) increased markedly in the mutant granulosa cells. Because disrupting Pten in granulosa cells leads to increased proliferation and survival, Pten was disrupted in the KrasG12D mutant mice. The Pten/Kras mutant mice were infertile but lacked granulosa cell tumors. By contrast, the Ptenfl/fl;KrasG12D;Amhr2-Cre mice developed aggressive ovarian surface epithelial cell tumors that did not occur in the Ptenfl/fl;KrasG12D;Cyp19-Cre or Ptenfl/fl;KrasG12D;Pgr-Cre mouse strains. These data document unequivocally that Amhr2-Cre is expressed in and mediates allelic recombination of oncogenic genes in ovarian surface epithelial cells. That KrasG12D/Pten mutant granulosa cells do not transform but rather undergo cell cycle arrest indicates that they resist the oncogenic insults of Kras/Pten by robust self-protecting mechanisms that silence the Kras gene and elevate PTEN and phosphorylated p38 MAPK.

Identification of QTL affecting seed mineral concentrations and content in the model legume Medicago truncatula

Increasing the amount of bioavailable micronutrients such as iron and zinc in plant foods for human consumption is an international goal, intended especially for developing countries where micronutrient deficiencies are an ongoing health risk. Legume seeds have the potential to provide the essential nutrients required by humans, but concentrations of several minerals are low when compared to other foods. In order to increase seed mineral concentrations, it is important to understand the genes and processes involved in mineral distribution within the plant. The main objectives of this study were to use a Medicago truncatula recombinant inbred population (Jemalong-6 x DZA 315.16) to determine loci governing seed mineral concentrations, seed mineral content, and average seed weight, and to use these loci to propose candidate genes whose expression might contribute to these traits. Ninety-three lines in 2004 and 169 lines in 2006 were grown for seed harvest and subsequent analysis of seed Ca, Cu, Fe, K, Mg, Mn, P, and Zn concentrations and content. Quantitative trait loci (QTL) cartographer was used to identify QTL using composite interval mapping (CIM). CIM identified 46 QTL for seed mineral concentration, 26 for seed mineral content, and 3 for average seed weight. At least one QTL was detected for each mineral trait, and colocation of QTL for several minerals was found in both years. Results comparing seed weight with seed mineral concentration and content QTL demonstrate that seed size can be an important determinant of seed mineral concentration. The identification, in this model legume, of transgressive segregation for nearly all the minerals suggests that allelic recombination of relevant mineral-related genes in agronomic legumes could be a successful strategy to increase seed mineral concentrations above current levels.

Allelic recombination between distinct genomic locations generates copy number diversity in human β-defensins

Beta-defensins are small secreted antimicrobial and signaling peptides involved in the innate immune response of vertebrates. In humans, a cluster of at least 7 of these genes shows extensive copy number variation, with a diploid copy number commonly ranging between 2 and 7. Using a genetic mapping approach, we show that this cluster is at not 1 but 2 distinct genomic loci approximately 5 Mb apart on chromosome band 8p23.1, contradicting the most recent genome assembly. We also demonstrate that the predominant mechanism of change in beta-defensin copy number is simple allelic recombination occurring in the interval between the 2 distinct genomic loci for these genes. In 416 meiotic transmissions, we observe 3 events creating a haplotype copy number not found in the parent, equivalent to a germ-line rate of copy number change of approximately 0.7% per gamete. This places it among the fastest-changing copy number variants currently known.

Sexual selection and intelligence: Does sexual reproduction drive the evolution of intelligence?

The basal hypothesis discussed here is the idea that brain architecture could be plastic on a very basal, genetic level due to sexual recombination and reassortment of alleles of genes related to brain development, e.g., neuronal cell adhesion molecules (NCAMs) and others. The role of sexual reassortment leads the study of brain development, species behavior and intelligence to a new version of the so-called “Red Queen Hypothesis”: using the mechanism described here, a kind of runaway selection mechanism seems to arise. Even if NCAMs are almost constant within an individual, they seem to act very differently at the population level and so the role of reassorting polymorphic NCAM- (and other) genes gets particularly clear. If several NCAM–NCAM combinations cause extreme behavior and intelligence variability in a population, these combinations also represent a use of sexual selection. This mechanism of NCAM allele assortment seems to be important for the process of speciation by mutual selection of individuals. Therefore NCAM variants and their associated behaviors are thought to be important for the development of intelligence, in that they promote the attraction of individuals with already high intelligence, leading to the speciation of super-intelligent groups.

Novel Safflower Germplasm with Increased Saturated Fatty Acid Content

ABSTRACTSafflower (Carthamus tinctorius L.) oil with high concentration of saturated fatty acids has potential applications in the food industry. So far only germplasm with increased stearic acid content (50–120 g kg−1), controlled by alleles at the locus St, has been developed. The objectives of the present research were to evaluate safflower germplasm for saturated fatty acid content, to isolate lines with increased levels of palmitic acid and stearic acid, to study the inheritance of both traits, and to evaluate the feasibility of recombining them. Germplasm evaluation and further selection led to the isolation of the line CR‐50 with increased palmitic acid content (98.2 ± 7.9 g kg−1 vs. 64.0 ± 3.4 g kg−1 in the check) and the line CR‐13 with increased stearic acid content (92.8 ± 9.2 g kg−1 vs. 22.2 ± 3.4 g kg−1 in the check). Inheritance studies including evaluation of F1, F2, and F3 seed generations from crosses with the nuclear male‐sterile line CL1, with conventional fatty acid profile, suggested that increased palmitic acid content was determined by additive alleles at a single locus Pa, whereas stearic acid content was controlled by partially recessive alleles at a single locus, probably St. Recombination of pa and st alleles produced transgressive segregants with 211.3 ± 14.6 g kg−1 saturated fatty acids, compared with 149.6 ± 14.0 g kg−1 in CR‐50, 159.7 ± 8.0 g kg−1 in CR‐13, and 89.3 ± 4.9 in the line CL1. These are the highest levels of saturated fatty acids reported so far in safflower.

Notch signaling in bile duct development: New insights raise new questions

Notch signaling in bile duct development: New insights raise new questions

The Collaborative Cross at Oak Ridge National Laboratory: developing a powerful resource for systems genetics.

Complex traits and disease comorbidity in humans and in model organisms are the result of naturally occurring polymorphisms that interact with each other and with the environment. To ensure the availability of resources needed to investigate biomolecular networks and systems-level phenotypes underlying complex traits, we have initiated breeding of a new genetic reference population of mice, the Collaborative Cross. This population has been designed to optimally support systems genetics analysis. Its novel and important features include a high level of genetic diversity, a large population size to ensure sufficient power in high-dimensional studies, and high mapping precision through accumulation of independent recombination events. Implementation of the Collaborative Cross has been ongoing at the Oak Ridge National Laboratory (ORNL) since May 2005. Production has been systematically managed using a software-assisted breeding program with fully traceable lineages, performed in a controlled environment. Currently, there are 650 lines in production, and close to 200 lines are now beyond their seventh generation of inbreeding. Retired breeders enter a high-throughput phenotyping protocol and DNA samples are banked for analyses of recombination history, allele drift and loss, and population structure. Herein we present a progress report of the Collaborative Cross breeding program at ORNL and a description of the kinds of investigations that this resource will support.

Excision of selectable genes from transgenic goat cells by a protein transducible TAT-Cre recombinase

The Cre/loxP site-specific recombination system is a widely used tool for genetic engineering of mammalian genomes. Recombination of loxP-modified alleles is often induced by introduction of foreign DNA vector expressing Cre into the cells. But the introduced DNA vector has the potential to integrate into the genome of the cells and continuous expression of Cre recombinase from the foreign vector has the potential to yield cytotoxicity and genotoxicity in various cells. In this study, we investigate the possibility of overcoming this limitation by using a cell-permeable TAT-Cre recombinase. We found that TAT-Cre treatment of transgenic goat fibroblast cells did not compromise the development competency of reconstructed embryos by using these TAT-Cre-treated cells as nuclear donor in nuclear transfer. Finally, we obtained two live cloned goats where a selectable gene cassette was removed. Our work not only provided an efficient protein transduction-based system for removing selectable genes from transgenic goats, but also presented strong evidence that no severe damage was made to the host cells during the process of protein transduction.

Delayed Development of Linezolid Resistance in Staphylococcus aureus following Exposure to Low Levels of Antimicrobial Agents

The development of resistance to linezolid (LZD) in gram-positive bacteria depends on the mutation of a single 23S rRNA gene, followed by homologous recombination and gene conversion of the other alleles. We sought to inhibit this process in Staphylococcus aureus using a range of antibacterial agents, including some that suppress recombination. A model for the rapid selection of LZD resistance was developed which allowed the selection of LZD-resistant mutants with G2576T mutations in all five copies of the 23S rRNA gene following only 5 days of subculture. The emergence of LZD-resistant isolates was delayed by exposing cultures to low concentrations of various classes of antibiotics. All antibiotic classes were effective in delaying the selection of LZD-resistant mutants and, with the exception of fusidic acid (FUS) and rifampin (RIF), prolonged the selection window from 5 to approximately 15 days. Inhibitors of DNA processing were no more effective than any other class of antibiotics at suppressing resistance development. However, the unrelated antimicrobials FUS and RIF were particularly effective at preventing the emergence of LZD resistance, prolonging the selection window from 5 to 25 days. The enhanced suppressive effect of FUS and RIF on the development of LZD resistance was lost in a recA-deficient host, suggesting that these drugs affect recA-dependent recombination. Furthermore, FUS and RIF were shown to be effective inhibitors of homologous recombination of a plasmid into the staphylococcal chromosome. We suggest that RIF or FUS in combination with LZD may have a role in preventing the emergence of LZD resistance.

Allelic recombination in the his-I locus of Neurospora crassa in isolates showing high and low frequency of intergenic recombination

From lines selected for high and low frequency of second division segregation (SDS) for asco in linkage group (LG) VI, one wild-type isolate of mating type (mt) A was picked out from the high and the low selection line, respectively. Both isolates were chosen on the basis of showing extreme SDS-frequencies, and both belonged to the last generation of selection. The two wild-type isolates were crossed with a strain carrying marker genes suitable for the analysis of allelic recombination in the his-I locus in LG V. The progeny of mt a was analysed for the influence of high and low line background on allelic recombination. The frequency of his-I prototrophs seems to be related to the high and low line background of the isolates. The two backgrounds showed no different influence on the recombination of markers flanking the his-I locus among his-I prototrophs. Variation of the frequency of his-I prototrophs not attributable to the rec-I locus is suggested. The data are interpreted according to Holliday's model of genetic recombination.

Variations of dendritic cell-specific intercellualar adhesion molecule-3-grabing nonintegrin neck region in HIV infected individuals

Variations of dendritic cell-specific intercellualar adhesion molecule-3-grabing nonintegrin neck region in HIV infected individuals

A Bayesian method for jointly estimating allele age and selection intensity

The problem of jointly estimating the intensity of past selection affecting an allele and the allele's age is formulated in a Bayesian framework. The prior distribution of allele age given its frequency is obtained from existing population genetics theory. The prior distribution of selection intensity is assumed to reflect the fact that positive selection on a new mutant is more likely to be weak than strong. The general approach is illustrated by the development of an importance sampling method applicable to low-frequency alleles. This method can be used either when the haplotypes of closely linked marker loci are known or when the lengths of linked ancestral chromosomal segments can be inferred. The method is illustrated with an application to the A-allele of G6PD in Africa. Because changes in allele frequency and recombination are both intrinsically stochastic, there are limits to the accuracy achievable with any method.