Genetic and molecular dissection of naturally occurring variation

Recent progress in plant genome analysis has made it possible to examine the naturally occurring allelic variation underlying complex traits. Many studies have described the genetic mapping of quantitative trait loci for several kinds of complex phenotypic traits. Some researchers have taken up the challenge of performing the molecular cloning of genes at these loci, and examples of cloning have recently been reported. Naturally occurring allelic variation could be a new resource for the functional analysis of plant genes.     

A naturally occurring cancer with molecular connectivity to human diseases

As Jessani et al. 1 point out development of cell and animal models that accurately depict human tumorigenesis remains a major goal of cancer research. Clam cancer offers significant advantages over traditional models for genotoxic and non-genotoxic preclinical analysis of treatments for human cancers with a similar molecular basis. The naturally occurring clam model closely resembles an out-breeding, human clinical population and provides both in vitro and in vivo alternatives to those generated from inbred mouse strains or by intentional exposure to known tumor viruses. Fly and worm in vivo models for adult human somatic cell cancers do not exist because their adult somatic cells do not divide. Clam cancer is the best characterized, naturally occurring malignancy with a known molecular basis remarkably similar to those observed in several unrelated human cancers where both genotoxic and non-genotoxic strategies can restore the function of wild-type p53. To further emphasize this point of view, we here demonstrate a p53-induced, mitochondrial-directed mechanism for promoting apoptosis in the clam cancer model that is similar to one recently identified in mammals. Discerning the molecular basis for naturally occurring diseases in non-traditional models and correlating these with related molecular mechanisms responsible for human diseases is a virtually unexplored aspect of toxico-proteomics and genomics and related drug discovery.     

A naturally occurring promoter polymorphism of the Arabidopsis FUM2 gene causes expression variation,and is associated with metabolic and growth traits

Fumarate and malate are known intermediates of the TCA cycle, a mitochondrial metabolic pathway generating NADH for respiration. Arabidopsis thaliana and other Brassicaceae contain an additional cytosolic fumarase (FUM2) that functions in carbon assimilation and nitrogen use. Here, we report the identification of a hitherto unknown FUM2 promoter insertion/deletion (InDel) polymorphism found between the Col‐0 and C24 accessions, which also divides a large number of Arabidopsis accessions carrying either the Col‐0 or the C24 allele. The polymorphism consists of two stretches of 2.1 and 3.8 kb, which are both absent from the promotor region of Col‐0 FUM2. By analysing mutants as well as mapping and natural populations with contrasting FUM2 alleles, the promotor insertion was linked to reduced FUM2 mRNA expression, reduced fumarase activity and reduced fumarate/malate ratio in leaves. In a large population of 174 natural accessions, the polymorphism was also found to be associated with the fumarate/malate ratio, malate and fumarate levels, and with dry weight at 15 days after sowing (DAS). The association with biomass production was confirmed in an even larger (251) accession population for dry weight at 22 DAS. The dominant Col‐0 allele that results in increased fumarate/malate ratios and enhanced biomass production is predominantly found in central/eastern European accessions, whereas the C24 type allele is prevalent on the Iberian Peninsula, west of the Rhine and in the British Isles. Our findings support the role of FUM2 in diurnal carbon storage, and point to a growth advantage of accessions carrying the FUM2 Col‐0 allele.     

S-layer variation in Bacillus stearothermophilus PV72 is based on DNA rearrangements between the chromosome and the naturally occurring megaplasmids

Bacillus stearothermophilus PV72 expresses different S-layer genes (sbsA and sbsB) under different growth conditions. No stretches of significant sequence identity between sbsA and sbsB were detected. In order to investigate S-layer gene regulation in B. stearothermophilus PV72, we characterized the upstream regulatory region of sbsA and sbsB by sequencing and primer extension analysis. Both genes are transcribed from unique but different promoters, independently of the growth phase. Localization of sbsB in the sbsA-expressing strain PV72/p6 revealed that the coding region of the second S-layer gene sbsB is located not on the chromosome but on a natural megaplasmid of the strain, whereas the upstream regulatory region of sbsB was exclusively detected on the chromosome of PV72/p6. For sbsB expression, the coding region has to be integrated into the chromosomally located expression site. After the switch to sbsB expression, the sbsA coding region was removed from the chromosome but could still be detected on the plasmid of the sbsB-expressing strain PV72/p2. The sbsA upstream regulatory region, however, remained on the chromosome. This is the first report of S-layer variation not caused by intrachromosomal DNA rearrangements, but where variant formation depends on recombinational events between the plasmid and the chromosome.     

Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA

The vaccinia virus genome is a single, linear, duplex DNA molecule whose complementary strands are naturally cross-linked. The molecular weight has been determined by contour length measurements from electron micrographs to be 122 ± 2.2 × 10⁶. Denaturation mapping techniques indicate that the nucleotide sequence arrangement of the DNA is unique. Two forms of cross-linked vaccinia DNA were observed in alkaline sucrose gradients. The relative S-values of the two cross-linked species were appropriate for a single-stranded circle and a linear single strand, each with a molecular weight twice that expected for an intact, linear, complementary strand of vaccinia DNA. The fraction of sheared vaccinia DNA able to “snap back” after denaturation suggested a minimum of two crosslinks per molecule. Full-length single-stranded circles were observed in the electron microscope after denaturation of vaccinia DNA. Partial denaturation produced single-stranded loops at the ends of all full-length molecules. Exposure of native vaccinia DNA to a single strand-specific endonuclease isolated from vaccinia virions caused disruption of the cross-links, as assayed by alkaline sedimentation, and produced free single-strand ends when partially denatured DNA was observed in the electron microscope. We conclude that vaccinia DNA contains two cross-links, one at or near (within 50 nucleotides) each end in a region of single-stranded DNA. Two models for the cross-links are presented.     

Genetic variation for an aphid wing polyphenism is genetically linked to a naturally occurring wing polymorphism

Many polyphenisms are examples of adaptive phenotypic plasticity where a single genotype produces distinct phenotypes in response to environmental cues. Such alternative phenotypes occur as winged and wingless parthenogenetic females in the pea aphid (Acyrthosiphon pisum). However, the proportion of winged females produced in response to a given environmental cue varies between clonal genotypes. Winged and wingless phenotypes also occur in males of the sexual generation. In contrast to parthenogenetic females, wing production in males is environmentally insensitive and controlled by the sex-linked, biallelic locus, aphicarus (api). Hence, environmental or genetic cues induce development of winged and wingless phenotypes at different stages of the pea aphid life cycle. We have tested whether allelic variation at the api locus explains genetic variation in the propensity to produce winged females. We assayed clones from an F2 cross that were heterozygous or homozygous for alternative api alleles for their propensity to produce winged offspring. We found that clones with different api genotypes differed in their propensity to produce winged offspring. The results indicate genetic linkage of factors controlling the female wing polyphenism and male wing polymorphism. This finding is consistent with the hypothesis that genotype by environment interaction at the api locus explains genetic variation in the environmentally cued wing polyphenism.     

A pre‐adaptive approach for tropical forest restoration during climate change using naturally occurring genetic variation in response to water limitation

Effective reforestation of degraded tropical forests depends on selecting planting material suited to the stressful environments typical at restoration sites that can be exacerbated by increased duration and intensity of dry spells expected with climate change. While reforestation efforts in nontropical systems are incorporating drought‐adapted genotypes into restoration programs to cope with drier conditions, such approaches have not been tested or implemented in tropical forests. As the first effort to examine genetic variation in plant response to drought in a tropical wet forest, we established a watering experiment using five replicated maternal lines (i.e. seedlings from different maternal trees) of five dipterocarp species native to Borneo. Apart from the expected species level variation in growth and herbivory (3‐fold variation in both cases), we also found intraspecific variation so that growth in some cases varied 2‐fold, and herbivory 3‐fold, among genetically different maternal lines. In two species we found that among‐maternal line variation in growth rate was negatively correlated with tolerance to water limitation, that is, the maternal lines that performed the best in the high water treatment lost proportionally more of their growth during water limitation. We argue that selection for tolerance to future drier conditions is not only likely to impact population genetics of entire forests, but likely extends from forest trees to the communities of canopy arthropods associated with these trees. In tropical reforestation efforts where increased drought is predicted from climate change, including plant material resilient to drier conditions may improve restoration effectiveness.     

DNA interaction with naturally occurring antioxidant flavonoids quercetin, kaempferol, and delphinidin

Flavonoids are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. However, there has been no information on the interactions of these antioxidants with individual DNA at molecular level. This study was designed to examine the interaction of quercetin (que), kaempferol (kae), and delphinidin (del) with calf-thymus DNA in aqueous solution at physiological conditions, using constant DNA concentration (6.5 mmol) and various drug/DNA(phosphate) ratios of 1/65 to 1. FTIR and UV-Visible difference spectroscopic methods are used to determine the drug binding sites, the binding constants and the effects of drug complexation on the stability and conformation of DNA duplex. Structural analysis showed quercetin, kaempferol, and delphinidin bind weakly to adenine, guanine (major groove), and thymine (minor groove) bases, as well as to the backbone phosphate group with overall binding constants K(que) = 7.25 x 10(4)M(-1), K(kae) = 3.60 x 10(4)M(-1), and K(del) = 1.66 x 10(4)M(-1). The stability of adduct formation is in the order of que>kae>del. Delphinidin with a positive charge induces more stabilizing effect on DNA duplex than quercetin and kaempferol. A partial B to A-DNA transition occurs at high drug concentrations.     

Linkage disequilibrium mapping of molecular polymorphisms at the scabrous locus associated with naturally occurring variation in bristle number in Drosophila melanogaster

We evaluated the hypothesis that the Drosophila melanogaster second chromosome gene scabrous (sca), a candidate sensory bristle number quantitative trait locus (QTL), contributes to naturally occurring variation in bristle number. Variation in abdominal and sternopleural bristle number was quantified for wild-derived sca alleles in seven genetic backgrounds: as homozygous second chromosomes (C2) in an isogenic background, homozygous lines in which approximately 20 cM including the sca locus had been introgressed into the isogenic background (sca BC), as C2 and sca BC heterozygotes and hemizygotes against a P element insertional sca allele and a P-induced sca deficiency in the same isogenic background, and as sca BC heterozygotes against the wild-type sca allele of isogenic strain. Molecular restriction map variation was determined for a 45 kb region including the sca locus, and single-stranded conformational polymorphism (SSCP) was examined for the third intron and parts of the third and fourth exons. Associations between each of the 27 molecular polymorphisms and bristle number were evaluated within each genotype and on the first principal component score determined from all seven genotypes, separately for each sex and bristle trait. Permutation tests were used to assess the empirical significance thresholds, accounting for multiple, correlated tests, and correlated markers. Three sites in regulatory regions were associated with female-specific variation in abdominal bristle number, one of which was an SSCP site in the region of the gene associated with regulation of sca in embryonic abdominal segments.     

Exploiting interactions among polymorphisms contributing to complex disease traits with boosted generative modeling.

Although there has been great success in identifying disease genes for simple, monogenic Mendelian traits, deciphering the genetic mechanisms involved in complex diseases remains challenging. One major approach is to identify configurations of interacting factors such as single nucleotide polymorphisms (SNPs) that confer susceptibility to disease. Traditional methods, such as the multiple dimensional reduction method and the combinatorial partitioning method, provide good tools to decipher such interactions amid a disease population with a single genetic cause. However, these traditional methods have not managed to resolve the issue of genetic heterogeneity, which is believed to be a very common phenomenon in complex diseases. There is rarely prior knowledge of the genetic heterogeneity of a disease, and traditional methods based on estimation over the entire population are unlikely to succeed in the presence of heterogeneity. We present a novel Boosted Generative Modeling (BGM) approach for structure-model the interactions leading to diseases in the context of genetic heterogeneity. Our BGM method bridges the ensemble and generative modeling approaches to genetic association studies under a case-control design. Generative modeling is employed to model the interaction network configuration and the causal relationships, while boosting is used to address the genetic heterogeneity problem. We perform our method on simulation data of complex diseases. The results indicate that our method is capable of modeling the structure of interaction networks among disease-susceptible loci and of addressing genetic heterogeneity issues where the traditional methods, such as multiple dimensional reduction method, fail to apply. Our BGM method provides an exploratory tool that identifies the variables (e.g., disease-susceptible loci) that are likely to correlate and contribute to the disease.     

Characterization and molecular mechanism of a naturally occurring metsulfuron-methyl resistant strain of Pseudomonas aeruginosa

Strain L36, naturally resistant to the herbicide metsulfuron-methyl (SM), was isolated and characterized with respect to the molecular mechanism of resistance. The isolate was identified as Pseudomonas aeruginosa based on bacterial morphology, physiology, cellular fatty acid, and 16S rRNA gene sequence. Minimal inhibitory concentrations of metsulfuron-methyl against the growth of L36 and wild type isolate PAO1 were 6.03 and 1.33 mM, respectively. L36 carried a nucleotide base change in the acetolactate synthase (ALS) gene that coded for a single amino acid mutation (Ala29 → Val29). The mutated ilvIH gene was functionally expressed, purified, and the kinetic properties of the purified ALS were tested. The mutant enzyme had K _m for pyruvate fourfold higher than the wild type enzyme, and K _i ^app for sulfonylureas some 30-fold higher. The A29 V mutation in the ALS resulted in the resistance of P. aeruginosa to sulfonylurea herbicides but not to imidazolinone herbicides.     

The molecular evolution and DNA profiling of toxic cyanobacteria

Rapid and sensitive methods for the detection and genetic characterization of cyanobacteria have been developed based on DNA amplification techniques. This article describes the molecular methods that have been used to characterize cyanobacteria and their use as tools to identify toxin-producing strains. Different species and strains were compared using restriction fragment length polymorphism (RFLP) of amplified fragments of the phycocyanin gene and the 16S-23S rRNA internal transcribed spacer.     

Integrating three comprehensive data sets shows that mitochondrial DNA variation is linked to species traits and paleogeographic events in European butterflies

Understanding the dynamics of biodiversity, including the spatial distribution of genetic diversity, is critical for predicting responses to environmental changes, as well as for effective conservation measures. This task requires tracking changes in biodiversity at large spatial scales and correlating with species functional traits. We provide three comprehensive resources to understand the determinants for mitochondrial DNA differentiation represented by (a) 15,609 COI sequences and (b) 14 traits belonging to 307 butterfly species occurring in Western‐Central Europe and (c) the first multi‐locus phylogenetic tree of all European butterfly species. By applying phylogenetic regressions we show that mitochondrial DNA spatial differentiation (as measured with G_ST, G′_ST, D and D_ST) is negatively correlated with species traits determining dispersal capability and colonization ability. Thanks to the high spatial resolution of the COI data, we also provide the first zoogeographic regionalization maps based on intraspecific genetic variation. The overall pattern obtained by averaging the spatial differentiation of all Western‐Central European butterflies shows that the paradigm of long‐term glacial isolation followed by rapid pulses of post‐glacial expansion has been a pervasive phenomenon in European butterflies. The results and the extensive data sets we provide here constitute the basis for genetically‐informed conservation plans for a charismatic group in a continent where flying insects are under alarming decline.