Comparison of MultiMap and TSP/CONCORDE for constructing radiation hybrid maps

Radiation hybrid (RH) map construction allows investigators to locate both type I and type II markers on a given genome map. The process is composed of two steps. The first consists of determining the pattern distribution of a set of markers within the different cell lines of an RH panel. This is mainly done by polymerase chain reaction (PCR) amplification and gel electrophoresis, and results in a series of numbers indicating the presence or the absence of each marker in each cell line. The second step consists of a comparison of these numbers, using various algorithms, to group and then order markers. Because different algorithms may provide (slightly) different orders, we have compared the merits of the MultiMap and TSP/CONCORDE packages using a data set of information currently under analysis for construction of the canine genome RH map.     

Mating system and early viability resistance to habitat fragmentation in a bird-pollinated eucalypt

Habitat fragmentation has been shown to disrupt ecosystem processes such as plant-pollinator mutualisms. Consequently, mating patterns in remnant tree populations are expected to shift towards increased inbreeding and reduced pollen diversity, with fitness consequences for future generations. However, mating patterns and phenotypic assessments of open-pollinated progeny have rarely been combined in a single study. Here, we collected seeds from 37 Eucalyptus incrassata trees from contrasting stand densities following recent clearance in a single South Australian population (intact woodland=12.6 trees ha⁻¹; isolated pasture=1.7 trees ha⁻¹; population area=10 km²). 649 progeny from these trees were genotyped at eight microsatellite loci. We estimated genetic diversity, spatial genetic structure, indirect contemporary pollen flow and mating patterns for adults older than the clearance events and open-pollinated progeny sired post-clearance. A proxy of early stage progeny viability was assessed in a common garden experiment. Density had no impact on mating patterns, adult and progeny genetic diversity or progeny growth, but was associated with increased mean pollen dispersal. Weak spatial genetic structure among adults suggests high historical gene flow. We observed preliminary evidence for inbreeding depression related to stress caused by fungal infection, but which was not associated with density. Higher observed heterozygosities in adults compared with progeny may relate to weak selection on progeny and lifetime-accumulated mortality of inbred adults. E. incrassata appears to be resistant to the negative mating pattern and fitness changes expected within fragmented landscapes. This pattern is likely explained by strong outcrossing and regular long-distance pollen flow.     

Genome-Wide Association Studies in an Isolated Founder Population from the Pacific Island of Kosrae

It has been argued that the limited genetic diversity and reduced allelic heterogeneity observed in isolated founder populations facilitates discovery of loci contributing to both Mendelian and complex disease. A strong founder effect, severe isolation, and substantial inbreeding have dramatically reduced genetic diversity in natives from the island of Kosrae, Federated States of Micronesia, who exhibit a high prevalence of obesity and other metabolic disorders. We hypothesized that genetic drift and possibly natural selection on Kosrae might have increased the frequency of previously rare genetic variants with relatively large effects, making these alleles readily detectable in genome-wide association analysis. However, mapping in large, inbred cohorts introduces analytic challenges, as extensive relatedness between subjects violates the assumptions of independence upon which traditional association test statistics are based. We performed genome-wide association analysis for 15 quantitative traits in 2,906 members of the Kosrae population, using novel approaches to manage the extreme relatedness in the sample. As positive controls, we observe association to known loci for plasma cholesterol, triglycerides, and C-reactive protein and to a compelling candidate loci for thyroid stimulating hormone and fasting plasma glucose. We show that our study is well powered to detect common alleles explaining ≥5% phenotypic variance. However, no such large effects were observed with genome-wide significance, arguing that even in such a severely inbred population, common alleles typically have modest effects. Finally, we show that a majority of common variants discovered in Caucasians have indistinguishable effect sizes on Kosrae, despite the major differences in population genetics and environment.     

Inhibition of fructose-1,6-bisphosphate aldolase from rabbit muscle and Bacillus stearothermophilus

Phosphoglycollohydroxamic acid and phosphoglycollamide are inhibitors of rabbit muscle fructose-1,6-bisphosphate aldolase. The binding dissociation constants determined by enzyme inhibition and protein fluorescence quenching suggest that two distinct enzyme inhibitor complexes may be formed. The binding dissociation constants of the two inhibitors to Bacillus stearothermophilus cobalt (II) fructose-1,6-bisphosphate aldolase have also been determined. The hydroxamic acid is an exceptionally potent inhibitor (Ki = 1.2 nM) probably due to direct chelation with Co(II) at the active site. The inhibition, however, is time-dependant and the association and dissociation constants have been estimated. Ethyl phosphoglycollate irreversibly inhibits rabbit muscle fructose-1,6-bisphosphate aldolase in the presence of sodium borohydride, presumably by forming a stable secondary amine through the active-site lysine reside. A new condensation assay for fructose-1,6-bisphosphate aldolases has been developed which is more sensitive than currently used assay procedures.     

Linkage mapping of the primary disease locus for collie eye anomaly

Collie eye anomaly (cea) is a hereditary ocular disorder affecting development of the choroid and sclera segregating in several breeds of dog, including rough, smooth, and Border collies and Australian shepherds. The disease is reminiscent of the choroidal hypoplasia phenotype observed in humans in conjunction with craniofacial or renal abnormalities. In dogs, however, the clinical phenotype can vary significantly; many dogs exhibit no obvious clinical consequences and retain apparently normal vision throughout life, while severely affected animals develop secondary retinal detachment, intraocular hemorrhage, and blindness. We report genetic studies establishing that the primary cea phenotype, choroidal hypoplasia, segregates as an autosomal recessive trait with nearly 100% penetrance. We further report linkage mapping of the primary cea locus to a 3.9-cM region of canine chromosome 37 (LOD = 22.17 at theta = 0.076), in a region corresponding to human chromosome 2q35. These results suggest the presence of a developmental regulatory gene important in ocular embryogenesis, with potential implications for other disorders of ocular vascularization.     

Interaction of fatty acids with recombinant rat intestinal and liver fatty acid-binding proteins

Intestinal enterocytes contain two homologous fatty acid-binding proteins, intestinal fatty acid-binding protein (I-FABP)2 and liver fatty acid-binding protein (L-FABP). Since the functional basis for this multiplicity is not known, the fatty acid-binding specificity of recombinant forms of both rat I-FABP and rat L-FABP was examined. A systematic comparative analysis of the 18 carbon chain length fatty acid binding parameters, using both radiolabeled (stearic, oleic, and linoleic) and fluorescent (trans-parinaric and cis-parinaric) fatty acids, was undertaken. Results obtained with a classical Lipidex-1000 binding assay, which requires separation of bound from free fatty acid, were confirmed with a fluorescent fatty acid-binding assay not requiring separation of bound and unbound ligand. Depending on the nature of the fatty acid ligand, I-FABP bound fatty acid had dissociation constants between 0.2 and 3.1 microM and a consistent 1:1 molar ratio. The dissociation constants for L-FABP bound fatty acids ranged between 0.9 and 2.6 microM and the protein bound up to 2 mol fatty acid per mole of protein. Both fatty acid-binding proteins exhibited relatively higher affinity for unsaturated fatty acids as compared to saturated fatty acids of the same chain length. cis-Parinaric acid or trans-parinaric acid (each containing four double bonds) bound to L-FABP and I-FABP were displaced in a competitive manner by non-fluorescent fatty acid. Hill plots of the binding of cis- and trans- parinaric acid to L-FABP showed that the binding affinities of the two sites were very similar and did not exhibit cooperativity. The lack of fluorescence self-quenching upon binding 2 mol of either trans- or cis-parinaric acid/mol L-FABP is consistent with the presence of two binding sites with dissimilar orientation in the L-FABP. Thus, the difference in binding capacity between I-FABP and L-FABP predicts a structurally different binding site or sites.     

Limited-host-range plasmid of Agrobacterium tumefaciens: molecular and genetic analyses of transferred DNA.

A tumor-inducing (Ti) plasmid from a strain of Agrobacterium tumefaciens that induces tumors on only a limited range of plants was characterized and compared with the Ti plasmids from strains that induce tumors on a wide range of plants. Whereas all wide-host-range Ti plasmids characterized to date contain closely linked oncogenic loci within a single transferred DNA (T-DNA) region, homology to these loci is divided into two widely separated T-DNA regions on the limited-host-range plasmid. These two plasmid regions, TA-DNA and TB-DNA, are separated by approximately 25 kilobases of DNA which is not maintained in the tumor. The TA-DNA region resembles a deleted form of the wide-host-range TL-DNA and contains a region homologous to the cytokinin biosynthetic gene. However, a region homologous to the two auxin biosynthetic loci of the wide-host-range plasmid mapped within the TB-DNA region. These latter genes play an important role in tumor formation because mutations in these loci result in a loss of virulence on Nicotiana plants. Furthermore, the TB-DNA region alone conferred tumorigenicity onto strains with an intact set of vir genes. Our results suggest that factors within both the T-DNA and the vir regions contribute to the expression of host range in Agrobacterium species. There was a tremendous variation among plants in susceptibility to tumor formation by various A. tumefaciens strains. This variation occurred not only among different plant species, but also among different varieties of plants within the same genus.     

Leaf morphology shift linked to climate change

Climate change is driving adaptive shifts within species, but research on plants has been focused on phenology. Leaf morphology has demonstrated links with climate and varies within species along climate gradients. We predicted that, given within-species variation along a climate gradient, a morphological shift should have occurred over time due to climate change. We tested this prediction, taking advantage of latitudinal and altitudinal variations within the Adelaide Geosyncline region, South Australia, historical herbarium specimens (n = 255) and field sampling (n = 274). Leaf width in the study taxon, Dodonaea viscosa subsp. angustissima, was negatively correlated with latitude regionally, and leaf area was negatively correlated with altitude locally. Analysis of herbarium specimens revealed a 2 mm decrease in leaf width (total range 1-9 mm) over 127 years across the region. The results are consistent with a morphological response to contemporary climate change. We conclude that leaf width is linked to maximum temperature regionally (latitude gradient) and leaf area to minimum temperature locally (altitude gradient). These data indicate a morphological shift consistent with a direct response to climate change and could inform provenance selection for restoration with further investigation of the genetic basis and adaptive significance of observed variation.     

Polarized distribution of Na, K-ATPase α-subunit isoforms in electrocyte membranes

We have previously demonstrated that Na⁺, K⁺-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the α subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of α isoforms (α1 and α2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na⁺, K⁺-ATPase activity 2.6-fold more sensitive to ouabain (I₅₀=1.0±0.1 μM) than the activity of innervated membranes (I₅₀=2.6±0.2 μM). As depicted in [³H]ouabain binding experiments, when the [³H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na⁺, K⁺-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of α1 and α2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na⁺, K⁺-ATPase α-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell.