The use of whole genome amplification in the study of human disease

The availability of large amounts of genomic DNA is of critical importance for many of the molecular biology assays used in the analysis of human disease. However, since the amount of patient tissue available is often limited and as particular foci of interest may consist of only a few hundred cells, the yield of DNA is often insufficient for extensive analysis. To address this problem, several whole genome amplification (WGA) methodologies have been developed. Initial WGA approaches were based on the polymerase chain reaction (PCR). However, recent reports have described the use of non-PCR-based linear amplification protocols for WGA. Using these methods, it is possible to generate microgram quantities of DNA starting with as little as 1mg of genomic DNA. This review will provide an overview of WGA approaches and summarize some of the uses for amplified DNA in various high-throughput genetic applications.     

The above-ground coarse wood productivity of 104 Neotropical forest plots

The net primary production of tropical forests and its partitioning between long‐lived carbon pools (wood) and shorter‐lived pools (leaves, fine roots) are of considerable importance in the global carbon cycle. However, these terms have only been studied at a handful of field sites, and with no consistent calculation methodology. Here we calculate above‐ground coarse wood carbon productivity for 104 forest plots in lowland New World humid tropical forests, using a consistent calculation methodology that incorporates corrections for spatial variations in tree‐size distributions and wood density, and for census interval length. Mean wood density is found to be lower in more productive forests. We estimate that above‐ground coarse wood productivity varies by more than a factor of three (between 1.5 and 5.5 Mg C ha^?1 a^?1) across the Neotropical plots, with a mean value of 3.1 Mg C ha^?1 a^?1. There appear to be no obvious relationships between wood productivity and rainfall, dry season length or sunshine, but there is some hint of increased productivity at lower temperatures. There is, however, also strong evidence for a positive relationship between wood productivity and soil fertility. Fertile soils tend to become more common towards the Andes and at slightly higher than average elevations, so the apparent temperature/productivity relationship is probably not a direct one. Coarse wood productivity accounts for only a fraction of overall tropical forest net primary productivity, but the available data indicate that it is approximately proportional to total above‐ground productivity. We speculate that the large variation in wood productivity is unlikely to directly imply an equivalent variation in gross primary production. Instead a shifting balance in carbon allocation between respiration, wood carbon and fine root production seems the more likely explanation.     

A novel MET-interacting protein shares high sequence similarity with RanBPM, but fails to stimulate MET-induced Ras/Erk signaling

MET is a receptor protein tyrosine kinase for hepatocyte growth factor, a multifunctional cytokine controlling cell growth, morphogenesis, and motility. In our previous study, RanBPM/RanBP9, whose name originated from its ability to interact with Ran, was identified as a MET-interacting protein. RanBPM/RanBP9 activates the Ras/Erk signaling pathway by serving as an adaptor protein of MET to recruit Sos. In this study, we identify a protein sharing a high amino acid sequence identity with RanBPM/RanBP9, especially in its SPRY domain, the region responsible for MET binding. This protein lacks the N-terminal poly-proline and poly-glutamine (Poly-PQ) stretch present in RanBPM/RanBP9 and has less homology with RanBPM/RanBP9 in its mid-region. We subsequently named this protein RanBP10 after demonstrating its interaction with Ran. We show that, like RanBPM/RanBP9, RanBP10 interacts with the tyrosine kinase domain of MET via its SPRY domain and these two proteins can compete with each other to bind to MET. Interestingly, unlike RanBPM/RanBP9, overexpression of RanBP10 cannot induce Erk1/2 phosphorylation and serum response element-luciferase (SRE-LUC) reporter gene expression. More importantly, co-transfection of RanBPM/RanBP9 and RanBP10 significantly represses SRE-LUC reporter gene expression induced by overexpression of RanBPM/RanBP9. Additional binding assays demonstrate that RanBP10 fails to interact with Sos, which explains its inability to activate the Ras/Erk pathway. Furthermore, we show that the N-terminus of RanBPM/RanBP9 with the Poly-PQ stretch is required for recruiting Sos and a truncated RanBPM/RanBP9 lacking this region fails to recruit Sos, indicating that the functional difference between RanBP10 and RanBPM/RanBP9 lies in their sequence difference in their N-termini.     

Using identity-by-descent information in affected sib pairs to increase the efficiency of genetic association studies.

The aim of this study was to determine whether identity-by-descent (IBD) information for affected sib pairs (ASPs) can be used to select a sample of cases for a genetic case-control study which will provide more power for detecting association with loci in a known linkage region. By modeling the expected frequency of the disease allele in ASPs showing IBD sharing of 0, 1, or 2 alleles, and considering additive, recessive, and dominant disease models, we show that cases selected from IBD 2 families are best for this purpose, followed by those selected from IBD 1 families; least useful are cases selected from IBD 0 families.     

Modelling the Species Distribution of Flat-Headed Cats (Prionailurus planiceps), an Endangered South-East Asian Small Felid

Background

The flat-headed cat (Prionailurus planiceps) is one of the world''s least known, highly threatened felids with a distribution restricted to tropical lowland rainforests in Peninsular Thailand/Malaysia, Borneo and Sumatra. Throughout its geographic range large-scale anthropogenic transformation processes, including the pollution of fresh-water river systems and landscape fragmentation, raise concerns regarding its conservation status. Despite an increasing number of camera-trapping field surveys for carnivores in South-East Asia during the past two decades, few of these studies recorded the flat-headed cat.

Methodology/Principal Findings

In this study, we designed a predictive species distribution model using the Maximum Entropy (MaxEnt) algorithm to reassess the potential current distribution and conservation status of the flat-headed cat. Eighty-eight independent species occurrence records were gathered from field surveys, literature records, and museum collections. These current and historical records were analysed in relation to bioclimatic variables (WorldClim), altitude (SRTM) and minimum distance to larger water resources (Digital Chart of the World). Distance to water was identified as the key predictor for the occurrence of flat-headed cats (>50% explanation). In addition, we used different land cover maps (GLC2000, GlobCover and SarVision LLC for Borneo), information on protected areas and regional human population density data to extract suitable habitats from the potential distribution predicted by the MaxEnt model. Between 54% and 68% of suitable habitat has already been converted to unsuitable land cover types (e.g. croplands, plantations), and only between 10% and 20% of suitable land cover is categorised as fully protected according to the IUCN criteria. The remaining habitats are highly fragmented and only a few larger forest patches remain.

Conclusion/Significance

Based on our findings, we recommend that future conservation efforts for the flat-headed cat should focus on the identified remaining key localities and be implemented through a continuous dialogue between local stakeholders, conservationists and scientists to ensure its long-term survival. The flat-headed cat can serve as a flagship species for the protection of several other endangered species associated with the threatened tropical lowland forests and surface fresh-water sources in this region.     

Prediction and functional analysis of the replication origin of the linear plasmid pSCL2 in Streptomyces clavuligerus

pSCL2 (120 kb), one of the linear plasmids found in Streptomyces clavuligerus NRRL3585, was isolated and partially sequenced. Computational analysis of the central region of pSCL2 revealed the presence of two open reading frames that appear to encode proteins highly homologous to RepL1 and RepL2, replication proteins from pSLA2-L, the large linear plasmid in Streptomyces rochei. The S. clavuligerus open reading frames were designated repC1 and repC2, encoding the proteins RepC1 (150 amino acids) and RepC2 (102 amino acids), respectively. The RepC and RepL proteins have identical translation features and very similar predicted secondary and tertiary structures. Functional analysis confirmed that RepC1 is essential for replication initiation of pSCL2, whereas RepC2 is dispensable but may play a role in copy number control. The RepC and RepL proteins do not show similarity to any other bacterial plasmid replication proteins. Three regions of DNA sequence, Box 1 (1050-850 bp), Box 2 (723-606 bp), and Box 3 (224-168 bp), located upstream of repC1, were also shown to be essential or very important for replication of pSCL2.     

RESPONSES OF FLORAL TRAITS TO SELECTION ON PRIMARY SEXUAL INVESTMENT IN SPERGULARIA MARINA: THE BATTLE BETWEEN THE SEXES

Two widespread assumptions underlie theoretical models of the evolution of sex allocation in hermaphroditic species: (1) resource allocations to male and female function are heritable; and (2) there is an intrinsic, genetically based negative correlation between male and female reproductive function. These assumptions have not been adequately tested in wild species, although a few studies have detected either genetic variation in pollen and ovule production per flower or evidence of trade-offs between male and female investment at the whole plant level. It may also be argued, however, that in highly autogamous, perfect-flowered plant taxa that exhibit genetic variation in gamete production, strong stabilizing selection for an efficient pollen:ovule ratio should result in a positive correlation among genotypes with respect to mean ovule and mean pollen production per flower. Here we report the results of a three-generation artificial selection experiment conducted on a greenhouse population of the autogamous annual plant Spergularia marina. Starting with a base population of 1200 individuals, we conducted intense mass selection for two generations, creating four selected lines (high and low ovule production per flower; high and low anther production per flower) and a control line. By examining the direct and correlated responses of several floral traits to selection on gamete production per flower, we evaluated the expectations that primary sexual investment would exhibit heritable variation and that resource-sharing, variation in resource-garnering ability, or developmental constraints mold the genetic correlations expressed among floral organs. The observed direct and correlated responses to selection on male and female gamete production revealed significant heritabilities of both ovule and anther production per flower and a significant negative genetic correlation between them. When plants were selected for increased ovules per flower over two generations, ovule production increased and anther production declined relative to the control line. Among plants selected for decreased anthers per flower, we observed a decline in anther production and an increase in ovule production relative to the control line. In contrast, the lines selected for low ovules per flower and for high anthers per flower exhibited no evidence for significant genetic correlations between male and female primary investment. Correlated responses to selection also indicate a genetically based negative correlation between the production of normal versus developmentally abnormal anthers (staminoid organs); a positive correlation between the production of ovules versus staminoid organs; and a positive correlation between the production of anthers and petals. The negative relationship between male versus female primary investment supports classical sex allocation theory, although the asymmetrical correlated responses to selection indicate that this relationship is not always expressed.     

Inflorescence architecture: A developmental genetics approach

We are characterizing a suiteof Pisum sativum mutants that alter inflorescence architecture to construct a model for the genetic regulation of inflorescence development in a plant with a compound raceme. Such a model, when compared with those created forAntirrhinum majus andArabidopsis thaliana, both of which have simple racemes, should provide insight into the evolution of the development of inflorescence architecture. The highly conserved nature of cloned genes that regulate reproductive development in plants and the morphological similarities among our mutants and those identified inA. majus andA. thaliana enhance the probability that a developmental genetics approach will be fruitful. Here we describe sixP. sativum mutants that affect morphologically and architecturally distinct aspects of the inflorescence, and we analyze interactions among these genes. Both vegetative and inflorescence growth of the primary axis is affected byUNIFOLIA TA, which is necessary for the function ofDETERMINATE (DET).DET maintains indeterminacy in the first-order axis. In its absence, the meristem differentiates as a stub covered with epidermal hairs.DET interacts withVEGETATIVE1 (VEG1).VEG1 appears essential for second-order inflorescence (I₂) development.veg1 mutants fail to flower or differentiate the I₂ meristem into a rudimentary stub,det veg1 double mutants produce true terminal flowers with no stubs, indicating that two genes must be eliminated for terminal flower formation inP. sativum, whereas elimination of a single gene accomplishes this inA. thaliana andA. majus. NEPTUNE also affects I₂ development by limiting to two the number of flowers produced prior to stub formation. Its role is independent ofDET, as indicated by the additive nature of the double mutantdet nep. UNI, BROC, and PIM all play roles in assigning floral meristem identity to the third-order branch.pim mutants continue to produce inflorescence branches, resulting in a highly complex architecture and aberrant flowers.uni mutants initiate a whorl of sepals, but floral organogenesis is aberrant beyond that developmental point, and the double mutantuni pim lacks identifiable floral organs. A wild-type phenotype is observed inbroc plants, butbroc enhancesthe pim phenotype in the double mutant, producing inflorescences that resemble broccoli. Collectively these genes ensure that only the third-order meristem, not higher- or lower-order meristems, generates floral organs, thus precisely regulating the overall architecture of the plant. Gene symbols used in this article: For clarity a common symbolization is used for genes of all species discussed in this article. Genes are symbolized with italicized capital letters. Mutant alleles are represented by lowercase, italicized letters. In both cases, the number immediately following the gene symbol differentiates among genes with the same symbol. If there are multiple alleles, a hyphen followed by a number is used to distinguish alleles. Protein products are represented by capital letters without italics.