Identification and biochemical analysis of novel olfactory-specific cytochrome P-450IIA and UDP-glucuronosyl transferase

Two major transmembranal polypeptides of bovine olfactory epithelium were identified by SDS electrophoretic analysis of Triton X-114 solubilized membranes. Both polypeptides were present in large amounts in membranes of the olfactory epithelium but were barely detectable in membranes of the nasal respiratory epithelium. Both polypeptides are enriched in the deciliated epithelium as compared with isolated cilia. One of them is a glycoprotein with an apparent molecular mass of 56 kDa (gp56); the other is an unglycosylated protein with an apparent molecular mass of 52 kDa (p52). Sequence analysis of peptides obtained by CNBr cleavage of purified gp56 indicates that it is highly homologous to UDP-glucuronosyl transferase (UDPGT). Parallel analysis shows that p52 is highly homologous to cytochrome P-450 sequences of the IIA subfamily. This protein is assigned the name P-450olf2. Polyclonal antibodies were raised against synthetic peptides corresponding to gp56 and p52 peptide sequences. Immunoblots with these antibodies reveal the following properties of gp56 and p52: (1) they are enriched in the microsomal fraction of the bovine olfactory epithelium; (2) they are possibly specific to the olfactory epithelium, as we could not detect reactivity in microsomes derived from respiratory epithelium or lung, and only a very small amount of basal reactivity was seen with liver microsomes; (3) cross-reacting proteins exist in microsomes derived from the rat olfactory epithelium. These results are consistent with a mechanism whereby the microsomal enzymes are involved in odorant modification and clearance from the nasal tissue.     

Distance‐driven species turnover in Bornean rainforests: homogeneity and heterogeneity in primary and post‐logging forests

Selective logging is practiced extensively within tropical rainforests of south‐east Asia, and its impact on local biodiversity is well documented. Little is known, however, about the impact of selective logging on patterns of spatial heterogeneity of species. We set out to test the hypothesis that selective logging will lead to a homogenization of the associated faunal assemblages, using moths (Lepidoptera) as our subject taxa. Large‐scale transects were established within primary and post‐logging lowland mixed dipterocarp rainforests around the Danum Valley Conservation Area and surroundings, Sabah, Malaysia (4°50′N–5°00′N and 117°35′E–117°45′E). Five study sites were located within each habitat with geometrically increasing inter‐site distances. Macro‐moths plus Pyraloidea were sampled by light trapping in 2007 and 2008. Vegetation state was also measured at each site. A clear distance–decay relationship (decreasing assemblage similarity with increasing geographic distances) was observed in primary forest but was absent in the post‐logging forest. Large, comparable numbers of macro‐moth species were found in both primary and post‐logging forests. There were no significant differences in moth assemblage composition between primary and post‐logging forests. There are important structural differences between primary and post‐logging forests reflected in the moth assemblages. A two‐stage hypothesis combining both neutral and niche concepts is probably the most parsimonious explanation of these results. First, the composition of the moth assemblage is almost certainly determined locally by the variety of plant–hosts available to larvae, with the plants representing important niche dimensions for the moth species. Second the turnover (or lack of same) in the underlying plant assemblage probably reflects clumping and, in turn, dispersal capacity of the commoner plants in each forest type. Although the impact of selective logging may be subtle, this study suggests that selective logging results in the spatial homogenization of macro‐moth assemblages.     

DEFOG: a practical scheme for deciphering families of genes

We developed a novel efficient scheme, DEFOG (for "deciphering families of genes"), for determining sequences of numerous genes from a family of interest. The scheme provides a powerful means to obtain a gene family composition in species for which high-throughput genomic sequencing data are not available. DEFOG uses two key procedures. The first is a novel algorithm for designing highly degenerate primers based on a set of known genes from the family of interest. These primers are used in PCR reactions to amplify the members of the gene family. The second combines oligofingerprinting of the cloned PCR products with clustering of the clones based on their fingerprints. By selecting members from each cluster, a low-redundancy clone subset is chosen for sequencing. We applied the scheme to the human olfactory receptor (OR) genes. OR genes constitute the largest gene superfamily in the human genome, as well as in the genomes of other vertebrate species. DEFOG almost tripled the size of the initial repertoire of human ORs in a single experiment, and only 7% of the PCR clones had to be sequenced. Extremely high degeneracies, reaching over a billion combinations of distinct PCR primer pairs, proved to be very effective and yielded only 0.4% nonspecific products.     

Identification and characterization of coding single-nucleotide polymorphisms within a human olfactory receptor gene cluster

Single-nucleotide polymorphisms (SNPs) were studied in 15 olfactory receptor (OR) coding regions, one control region and two noncoding sequences all residing within a 412 kb OR gene cluster on human chromosome 17p13.3, as well as in other G-protein coupled receptors (GPCRs). A total of 26 SNPs were identified in ORs, 21 of which are coding SNPs (cSNPs). The mean nucleotide diversity of OR coding regions was 0.078% (ranging from 0 to 0.16%), which is about twice higher than that of other GPCRs, and similar to the nucleotide diversity levels of noncoding regions along the human genome. The high polymorphism level in the OR coding regions might be due to a weak positive selection pressure acting on the OR genes. In two cases, OR genes have been found to share the same cSNP. This could be explained by recent gene conversion events, which might be a part of a concerted evolution mechanism acting on the OR superfamily. Using the genotype data of 85 unrelated individuals in 15 SNPs, we found linkage disequilibrium (LD) between pairs of SNPs located on the centromeric part of the cluster. On the other hand, no LD was found between SNPs located on the telomeric part of the cluster, suggesting the presence of several hot-spots for recombination within this cluster. Thus, different regions of this gene cluster may have been subject to different recombination rates.     

The high value of logged tropical forests: lessons from northern Borneo

The carbon storage and conservation value of old-growth tropical forests is clear, but the value of logged forest is less certain. Here we analyse >100,000 observations of individuals from 11 taxonomic groups and >2,500 species, covering up to 19?years of post-logging regeneration, and quantify the impacts of logging on carbon storage and biodiversity within lowland dipterocarp forests of Sabah, Borneo. We estimate that forests lost ca. 53% of above-ground biomass as a result of logging but despite this high level of degradation, logged forest retained considerable conservation value: floral species richness was higher in logged forest than in primary forest and whilst faunal species richness was typically lower in logged forest, in most cases the difference between habitats was no greater than ca. 10%. Moreover, in most studies >90% of species recorded in primary forest were also present in logged forest, including species of conservation concern. During recovery, logged forest accumulated carbon at five times the rate of natural forest (1.4 and 0.28?Mg?C?ha^?1?year^?1, respectively). We conclude that allowing the continued regeneration of extensive areas of Borneo??s forest that have already been logged, and are at risk of conversion to other land uses, would provide a significant carbon store that is likely to increase over time. Protecting intact forest is critical for biodiversity conservation and climate change mitigation, but the contribution of logged forest to these twin goals should not be overlooked.     

Asymmetric boundary shifts of tropical montane Lepidoptera over four decades of climate warming

Aim To estimate whether species have shifted at equal rates at their leading edges (cool boundaries) and trailing edges (warm boundaries) in response to climate change. We provide the first such evidence for tropical insects, here examining elevation shifts for the upper and lower boundaries shifts of montane moths. Threats to species on tropical mountains are considered. Location Mount Kinabalu, Sabah, Malaysia. Methods We surveyed Lepidoptera (Geometridae) on Mount Kinabalu in 2007, 42 years after the previous surveys in 1965. Changes in species upper and lower boundaries, elevational extents and range areas were assessed. We randomly subsampled the data to ensure comparable datasets between years. Estimated shifts were compared for endemic versus more widespread species, and for species that reached their range limits at different elevations. Results Species that reached their upper limits at 2500–2700 m (n= 28 species, 20% of those considered) retreated at both their lower and upper boundaries, and hence showed substantial average range contractions (?300 m in elevational extent and ?45 km² in estimated range area). These declines may be associated with changes in cloud cover and the presence of ecological barriers (geological and vegetation transitions) which impede uphill movement. Other than this group, most species (n= 109, 80% of the species considered) expanded their upper boundaries upwards (by an average of 152 m) more than they retreated at their lower boundaries (77 m). Main conclusions Without constraints, leading margins shifted uphill faster than trailing margins retreated, such that many species increased their elevational extents. However, this did not result in increases in range area because the area of land available declines with increasing elevation. Species close to a major ecological/geological transition zone on the mountain flank declined in their range areas. Extinction risk may increase long before species reach the summit, even when undisturbed habitats are available.     

High-resolution copy-number variation map reflects human olfactory receptor diversity and evolution

Olfactory receptors (ORs), which are involved in odorant recognition, form the largest mammalian protein superfamily. The genomic content of OR genes is considerably reduced in humans, as reflected by the relatively small repertoire size and the high fraction ( approximately 55%) of human pseudogenes. Since several recent low-resolution surveys suggested that OR genomic loci are frequently affected by copy-number variants (CNVs), we hypothesized that CNVs may play an important role in the evolution of the human olfactory repertoire. We used high-resolution oligonucleotide tiling microarrays to detect CNVs across 851 OR gene and pseudogene loci. Examining genomic DNA from 25 individuals with ancestry from three populations, we identified 93 OR gene loci and 151 pseudogene loci affected by CNVs, generating a mosaic of OR dosages across persons. Our data suggest that approximately 50% of the CNVs involve more than one OR, with the largest CNV spanning 11 loci. In contrast to earlier reports, we observe that CNVs are more frequent among OR pseudogenes than among intact genes, presumably due to both selective constraints and CNV formation biases. Furthermore, our results show an enrichment of CNVs among ORs with a close human paralog or lacking a one-to-one ortholog in chimpanzee. Interestingly, among the latter we observed an enrichment in CNV losses over gains, a finding potentially related to the known diminution of the human OR repertoire. Quantitative PCR experiments performed for 122 sampled ORs agreed well with the microarray results and uncovered 23 additional CNVs. Importantly, these experiments allowed us to uncover nine common deletion alleles that affect 15 OR genes and five pseudogenes. Comparison to the chimpanzee reference genome revealed that all of the deletion alleles are human derived, therefore indicating a profound effect of human-specific deletions on the individual OR gene content. Furthermore, these deletion alleles may be used in future genetic association studies of olfactory inter-individual differences.     

Legacies in Switchgrass Resistance to and Recovery from Drought Suggest That Good Years Can Sustain Plants Through Bad Years

The warm-season perennial switchgrass (Panicum virgatum) is a candidate bioenergy crop. To be successful, switchgrass production must be maintained on low-quality landscapes with minimal inputs while facing future climates that are expected to be more extreme and more variable. We propose that antecedent rainfall constrains how plants respond to drought, as well as subsequently recover from drought. To test this idea, we examined how six switchgrass genotypes responded to a 1-year severe drought and then recovered under normal rainfall in the following year. These plants had previously grown for 3 years under a range of dry to wet rainfall levels in a shallow-soil common garden with no fertilizer. Plants previously exposed to drought produced less biomass, and basal area after the severe drought was relieved compared to previously well-watered plants. In addition, there were legacy effects caused by plant size: plants that were larger pre-drought were more likely to survive the severe drought, and plants that were larger during the severe drought recovered more biomass, basal area, and tillers post-drought. Although genotypes differed somewhat in their responses, the size constraint was consistent across genotypes. These findings suggest that we can establish more drought-resilient switchgrass stands by, for example, planning for initial irrigation or planting during a wet year to allow plants to grow larger prior to experiencing drought. Additional studies are needed to understand whether these rainfall and size legacies persist or are transient.     

Effect of slice thickness on brain magnetic resonance image texture analysis

Background  

The accuracy of texture analysis in clinical evaluation of magnetic resonance images depends considerably on imaging arrangements and various image quality parameters. In this paper, we study the effect of slice thickness on brain tissue texture analysis using a statistical approach and classification of T1-weighted images of clinically confirmed multiple sclerosis patients.     

Systematic inference of copy-number genotypes from personal genome sequencing data reveals extensive olfactory receptor gene content diversity

Copy-number variations (CNVs) are widespread in the human genome, but comprehensive assignments of integer locus copy-numbers (i.e., copy-number genotypes) that, for example, enable discrimination of homozygous from heterozygous CNVs, have remained challenging. Here we present CopySeq, a novel computational approach with an underlying statistical framework that analyzes the depth-of-coverage of high-throughput DNA sequencing reads, and can incorporate paired-end and breakpoint junction analysis based CNV-analysis approaches, to infer locus copy-number genotypes. We benchmarked CopySeq by genotyping 500 chromosome 1 CNV regions in 150 personal genomes sequenced at low-coverage. The assessed copy-number genotypes were highly concordant with our performed qPCR experiments (Pearson correlation coefficient 0.94), and with the published results of two microarray platforms (95-99% concordance). We further demonstrated the utility of CopySeq for analyzing gene regions enriched for segmental duplications by comprehensively inferring copy-number genotypes in the CNV-enriched >800 olfactory receptor (OR) human gene and pseudogene loci. CopySeq revealed that OR loci display an extensive range of locus copy-numbers across individuals, with zero to two copies in some OR loci, and two to nine copies in others. Among genetic variants affecting OR loci we identified deleterious variants including CNVs and SNPs affecting ~15% and ~20% of the human OR gene repertoire, respectively, implying that genetic variants with a possible impact on smell perception are widespread. Finally, we found that for several OR loci the reference genome appears to represent a minor-frequency variant, implying a necessary revision of the OR repertoire for future functional studies. CopySeq can ascertain genomic structural variation in specific gene families as well as at a genome-wide scale, where it may enable the quantitative evaluation of CNVs in genome-wide association studies involving high-throughput sequencing.