High-throughput amplicon sequencing and stream benthic bacteria: identifying the best taxonomic level for multiple-stressor research.

Disentangling the individual and interactive effects of multiple stressors on microbial communities is a key challenge to our understanding and management of ecosystems. Advances in molecular techniques allow studying microbial communities in situ and with high taxonomic resolution. However, the taxonomic level which provides the best trade-off between our ability to detect multiple-stressor effects versus the goal of studying entire communities remains unknown. We used outdoor mesocosms simulating small streams to investigate the effects of four agricultural stressors (nutrient enrichment, the nitrification inhibitor dicyandiamide (DCD), fine sediment and flow velocity reduction) on stream bacteria (phyla, orders, genera, and species represented by Operational Taxonomic Units with 97% sequence similarity). Community composition was assessed using amplicon sequencing (16S rRNA gene, V3-V4 region). DCD was the most pervasive stressor, affecting evenness and most abundant taxa, followed by sediment and flow velocity. Stressor pervasiveness was similar across taxonomic levels and lower levels did not perform better in detecting stressor effects. Community coverage decreased from 96% of all sequences for abundant phyla to 28% for species. Order-level responses were generally representative of responses of corresponding genera and species, suggesting that this level may represent the best compromise between stressor sensitivity and coverage of bacterial communities.

Species of cyanolichens from Pseudocyphellaria with indistinguishable ITS sequences have different photobionts.

• Cyanobacteria were isolated from bipartite cyanolichen species of Pseudocyphellaria and the identity of the major photobionts established. The specificity of the cyanobacterial-fungal association was also examined. • Comparison of 16S rRNA gene sequences distinguished cyanobacterial and green algal isolates, and both 16S rRNA gene and tRNALeu  (UAA) intron sequences of isolates and lichen thalli identified candidate photobionts. In addition, the genetic diversity of both the cyanobiont and mycobiont was investigated using the comparison of tRNALeu  (UAA) intron sequences and ITS sequences, respectively. • The 16S rRNA gene sequences identified two species-specific photobionts with similar sequences; however, the tRNALeu  (UAA) intron sequences unambiguously discriminated between the two symbiotic cyanobacterial strains. Moreover, the fungal ITS sequences of the two corresponding lichens, Pseudocyphellaria crocata and Pseudocyphellaria neglecta, showed little variation. • The cyanobacterial-fungal associations of P. crocata and P. neglecta were specific for all samples. However, the similarity of the ITS sequences raised the possibility that they represent the same species and that their different morphology is influenced by the cyanobacterial symbiont.

The sheep genome contributes to localization of control elements in a human gene with complex regulatory mechanisms.

Genes that show complex tissue-specific and temporal control by regulatory elements located outside their promoters present a considerable challenge to identify the sequences involved. The rapid accumulation of genomic sequence information for a number of species has enabled a comparative phylogenetic approach to find important regulatory elements. For some genes, which show a similar pattern of expression in humans and rodents, genomic sequence information for these two species may be sufficient. Others, such as the cystic fibrosis transmembrane conductance regulator (CFTR) gene, show significant divergence in expression patterns between mouse and human, necessitating phylogenetic approaches involving additional species. The ovine CFTR gene has a temporal and spatial expression pattern that is very similar to that of human CFTR. Comparative genomic sequence analysis of ovine and human CFTR identified high levels of homology between the core elements in several potential regulatory elements defined as DNase I hypersensitive sites in human CFTR. These data provide a case for the power of an artiodactyl genome to contribute to the understanding of human genetic disease.

The gene for X-linked hypophosphataemic rickets maps to a 200-300kb region in Xp22.1, and is located on a single YAC containing a putative vitamin D response element (VDRE).

The location of the HYP gene, which determines X-linked hypophosphataemic rickets, has been refined considerably by linkage analysis, and three new microsatellite primers isolated, Cap32 (DXS7473), Cap29 (DXS7474) and 7v2 (DXS7475). The locations of four other markers have also been determined (DXS1226, AFMa176zb1, AFMa152wc5, and AFM346azc1). Markers Cap29 and Cap32 are the closest distal markers to the gene with zetamax=11.93, thetamax= 0.018 and zetamax=12.03, thetamax = 0.015 respectively. Both Cap29 and Cap32 are proximal to DXS365 and AFMa176zb1, as deduced by screening non-chimaeric yeast artificial chromosomes (YACs) from a contig spanning the HYP gene. A single crossover places AFMa176zbl distal to the disease gene. There are no recombinations between 7v2 and HYP (zetamax=12.9, thetamax=0.0), or between 7v2 and adjacent markers Cap32, Cap29, AFMa176zb1, DXS1683 and DXS365. However screening of YAC clones encompassing the HYP gene and also P1 clones localises 7v2 distal to Cap29 and Cap32, and proximal to DXS443. Marker DXS1226 is placed outside the region containing the gene, and is located proximal to DXS274 as confirmed by a crossover for this marker and DXS41 against HYP and its presence on YAC 83B05. Genetic mapping of CEPH pedigrees, and screening of YACs places AFMa152wc5 and AFMa346zcl between DXS1683 and DXS1052. The following gene marker map presents the best order for the HYP region: Xptel-DXS43-DXS999-DXS443-(DXS365/DXS74 75/AFMa176zb1)-(DXS7474/DXS7473)-HYP- DXS1683-(AFMa152wc5/AFMa346zc1)-DXS1052-DXS 274 -(DXS41/DXS1226)-Xcen. The distance between the cluster of distal flanking markers Cap29 (DXS7474), Cap32 (DXS7473), and DXS1683 is approximately 300 kb, as deduced from physical map data from a YAC contig spanning the gene. Thus the gene for HYP is contained within a single YAC (900AO472). Of further interest, is the location of a putative vitamin D response element (VDRE) on this YAC.