Following domestication, livestock breeds have experienced intense selection pressures for the development of desirable traits. This has resulted in a large diversity of breeds that display variation in many phenotypic traits, such as coat colour, muscle composition, early maturity, growth rate, body size, reproduction, and behaviour. To better understand the relationship between genomic composition and phenotypic diversity arising from breed development, the genomes of 13 traditional and commercial European pig breeds were scanned for signatures of diversifying selection using the Porcine60K SNP chip, applying a between-population (differentiation) approach. Signatures of diversifying selection between breeds were found in genomic regions associated with traits related to breed standard criteria, such as coat colour and ear morphology. Amino acid differences in the EDNRB gene appear to be associated with one of these signatures, and variation in the KITLG gene may be associated with another. Other selection signals were found in genomic regions including QTLs and genes associated with production traits such as reproduction, growth, and fat deposition. Some selection signatures were associated with regions showing evidence of introgression from Asian breeds. When the European breeds were compared with wild boar, genomic regions with high levels of differentiation harboured genes related to bone formation, growth, and fat deposition. 相似文献
In preclinical cancer studies, three-dimensional (3D) cell spheroids and aggregates are preferred over monolayer cell cultures due to their architectural and functional similarity to solid tumors. We performed a proof-of-concept study to generate physiologically relevant and predictive preclinical models using non–small cell lung adenocarcinoma, and colon and colorectal adenocarcinoma cell line-derived 3D spheroids and aggregates. Distinct panels were designed to determine the expression profiles of frequently studied biomarkers of the two cancer subtypes. The lung adenocarcinoma panel included ALK, EGFR, TTF-1, and CK7 biomarkers, and the colon and colorectal adenocarcinoma panel included BRAF V600E, MSH2, MSH6, and CK20. Recent advances in immunofluorescence (IF) multiplexing and imaging technology enable simultaneous detection and quantification of multiple biomarkers on a single slide. In this study, we performed IF staining of multiple biomarkers per section on formalin-fixed paraffin-embedded 3D spheroids and aggregates. We optimized protocol parameters for automated IF and demonstrated staining concordance with automated chromogenic immunohistochemistry performed with validated protocols. Next, post-acquisition spectral unmixing of the captured fluorescent signals were utilized to delineate four differently stained biomarkers within a single multiplex IF image, followed by automated quantification of the expressed markers. This workflow has the potential to be adapted to preclinical high-throughput screening and drug efficacy studies utilizing 3D spheroids from cancer cell lines and patient-derived organoids. The process allows for cost, time, and resource savings through concurrent staining of several biomarkers on a single slide, the ability to study the interactions of multiple expressed proteins within a single region of interest, and enable quantitative assessment of biomarkers in cancer cells. 相似文献
Endonucleases play a crucial role as reagents in laboratory research and diagnostics. Here, metagenomics was used to functionally screen a fosmid library for endonucleases. A fosmid library was constructed using metagenomic DNA isolated from soil sampled from the unique environment of the Kogelberg Nature Reserve in the Western Cape of South Africa. The principle of acquired immunity against phage infection was used to develop a plate-based screening technique for the isolation of restriction endonucleases from the library. Using next-generation sequencing and bioinformatics tools, sequence data were generated and analysed, revealing 113 novel open reading frames (ORFs) encoding putative endonuclease genes and ORFs of unknown identity and function. One endonuclease designated Endo52 was selected from the putative endonuclease ORFs and was recombinantly produced in Escherichia coli Rosetta? (DE3) pLysS. Endo52 was purified by immobilised metal affinity chromatography and yielded 0.437 g per litre of cultivation volume. Its enzyme activity was monitored by cleaving lambda DNA and pUC19 plasmid as substrates, and it demonstrated non-specific endonuclease activity. In addition to endonuclease-like genes, the screen identified several unknown genes. These could present new phage resistance mechanisms and are an opportunity for future investigations.
The maintenance of gene flow in species that have experienced population contractions and are geographically fragmented is important to the maintenance of genetic variation and evolutionary potential; thus, gene flow is also important to conservation and management of these species. For example, the Reddish Egret (Egretta rufescens) has recovered after severe population reductions during the 19th and 20th centuries, but population numbers remain below historical levels. In this study, we characterized gene flow among management units of the Reddish Egret by using ten nuclear microsatellite markers and part of the mitochondrial (mtDNA) control region from 176 nestlings captured at eight localities in Mexico (Baja California, Chiapas, Tamaulipas, and Yucatan), the USA (Texas, Louisiana, and Florida), and the Bahamas. We found evidence of population structure and that males disperse more often and across longer distances compared with females, which is congruent with previous banding and telemetry data. The maternally inherited mtDNA and biparentally inherited microsatellite data supported slightly different MU models; however, when interpreted together, a four MU model that considered population structure and geographic proximity was most optimal. Namely, MU 1 (Baja California); MU 2 (Chiapas); MU 3 (Yucatan, Tamaulipas, Texas, and Louisiana); and MU 4 (Florida and the Bahamas). Regions outside our sampled localities (e.g., the Greater Antilles and South America) require additional sampling to fully understand gene flow and movement of individuals across the species’ entire range. However, the four MUs we have defined group nesting localities into genetically similar subpopulations, which can guide future management plans. 相似文献
Landscape genomics studies focus on identifying candidate genes under selection via spatial variation in abiotic environmental variables, but rarely by biotic factors (i.e., disease). The Tasmanian devil (Sarcophilus harrisii) is found only on the environmentally heterogeneous island of Tasmania and is threatened with extinction by a transmissible cancer, devil facial tumor disease (DFTD). Devils persist in regions of long-term infection despite epidemiological model predictions of species’ extinction, suggesting possible adaptation to DFTD. Here, we test the extent to which spatial variation and genetic diversity are associated with the abiotic environment (i.e., climatic variables, elevation, vegetation cover) and/or DFTD. We employ genetic-environment association analyses using 6886 SNPs from 3287 individuals sampled pre- and post-disease arrival across the devil's geographic range. Pre-disease, we find significant correlations of allele frequencies with environmental variables, including 365 unique loci linked to 71 genes, suggesting local adaptation to abiotic environment. The majority of candidate loci detected pre-DFTD are not detected post-DFTD arrival. Several post-DFTD candidate loci are associated with disease prevalence and were in linkage disequilibrium with genes involved in tumor suppression and immune response. Loss of apparent signal of abiotic local adaptation post-disease suggests swamping by strong selection resulting from the rapid onset of DFTD. 相似文献
In target tissues, cortisol is metabolised by two 11β-hydroxysteroid dehydrogenase (11βHSD) isoenzymes, namely 11βHSD1 and 11βHSD2, both of which are co-expressed in the boar testis and reproductive tract. The present study has assessed whether cortisol-cortisone metabolism in boar testis and caput epididymidis can be regulated via the gonadotrophin-cAMP signalling pathway. 11βHSD activities were measured by using a radiometric conversion assay in static tissue culture. In both testis and caput epididymidis, the net reduction of cortisone but not the net oxidation of cortisol, was significantly decreased by luteinising hormone (by 53?±?20% and 45?±?9%, respectively, P?<?0.05), forskolin (by 60?±?7% and 57?±?9%, respectively, P?<?0.01) and 8-bromo-cAMP (by 54?±?4% and 64?±?1%, respectively, P?<?0.01). This suppression of 11-ketosteroid reductase activity in the boar testis by forskolin could be attenuated by the protein kinase A (PKA) inhibitor, H89. Hence, within the boar testis and the caput epididymidis, the local actions of glucocorticoids are modulated by gonadotrophin-cAMP-PKA signalling via their selective effects on the reductase activity of 11βHSD. 相似文献
Gene synthesis attempts to assemble user-defined DNA sequences with base-level precision. Verifying the sequences of construction intermediates and the final product of a gene synthesis project is a critical part of the workflow, yet one that has received the least attention. Sequence validation is equally important for other kinds of curated clone collections. Ensuring that the physical sequence of a clone matches its published sequence is a common quality control step performed at least once over the course of a research project. GenoREAD is a web-based application that breaks the sequence verification process into two steps: the assembly of sequencing reads and the alignment of the resulting contig with a reference sequence. GenoREAD can determine if a clone matches its reference sequence. Its sophisticated reporting features help identify and troubleshoot problems that arise during the sequence verification process. GenoREAD has been experimentally validated on thousands of gene-sized constructs from an ORFeome project, and on longer sequences including whole plasmids and synthetic chromosomes. Comparing GenoREAD results with those from manual analysis of the sequencing data demonstrates that GenoREAD tends to be conservative in its diagnostic. GenoREAD is available at www.genoread.org. 相似文献