Sampling the skin transcriptome of the North Atlantic right whale.
ABSTRACT As an initial step in defining the transcriptome of the North Atlantic right whale (Eubalaena glacialis) and developing functional genomic tools to study right whale health at the molecular physiological level, a cDNA library has been constructed from a skin biopsy. 2496 randomly selected clones (expressed sequence tags, ESTs) have been sequenced, and genes identified as important in the response to stress and immune challenges have been cloned by targeted RT-PCR from skin cDNA. The analysis of the EST collection (archived at www.marinegenomics.org and GenBank) showed a 34.79% redundancy, yielding 1578 unigenes and 27 potential microsatellite markers. 96 genes were cloned by targeted PCR; moreover, 52 of these genes are stress and immune function related. A Gene Ontology analysis of the unigene collection indicates that the skin is a rich source of expressed genes with diverse functions, suggesting an important role in multiple physiological processes including those related to immunity and stress response.
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ABSTRACT: Despite international success in reducing ozone-depleting emissions, ultraviolet radiation (UV) is not expected to decrease for several decades. Thus, it is pressing to implement tools that allow investigating the capacity of wildlife to respond to excessive UV, particularly species like cetaceans that lack anatomical or physiological protection. One approach is to examine epidermal expression of key genes involved in genotoxic stress response pathways. However, quantitation of mRNA transcripts requires previous standardization, with accurate selection of control and target genes. The latter is particularly important when working with environmental stressors such as UV that can activate numerous genes. Using 20 epidermal biopsies from blue, fin and sperm whale, we found that the genes encoding the ribosomal proteins L4 and S18 (RPL4 and RPS18) were the most suitable to use as controls, followed by the genes encoding phosphoglycerate kinase 1 (PGK1) and succinate dehydrogenase complex subunit A (SDHA). A careful analysis of the transcription pathways known to be activated by UV-exposure in humans and mice led us to select as target genes those encoding for i) heat shock protein 70 (HSP70) an indicator of general cell stress, ii) tumour suppressor protein P53 (P53), a transcription factor activated by UV and other cell stressors, and iii) KIN17 (KIN), a cell cycle protein known to be up-regulated following UV exposure. These genes were successfully amplified in the three species and quantitation of their mRNA transcripts was standardised using RPL4 and RPS18. Using a larger sample set of 60 whale skin biopsies, we found that the target gene with highest expression was HSP70 and that its levels of transcription were correlated with those of KIN and P53. Expression of HSP70 and P53 were both related to microscopic sunburn lesions recorded in the whales' skin. This article presents groundwork data essential for future qPCR-based studies on the capacity of wildlife to resolve or limit UV-induced damage. The proposed target genes are HSP70, P53 and KIN, known to be involved in genotoxic stress pathways, and whose expression patterns can be accurately assessed by using two stable control genes, RPL4 and RPS18.BMC Research Notes 07/2013; 6(1):264. DOI:10.1186/1756-0500-6-264
American journal of men's health 10/2010; 7(3):307-307. DOI:10.1016/j.jomh.2010.09.084 · 1.15 Impact Factor
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ABSTRACT: Background Transcriptomes are powerful resources, providing a window on the expressed portion of the genome that can be generated rapidly and at low cost for virtually any organism. However, because many genes have tissue-specific expression patterns, developing a complete transcriptome usually requires a 'discovery pool' of individuals to be sacrificed in order to harvest mRNA from as many different types of tissue as possible. This hinders transcriptome development in large, charismatic and endangered species, many of which stand the most to gain from such approaches. To circumvent this problem in a model pinniped species, we 454 sequenced cDNA from testis, heart, spleen, intestine, kidney and lung tissues obtained from nine adult male Antarctic fur seals (Arctocephalus gazella) that died of natural causes at Bird Island, South Georgia. Results After applying stringent quality control criteria based on length and annotation, we obtained 12,397 contigs which, in combination with 454 data previously obtained from skin, gave a total of 23,096 unique contigs. Homology was found to 77.0% of dog (Canis lupus familiaris) transcripts, suggesting that the combined assembly represents a substantial proportion of this species' transcriptome. Moreover, only 0.5% of transcripts revealed sequence similarity to bacteria, implying minimal contamination, and the percentage of transcripts involved in cell death was low at 2.6%. Transcripts with immune-related annotations were almost five-fold enriched relative to skin and represented 13.2% of all spleen-specific contigs. By reference to the dog, we also identified transcripts revealing homology to five class I, ten class II and three class III genes of the Major Histocompatibility Complex and derived the putative genomic distribution of 17,121 contigs, 2,119 in silico mined microsatellites and 9,382 single nucleotide polymorphisms. Conclusions Our findings suggest that transcriptome development based on samples collected post mortem may greatly facilitate genomic studies, not only of marine mammals but also more generally of species that are of conservation concern.BMC Genomics 01/2013; 14(1):52. DOI:10.1186/1471-2164-14-52 · 4.04 Impact Factor