RESUMEN
A purpose-designed microarray platform (Stressgenes, Phase 1) was utilised to investigate the changes in gene expression within the liver of rainbow trout during exposure to a prolonged period of confinement. Tissue and blood samples were collected from trout at intervals up to 648 h after transfer to a standardised confinement stressor, together with matched samples from undisturbed control fish. Plasma ACTH, cortisol, glucose and lactate were analysed to confirm that the neuroendocrine response to confinement was consistent with previous findings and to provide a phenotypic context to assist interpretation of gene expression data. Liver samples for suppression subtractive hybridisation (SSH) library construction were selected from within the experimental groups comprising "early" stress (2-48 h) and "late" stress (96-504 h). In order to reduce redundancy within the four SSH libraries and yield a higher number of unique clones an additional subtraction was carried out. After printing of the arrays a series of 55 hybridisations were executed to cover 6 time points. At 2 h, 6 h, 24 h, 168 h and 504 h 5 individual confined fish and 5 individual control fish were used with control fish only at 0 h. A preliminary list of 314 clones considered differentially regulated over the complete time course was generated by a combination of data analysis approaches and the most significant gene expression changes were found to occur during the 24 h to 168 h time period with a general approach to control levels by 504 h. Few changes in expression were apparent over the first 6 h. The list of genes whose expression was significantly altered comprised predominantly genes belonging to the biological process category (response to stimulus) and one cellular component category (extracellular region) and were dominated by so-called acute phase proteins. Analysis of the gene expression profile in liver tissue during confinement revealed a number of significant clusters. The major patterns comprised genes that were up-regulated at 24 h and beyond, the primary examples being haptoglobin, beta-fibrinogen and EST10729. Two representative genes from each of the six k-means clusters were validated by qPCR. Correlations between microarray and qPCR expression patterns were significant for most of the genes tested. qPCR analysis revealed that haptoglobin expression was up-regulated approximately 8-fold at 24 h and over 13-fold by 168 h.
RESUMEN
Bifidobacterium pseudolongum subsp. globosum DPC479 is an intestinally-derived strain which contains a plasmid, pASV479, 4.8 kb in size. This plasmid has a G + C content of 59% and contains six open reading frames (ORFs), four of which are cryptic. The other two ORFs have 47% and 54% identity, respectively, to the replication and FtsK-like proteins found in a Bifidobacterium breve NCFB 2258 plasmid, indicating that these plasmids, though isolated from differing Bifidobacterium species, are related. Using this plasmid as a backbone, an expression vector, pBIFRIBO, was constructed which exploits a bifidobacteria rRNA promoter.
Asunto(s)
Bifidobacterium/genética , Vectores Genéticos/genética , Plásmidos/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Emparejamiento Base , Secuencia de Bases , Cromosomas Bacterianos , Secuencia Conservada , ADN Bacteriano/química , ADN Bacteriano/genética , Glucuronidasa/análisis , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Mapeo Físico de Cromosoma , Regiones Promotoras Genéticas , ARN Bacteriano/genética , ARN Ribosómico/genética , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
A homologue of IkappaBalpha, the alpha member of the IkappaB family of NF-kappaB inhibitors, was identified in a Rainbow trout suppression subtractive hybridization library enriched in sequences up-regulated in cultured leukocytes after lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFalpha) stimulation. The full-length cDNA was isolated and sequenced. The predicted amino acid sequence is 61.5% similar and 54% identical to human IkappaBalpha, while only 42% similar and 35% identical to IkappaBbeta, and 38% similar and 32% identical to IkappaBvarepsilon. Rainbow trout IkappaBalpha contains a central ankyrin repeat domain required for its interaction with NF-kappaB and a putative PEST-like sequence in the C-terminus. Expression of IkappaBalpha is up-regulated by LPS and TNFalpha treatment, two known activators of NF-kappaB, suggesting the existence of an autoregulatory loop in fish, as is the case for mammals. These results confirm the existence of the NF-kappaB signalling pathway in fish and suggest a similar functional interaction between IkappaBalpha and NF-kappaB.
Asunto(s)
Proteínas I-kappa B/genética , FN-kappa B/antagonistas & inhibidores , Oncorhynchus mykiss/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Humanos , Proteínas I-kappa B/química , Proteínas I-kappa B/fisiología , Lipopolisacáridos/farmacología , Ratones , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , Fosforilación , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
A homologue of mammalian type II interleukin-1 receptor (IL-1RII) was isolated from a rainbow trout cDNA library by differential hybridization using a suppression subtractive hybridization generated probe enriched for sequences upregulated after immune stimulation. The trout cDNA has an ORF encoding 441 amino acids, and represents the first piscine IL-1 receptor described. The predicted amino-acid sequence has 29 and 26% identity with human and mouse IL-1RII, respectively. The trout IL-1 receptor has a domain organization similar to that of mammalian type II receptor, with a short cytoplasmic tail of 24 amino acids. These results suggest that type II receptor is also present in lower vertebrates, and therefore the duplication of an ancestral gene that generated type I and type II IL-1 receptors occurred prior to the time mammals emerged.