Leigh Syndrome (LS) is a severe neurological disorder characterized by bilaterally symmetrical necrotic lesions in subcortical brain regions that is commonly associated with systemic cytochrome c oxidase (COX) deficiency. COX deficiency is an autosomal recessive trait and most patients belong to a single genetic complementation group. DNA sequence analysis of the genes encoding the structural subunits of the COX complex has failed to identify a pathogenic mutation. Using microcell-mediated chromosome transfer, we mapped the gene defect in this disorder to chromosome 9q34 by complementation of the respiratory chain deficiency in patient fibroblasts. Analysis of a candidate gene (SURF1) of unknown function revealed several mutations, all of which predict a truncated protein. These data suggest a role for SURF1 in the biogenesis of the COX complex and define a new class of gene defects causing human neurodegenerative disease.

Background: Fast, efficiently growing animals have increased protein synthesis and/or reduced protein degradation relative to slow, inefficiently growing animals. Consequently, minimizing the energetic cost of protein turnover is a strategic goal for enhancing animal growth. Characterization of gene expression profiles associated with protein turnover would allow us to identify genes that could potentially be used as molecular biomarkers to select for germplasm with improved protein accretion.

Fast growth is an important and highly desired trait, which affects the profitability of food animal production, with feed costs accounting for the largest proportion of production costs. Traditional phenotype-based selection is typically used to select for growth traits; however, genetic improvement is slow over generations. Single nucleotide polymorphisms (SNPs) explain 90% of the genetic differences between individuals; therefore, they are most suitable for genetic evaluation and strategies that employ molecular genetics for selective breeding. SNPs found within or near a coding sequence are of particular interest because they are more likely to alter the biological function of a protein. We aimed to use SNPs to identify markers and genes associated with genetic variation in growth. RNA-Seq whole-transcriptome analysis of pooled cDNA samples from a population of rainbow trout selected for improved growth versus unselected genetic cohorts (10 fish from 1 full-sib family each) identified SNP markers associated with growth-rate. The allelic imbalances (the ratio between the allele frequencies of the fast growing sample and that of the slow growing sample) were considered at scores >5.0 as an amplification and <0.2 as loss of heterozygosity. A subset of SNPs (n = 54) were validated and evaluated for association with growth traits in 778 individuals of a three-generation parent/offspring panel representing 40 families. Twenty-two SNP markers and one mitochondrial haplotype were significantly associated with growth traits. Polymorphism of 48 of the markers was confirmed in other commercially important aquaculture stocks. Many markers were clustered into genes of metabolic energy production pathways and are suitable candidates for genetic selection. The study demonstrates that RNA-Seq at low sequence coverage of divergent populations is a fast and effective means of identifying SNPs, with allelic imbalances between phenotypes. This technique is suitable for marker development in non-model species lacking complete and well-annotated genome reference sequences.

Nuclear proteins such as transcription and chromatin remodeling factors are required for initiation of transcription in early embryos before embryonic genome activation. The nuclear transport of these proteins is mediated by transport factors such as importins. Through analysis of expressed sequence tags from a bovine oocyte cDNA library, we identified a new member of the importin alpha family (named importin alpha8). The cloned cDNA for bovine importin alpha8 (KPNA7) is 1817 base pair in length, encoding a protein of 522 amino acids that contains a conserved importin beta-binding domain and seven armadillo motifs. The RT-PCR analysis revealed that KPNA7 mRNA is specifically expressed in ovaries and mature oocytes. Real-time PCR demonstrated that KPNA7 expression in germinal vesicle (GV) oocytes is 33 to 2396 times higher than that of other importin alpha genes and that KPNA7 mRNA is abundant in GV and metaphase II oocytes, as well as in early-stage embryos collected before embryonic genome activation, but is barely detectable in morula- and blastocyst-stage embryos. Similarly, expression of KPNA7 protein is very high in oocytes and early embryos but is low in blastocysts. A glutathione S-transferase pull-down assay revealed that KPNA7 has a strong binding affinity for the nuclear protein nucleoplasmin 2 relative to that of other importin alphas. RNA interference experiments demonstrated that knockdown of KPNA7 in early embryos results in a decreased proportion of embryos developing to the blastocyst stage. These results suggest that KPNA7 may have an important role in the transport of essential nuclear proteins required for early embryogenesis.

Salem M, Kenney PB, Rexroad CE 3rd, Yao J. Microarray gene expression analysis in atrophying rainbow trout muscle: a unique nonmammalian muscle degradation model. Physiol Genomics 28: 33-45, 2006. First published August 1, 2006; doi:10.1152/physiolgenomics.00114.2006.-Muscle atrophy is a physiological response to diverse physiological and pathological conditions that trigger muscle deterioration through specific cellular mechanisms. Despite different signals, the biochemical changes in atrophying muscle share many common cascades. Muscle deterioration as a physiological response to the energetic demands of fish vitellogenesis represents a unique model for studying the mechanisms of muscle degradation in non-mammalian animals. A salmonid microarray, containing 16,006 cDNAs, was used to study the transcriptome response to atrophy of fast-switch muscles from gravid rainbow trout compared with sterile fish. Eighty-two unique transcripts were upregulated and 120 transcripts were downregulated in atrophying muscles. Transcripts having gene ontology identifiers were grouped according to their functions. Muscle deterioration was associated with elevated expression of genes involved in the catheptic and collagenase proteolytic pathways; the aerobic production, buffering, and utilization of ATP; and growth arrest; whereas atrophying muscle showed downregulation of genes encoding a serine proteinase inhibitor, enzymes of anaerobic respiration, muscle proteins as well as genes required for RNA and protein biosynthesis/processing. Therefore, gene transcription of the trout muscle atrophy changed in a manner similar to mammalian muscle atrophy. These changes result in an arrest of normal cell growth, protein degradation, and decreased glycolytic cellular respiration that is characteristic of the fast-switch muscle. For the first time, other changes/mechanisms unique to fish were discussed including genes associated with muscle atrophy. atrophy; Oncorhynchus mykiss SEVERAL PHYSIOLOGICAL and pathological conditions can cause muscle atrophy by triggering unique responses through distinct cellular stimuli. Transcriptional mechanisms of muscle wastage are well characterized at the transcriptome level in mammalian models as a response to nutritional restriction (7), fasting, severe diseases, including cancer, renal failure, diabetes (41, 42), and sepsis (42), as well as muscle disuse or denervation (4, 57). Some studies have dealt with fish muscle atrophy at the level of individual genes/mechanisms (46, 51, 58, 60 -62). The molecular basis of mammalian muscle atrophy has received considerable attention in the literature (25,30). Losing muscle mass results from elevated rate of proteolysis and decreased rate of protein synthesis. Despite the significant amount of literature (30,32,42), the specific roles of the proteolytic systems in degrading mammalian muscle still are unclear. Moreover, we recently showed that fish use different muscle degrading proteolytic strategies compared with mammals (58). The ubiquitin-proteasome pathway,...

BackgroundTo enhance capabilities for genomic analyses in rainbow trout, such as genomic selection, a large suite of polymorphic markers that are amenable to high-throughput genotyping protocols must be identified. Expressed Sequence Tags (ESTs) have been used for single nucleotide polymorphism (SNP) discovery in salmonids. In those strategies, the salmonid semi-tetraploid genomes often led to assemblies of paralogous sequences and therefore resulted in a high rate of false positive SNP identification. Sequencing genomic DNA using primers identified from ESTs proved to be an effective but time consuming methodology of SNP identification in rainbow trout, therefore not suitable for high throughput SNP discovery. In this study, we employed a high-throughput strategy that used pyrosequencing technology to generate data from a reduced representation library constructed with genomic DNA pooled from 96 unrelated rainbow trout that represent the National Center for Cool and Cold Water Aquaculture (NCCCWA) broodstock population.ResultsThe reduced representation library consisted of 440 bp fragments resulting from complete digestion with the restriction enzyme HaeIII; sequencing produced 2,000,000 reads providing an average 6 fold coverage of the estimated 150,000 unique genomic restriction fragments (300,000 fragment ends). Three independent data analyses identified 22,022 to 47,128 putative SNPs on 13,140 to 24,627 independent contigs. A set of 384 putative SNPs, randomly selected from the sets produced by the three analyses were genotyped on individual fish to determine the validation rate of putative SNPs among analyses, distinguish apparent SNPs that actually represent paralogous loci in the tetraploid genome, examine Mendelian segregation, and place the validated SNPs on the rainbow trout linkage map. Approximately 48% (183) of the putative SNPs were validated; 167 markers were successfully incorporated into the rainbow trout linkage map. In addition, 2% of the sequences from the validated markers were associated with rainbow trout transcripts.ConclusionThe use of reduced representation libraries and pyrosequencing technology proved to be an effective strategy for the discovery of a high number of putative SNPs in rainbow trout; however, modifications to the technique to decrease the false discovery rate resulting from the evolutionary recent genome duplication would be desirable.

BackgroundRainbow trout are important fish for aquaculture and recreational fisheries and serves as a model species for research investigations associated with carcinogenesis, comparative immunology, toxicology and evolutionary biology. However, to date there is no genome reference sequence to facilitate the development of molecular technologies that utilize high-throughput characterizations of gene expression and genetic variation. Alternatively, transcriptome sequencing is a rapid and efficient means for gene discovery and genetic marker development. Although a large number (258,973) of EST sequences are publicly available, the nature of rainbow trout duplicated genome hinders assembly and complicates annotation.ResultsHigh-throughput deep sequencing of the Swanson rainbow trout doubled-haploid transcriptome using 454-pyrosequencing technology yielded ~1.3 million reads with an average length of 344 bp, a total of 447 million bases. De novo assembly of the sequences yielded 151,847 Tentative Consensus (TC) sequences (average length of 662 bp) and 224,391 singletons. A combination assembly of both the 454-pyrosequencing ESTs and the pre-existing sequences resulted in 161,818 TCs (average length of 758 bp) and 261,071 singletons. Gene Ontology analysis of the combination assembly showed high similarities to transcriptomes of other fish species with known genome sequences.ConclusionThe 454 library significantly increased the suite of ESTs available for rainbow trout, allowing improved assembly and annotation of the transcriptome. Furthermore, the 454 sequencing enables functional genome research in rainbow trout, providing a wealth of sequence data to serve as a reference transcriptome for future studies including identification of paralogous sequences and/or allelic variation, digital gene expression and proteomic research.

The present study was undertaken to discover molecular markers in bovine cumulus cells predictive of oocyte competence and to elucidate their functional significance. Differences in RNA transcript abundance in cumulus cells harvested from oocytes of adult versus prepubertal animals (a model of poor oocyte quality) were identified by microarray analysis. Four genes of interest encoding for the lysosomal cysteine proteinases cathepsins B, S, K, and Z and displaying greater transcript abundance in cumulus cells surrounding oocytes harvested from prepubertal animals were chosen for further investigation. Greater mRNA abundance for such genes in cumulus cells of prepubertal oocytes was confirmed by real-time RT-PCR. Elevated transcript abundance for cathepsins B, S, and Z also was observed in cumulus cells surrounding adult metaphase II oocytes that developed to the blastocyst stage at a low percentage following parthenogenetic activation versus those that developed at a high percentage. Functional significance of cumulus cell cathepsin expression to oocyte competence was confirmed by treatment of cumulus-oocyte complexes during in vitro oocyte maturation with a cell-permeable cysteine proteinase (cathepsin) inhibitor. Inhibitor treatment decreased apoptotic nuclei in the cumulus layer and enhanced development of parthenogenetically activated and in vitro-fertilized adult oocytes to the blastocyst stage. Stimulatory effects of inhibitor treatment during meiotic maturation on subsequent embryonic development were not observed when oocytes were matured in the absence of cumulus cells. The present results support a functional role for cumulus cell cathepsins in compromised oocyte competence and suggest that cumulus cell cathepsin mRNA abundance may be predictive of oocyte quality.