RESUMO
Carotenoids are primarily responsible for the characteristic red flesh coloration of salmon. Flesh coloration is an economically and evolutionarily significant trait that varies inter- and intra-specifically, yet the underlying genetic mechanism is unknown. Chinook salmon (Oncorhynchus tshawytscha) represents an ideal system to study carotenoid variation as, unlike other salmonids, they exhibit extreme differences in carotenoid utilization due to genetic polymorphisms. Here, we crossed populations of Chinook salmon with fixed differences in flesh coloration (red versus white) for a genome-wide association study to identify loci associated with pigmentation. Here, the beta-carotene oxygenase 2-like (BCO2-l) gene was significantly associated with flesh colour, with the most significant single nucleotide polymorphism explaining 66% of the variation in colour. BCO2 gene disruption is linked to carotenoid accumulation in other taxa, therefore we hypothesize that an ancestral mutation partially disrupting BCO2-l activity (i.e. hypomorphic mutation) allowed the deposition and accumulation of carotenoids within Salmonidae. Indeed, we found elevated transcript levels of BCO2-l in white Chinook salmon relative to red. The long-standing mystery of why salmon are red, while no other fishes are, is thus probably explained by a hypomorphic mutation in the proto-salmonid at the time of divergence of red-fleshed salmonid genera (approx. 30 Ma).
Assuntos
Carotenoides/metabolismo , Pigmentação/genética , Salmão/fisiologia , Animais , Aptidão Genética , Polimorfismo GenéticoRESUMO
We report here the comparative DNA sequence analysis of nearly 100 kilobases of contiguous DNA in the C delta to C alpha region of the alpha/delta T cell receptor loci (TCRAC/TCRDC) of mouse and man. This analysis--the largest genomic sequence comparison so far--provides new insights into the functions of the T cell receptor genes as well as the surrounding chromosome structure through the identification of actively conserved DNA sequences. In this comparison we have identified a very high level of organizational and noncoding sequence similarity (approximately 71%) in contrast to previous findings in the beta-globin gene cluster. This observation begins to question the notion that much of the chromosomal non-coding sequence is junk.
Assuntos
Camundongos/genética , Receptores de Antígenos de Linfócitos T/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Genes , Humanos , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
It has been hypothesized that salmonids use olfactory cues to return to their natal rivers and streams. The key components of the molecular pathways involved in imprinting and homing, however, are still unknown. Aquatic chemical cues are received through the nares and into the nasal cavity that contains a single olfactory organ, the olfactory rosette. The olfactory rosette contains sensory neurons, each of which is thought to express only one olfactory receptor. If odorants are involved in salmonid homing migration then olfactory receptors should play a critical role in the dissipation of information from the environment to the fish. Therefore, to understand the molecular basis for imprinting and homing in Atlantic salmon Salmo salar it is important to identify and characterize the repertoire of olfactory receptors in this species. The first public assembly of the S. salar genome was searched for genes encoding three of the superfamilies of fish olfactory receptors: V2R-like (olfc), V1R-like (ora) and main olfactory receptor (mor). A further six ora genes were added to ora1 and ora2, which had been described previously. In addition, 48 putative mors were identified, 24 of which appear to be functional based on their gene structures and predicted amino-acid sequences. Phylogenetic analyses were then used to compare these S. salar olfactory receptor genes with those of zebrafish Danio rerio, two pufferfish species Takifugu rubripes and Tetraodon nigroviridis, medaka Oryzias latipes and three-spined stickleback Gasterosteus aculeatus.
Assuntos
Proteínas de Peixes/genética , Receptores Odorantes/genética , Salmo salar/genética , Animais , Mapeamento Cromossômico , Bases de Dados Genéticas , Filogenia , Análise de Sequência de ProteínaRESUMO
Males are the heterogametic sex in salmonid fishes. In brown trout (Salmo trutta) the sex-determining locus, SEX, has been mapped to the end of linkage group BT-28, which corresponds to linkage group AS-8 and chromosome SSA15 in Atlantic salmon (Salmo salar). We set out to identify the sex chromosomes in brown trout. We isolated Atlantic salmon BAC clones containing microsatellite markers that are on BT-28 and also on AS-8, and used these BACs as probes for fluorescent in situ hybridization (FISH) analysis. SEX is located on the short arm of a small subtelocentric/acrocentric chromosome in brown trout, which is consistent with linkage analysis. The acrocentric chromosome SSA15 in Atlantic salmon appears to have arisen by a centric fusion of 2 small acrocentric chromosomes in the common ancestor of Salmo sp. We speculate that the fusion process that produced Atlantic salmon chromosome SSA15 disrupted the ancestral sex-determining locus in the Atlantic salmon lineage, providing the impetus either for the relocation of SEX or selection pressure for a novel sex-determining gene to arise in this species. Thus, the sex-determining genes may differ in Atlantic salmon and brown trout.
Assuntos
Oncorhynchus mykiss/genética , Salmo salar/genética , Salmonidae/genética , Cromossomos Sexuais , Animais , Feminino , MasculinoRESUMO
It has been hypothesized that salmonids use olfactory cues to return to their natal rivers and streams. However, the key components of the molecular pathway involved in imprinting and homing are still unknown. If odorants are involved in salmon homing migration, then olfactory receptors should play a critical role in the dissipation of information from the environment to the fish. Therefore, we examined the expression profiles of a suite of genes encoding olfactory receptors and other olfactory-related genes in the olfactory rosettes of different life stages in two anadromous and one non-anadromous wild Atlantic salmon populations from Newfoundland, Canada. We identified seven differentially expressed OlfC genes in juvenile anadromous salmon compared to returning adults in both populations of anadromous Atlantic salmon. The salmon from the Campbellton River had an additional 10 genes that were differentially expressed in juveniles compared to returning adults. There was no statistically significant difference in gene expression of any of the genes in the non-anadromous population (P < 0.01). The function of the OlfC gene products is not clear, but they are predicted to be amino acid receptors. Other studies have suggested that salmon use amino acids for imprinting and homing. This study, the first to examine the expression of olfactory-related genes in wild North American Atlantic salmon, has identified seven OlfC genes that may be involved in the imprinting and homeward migration of anadromous Atlantic salmon.
Assuntos
Proteínas de Peixes/metabolismo , Receptores Odorantes/metabolismo , Salmo salar/metabolismo , Migração Animal , Animais , Proteínas de Peixes/genética , Comportamento de Retorno ao Território Vital , Fixação Psicológica Instintiva/fisiologia , Terra Nova e Labrador , Salmo salar/crescimento & desenvolvimento , Salmo salar/fisiologiaRESUMO
Animal domestication and artificial selection give rise to gradual changes at the genomic level in populations. Subsequent footprints of selection, known as selection signatures or selective sweeps, have been traced in the genomes of many animal livestock species by exploiting variation in linkage disequilibrium patterns and/or reduction of genetic diversity. Domestication of most aquatic species is recent in comparison with land animals, and salmonids are one of the most important fish species in aquaculture. Coho salmon (Oncorhynchus kisutch), cultivated primarily in Chile, has been subjected to breeding programs to improve growth, disease resistance traits, and flesh color. This study aimed to identify selection signatures that may be involved in adaptation to culture conditions and traits of productive interest. To do so, individuals of two domestic populations cultured in Chile were genotyped with 200 thousand SNPs, and analyses were conducted using iHS, XP-EHH and CLR. Several signatures of selection on different chromosomal regions were detected across both populations. Some of the identified regions under selection contained genes such anapc2, alad, chp2 and myn, which have been previously associated with body weight in Atlantic salmon, or sec24d and robo1, which have been associated with resistance to Piscirickettsia salmonis in coho salmon. Findings in our study can contribute to an integrated genome-wide map of selection signatures, to help identify the genetic mechanisms of phenotypic diversity in coho salmon.
Assuntos
Aquicultura , Estudo de Associação Genômica Ampla , Oncorhynchus kisutch/genética , Animais , Doenças dos Peixes/microbiologia , Genótipo , Humanos , Oncorhynchus kisutch/microbiologia , Fenótipo , Piscirickettsia/patogenicidade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The phylogenetic relationships among the three subfamilies (Salmoninae, Coregoninae and Thymallinae) in the Salmonidae have not been addressed extensively at the molecular level. In this study, the whole mitochondrial genomes of two Thymallinae species, Thymallus arcticus and Thymallus thymallus were sequenced, and the published mitochondrial genome sequences of other salmonids were used for Bayesian and maximum-likelihood phylogenetic analyses. These results support an ancestral Coregoninae, branching within the Salmonidae, with Thymallinae as the sister group to Salmoninae.
Assuntos
Genoma Mitocondrial/genética , Filogenia , Salmonidae/classificação , Salmonidae/genética , Animais , Dados de Sequência MolecularRESUMO
The human beta T cell receptor (TCR) locus, comprising a complex family of genes, has been sequenced. The locus contains two types of coding elements--TCR elements (65 variable gene segments and two clusters of diversity, joining, and constant segments) and eight trypsinogen genes --that constitute 4.6 percent of the DNA. Genome-wide interspersed repeats and locus-specific repeats span 30 and 47 percent, respectively, of the 685-kilobase sequence. A comparison of the germline variable elements with their approximately 300 complementary DNA counterparts reveals marked differential patterns of variable gene expression, the importance of exonuclease activity in generating TCR diversity, and the predominant tendency for only functional variable elements to be present in complementary DNA libraries.
Assuntos
Família Multigênica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , Cromossomos Humanos Par 7 , Cromossomos Humanos Par 9 , DNA Complementar/genética , Éxons , Variação Genética , Humanos , Íntrons , Dados de Sequência Molecular , Polimorfismo Genético , Regiões Promotoras Genéticas , Pseudogenes , Splicing de RNA , Sequências Repetitivas de Ácido Nucleico , Translocação Genética , Tripsinogênio/genéticaRESUMO
DNA sequence analysis is a multistage process that includes the preparation of DNA, its fragmentation and base analysis, and the interpretation of the resulting sequence information. New technological advances have led to the automation of certain steps in this process and have raised the possibility of large-scale DNA sequencing efforts in the near future [for example, 1 million base pairs (Mb) per year]. New sequencing methodologies, fully automated instrumentation, and improvements in sequencing-related computational resources may render genome-size sequencing projects (100 Mb or larger) feasible during the next 5 to 10 years.
Assuntos
Sequência de Bases , DNA/química , Projeto Genoma Humano , Animais , Automação , Humanos , Espectrometria de Massas , Microscopia/instrumentação , Hibridização de Ácido Nucleico , RobóticaRESUMO
Several important cultured marine fish are highly susceptible to an ectoparasitic condition known as amoebic gill disease (AGD). In AGD-affected fish, modulation of IL-1beta, p53 and p53-regulated transcripts is restricted to the (multi)focal AGD-associated gill lesions. To determine whether this lesion-restricted modulation of transcripts occurs on a transcriptome-wide scale and to identify mechanisms that underpin the susceptibility of fish to AGD, we compared the transcriptome of AGD lesions with "normal" tissue from AGD-affected and healthy individuals. Global gene expression profiling using a 16K salmonid microarray, revealed a total of 176 significantly regulated annotated features and of those, the modulation of 99 (56%) was lesion-restricted. Annotated transcripts were classified according to functional gene ontology. Within the immune response category, transcripts were almost universally down-regulated. In AGD-affected tissue, significant, coordinated down-regulation of the major histocompatibility complex class I (MHC I) pathway-related genes occurred during the later stages of infection and appeared to be mediated by down-regulation of interferon-regulatory factor (IRF)-1, independent of interferon-alpha, interferon-gamma and IRF-2 expression. Within this micro-environment, suppression of the MHC I and possibly the MHC II pathways may inhibit the development of acquired immunity and could explain the unusually high susceptibility of Atlantic salmon to AGD.
Assuntos
Amebíase/veterinária , Amébidos , Apresentação de Antígeno/genética , Doenças dos Peixes/imunologia , Brânquias/imunologia , Salmo salar , Amebíase/genética , Amebíase/imunologia , Amebíase/parasitologia , Animais , Regulação para Baixo , Doenças dos Peixes/genética , Doenças dos Peixes/parasitologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica , Genes MHC Classe I , Genes MHC da Classe II , Brânquias/metabolismo , Brânquias/parasitologia , Fator Regulador 1 de Interferon/metabolismo , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Salmo salar/genética , Salmo salar/imunologia , Salmo salar/parasitologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Genomic resources in rainbow smelt (Osmerus mordax) enable us to examine the genome duplication process in salmonids and test hypotheses relating to the fate of duplicated genes. They further enable us to pursue physiological and ecological studies in smelt. A bacterial artificial chromosome library containing 52,410 clones with an average insert size of 146 kb was constructed. This library represents an 11-fold average coverage of the rainbow smelt (O. mordax) genome. In addition, several complementary deoxyribonucleic acid libraries were constructed, and 36,758 sequences were obtained and combined into 12,159 transcripts. Over half of these transcripts have been identified, several of which have been associated with cold adaptation. These basic resources show high levels of similarity (86%) to salmonid genes and provide initial support for genome duplication in the salmonid ancestor. They also facilitate identification of genes important to fish and direct us toward new technologies for other studies in fish biology.
Assuntos
Etiquetas de Sequências Expressas , Biblioteca Genômica , Osmeriformes/genética , Animais , Cromossomos Artificiais Bacterianos/genética , Temperatura Baixa , Bases de Dados Genéticas , Proteínas de Peixes/genética , Biblioteca Gênica , Dados de Sequência MolecularRESUMO
Three patterns of DNA sequence conservation have been identified from five human and rodent genomic sequence comparisons. First, a divergent pattern was observed in the noncoding sequences of the beta-globin and gamma-crystallin gene clusters, and second, a highly conserved pattern was observed in the noncoding regions of the T cell receptor C alpha-C delta, and the alpha- and beta-myosin-heavy-chain genes. A third, mixed pattern has also been found in the immunoglobulin IgH C mu-C delta gene region. These three patterns of genomic evolution pose the fascinating possibility that large portions of the genome evolve at different rates.
Assuntos
Evolução Molecular , Hominidae/genética , Modelos Genéticos , Roedores/genética , Homologia de Sequência do Ácido Nucleico , Animais , Cristalinas/genética , Genes de Imunoglobulinas , Globinas/genética , Humanos , Cadeias Pesadas de Miosina/genética , Receptores de Antígenos de Linfócitos T/genética , Especificidade da EspécieRESUMO
As more salmon gene expression data has become available, the cDNA microarray platform has emerged as an appealing alternative in ecotoxicological screening of single chemicals and environmental samples relevant to the aquatic environment. This study was performed to validate biomarker gene responses of in vitro cultured rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to model chemicals, and to investigate effects of mixture toxicity in a synthetic mixture. Chemicals used for 24h single chemical- and mixture exposures were 10 nM 17alpha-ethinylestradiol (EE2), 0.75 nM 2,3,7,8-tetrachloro-di-benzodioxin (TCDD), 100 microM paraquat (PQ) and 0.75 microM 4-nitroquinoline-1-oxide (NQO). RNA was isolated from exposed cells, DNAse treated and quality controlled before cDNA synthesis, fluorescent labelling and hybridisation to a 16k salmonid microarray. The salmonid 16k cDNA array identified differential gene expression predictive of exposure, which could be verified by quantitative real time PCR. More precisely, the responses of biomarker genes such as cytochrome p4501A and UDP-glucuronosyl transferase to TCDD exposure, glutathione reductase and gammaglutamyl cysteine synthetase to paraquat exposure, as well as vitellogenin and vitelline envelope protein to EE2 exposure validated the use of microarray applied to RNA extracted from in vitro exposed hepatocytes. The mutagenic compound NQO did not result in any change in gene expression. Results from exposure to a synthetic mixture of the same four chemicals, using identical concentrations as for single chemical exposures, revealed combined effects that were not predicted by results for individual chemicals alone. In general, the response of exposure to this mixture led to an average loss of approximately 60% of the transcriptomic signature found for single chemical exposure. The present findings show that microarray analyses may contribute to our mechanistic understanding of single contaminant mode of action as well as mixture effects, but that its use in screening of complex environmental samples will need to be further evaluated.
Assuntos
Etinilestradiol/toxicidade , Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Compostos Heterocíclicos/toxicidade , Oncorhynchus mykiss/genética , Poluentes Químicos da Água/toxicidade , Animais , Primers do DNA/química , Regulação para Baixo , Sinergismo Farmacológico , Perfilação da Expressão Gênica/veterinária , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Oncorhynchus mykiss/fisiologia , Análise de Componente Principal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Toxicogenética , Regulação para CimaRESUMO
Polychlorinated biphenyls (PCBs) have been associated with a number of toxic effects in marine mammals such as endocrine disruption and immunotoxicity that, in turn, are widely thought to have contributed to population level impacts including reproductive failure and outbreaks of disease. In this study, the dietary hormone vitamin A and expression levels of one of its receptors, retinoic acid receptor alpha (RARalpha), were used as biomarkers of PCB-associated health effects in harbour seals. Harbour seal pups (n=24) were live-captured in coastal British Columbia, Canada, and Washington State, USA, and sampled for whole blood (to obtain peripheral blood mononuclear cells, PBMCs) and blood plasma, as well as biopsies of blubber and skin. Concentrations of circulatory vitamin A (retinol) in plasma and stored vitamin A in blubber were negatively associated with blubber PCB concentrations (R=-0.518, p=0.013 and R=-0.645, p=0.009, respectively). However, vitamin A concentrations in skin, an important target tissue, remained constant, which likely reflects a compensatory transfer from blubber to maintain physiological functions. In addition, we characterized the harbour seal RARalpha, and investigated its expression levels as a potential biomarker in seals. RARalpha expression in blubber, but not on PBMCs, was elevated in more contaminated animals (R=0.580, p=0.009). This may represent a direct contaminant-related effect, or, a compensation for the contaminant-related disruption of (circulatory and/or blubber) hormone levels. Since vitamin A is critical to developmental, reproductive and immunological health, our observations of a contaminant-related disruption of its physiology in free-ranging seals may portend population level consequences. Vitamin A concentrations and RARalpha expression levels can therefore represent relevant and sensitive biomarkers of PCB-associated toxic effects in toxicological studies of marine mammals.
Assuntos
Phoca/fisiologia , Bifenilos Policlorados/toxicidade , Receptores do Ácido Retinoico/efeitos dos fármacos , Vitamina A/análise , Poluentes Químicos da Água/toxicidade , Tecido Adiposo/química , Animais , Sequência de Bases , Biomarcadores/análise , Primers do DNA/química , Feminino , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica/veterinária , Masculino , Dados de Sequência Molecular , Bifenilos Policlorados/análise , Receptores do Ácido Retinoico/análise , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/química , Estatística como AssuntoRESUMO
We have integrated data from linkage mapping, physical mapping and karyotyping to gain a better understanding of the sex-determining locus, SEX, in Atlantic salmon (Salmo salar). SEX has been mapped to Atlantic salmon linkage group 1 (ASL1) and is associated with several microsatellite markers. We have used probes designed from the flanking regions of these sex-linked microsatellite markers to screen a bacterial artificial chromosome (BAC) library, representing an 11.7x coverage of the Atlantic salmon genome, which has been HindIII fingerprinted and assembled into contigs. BACs containing sex-linked microsatellites and their related contigs have been identified and representative BACs have been placed on the Atlantic salmon chromosomes by fluorescent in situ hybridization (FISH). This identified chromosome 2, a large metacentric, as the sex chromosome. By positioning several BACs on this chromosome by FISH, it was possible to orient ASL1 with respect to chromosome 2. The region containing SEX appears to lie on the long arm between marker Ssa202DU and a region of heterochromatin identified by DAPI staining. BAC end-sequencing of clones within sex-linked contigs revealed five hitherto unmapped genes along the sex chromosome. We are using an in silico approach coupled with physical probing of the BAC library to extend the BAC contigs to provide a physical map of ASL1, with a view to sequencing chromosome 2 and, in the process, identifying the sex-determining gene.
Assuntos
Mapeamento Cromossômico , Salmo salar/genética , Animais , Sequência de Bases , Cromossomos Artificiais Bacterianos , Primers do DNA , Feminino , Hibridização in Situ Fluorescente , Masculino , Repetições de Microssatélites , Proteínas/genética , Processos de Determinação SexualRESUMO
Chromosome 7q22 has been the focus of many cytogenetic and molecular studies aimed at delineating regions commonly deleted in myeloid leukemias and myelodysplastic syndromes. We have compared a gene-dense, GC-rich sub-region of 7q22 with the orthologous region on mouse chromosome 5. A physical map of 640 kb of genomic DNA from mouse chromosome 5 was derived from a series of overlapping bacterial artificial chromosomes. A 296 kb segment from the physical map, spanning ACHE: to Tfr2, was compared with 267 kb of human sequence. We identified a conserved linkage of 12 genes including an open reading frame flanked by ACHE: and Asr2, a novel cation-chloride cotransporter interacting protein Cip1, Ephb4, Zan and Perq1. While some of these genes have been previously described, in each case we present new data derived from our comparative sequence analysis. Adjacent unfinished sequence data from the mouse contains an orthologous block of 10 additional genes including three novel cDNA sequences that we subsequently mapped to human 7q22. Methods for displaying comparative genomic information, including unfinished sequence data, are becoming increasingly important. We supplement our printed comparative analysis with a new, Web-based program called Laj (local alignments with java). Laj provides interactive access to archived pairwise sequence alignments via the WWW. It displays synchronized views of a dot-plot, a percent identity plot, a nucleotide-level local alignment and a variety of relevant annotations. Our mouse-human comparison can be viewed at http://web.uvic.ca/~bioweb/laj.html. Laj is available at http://bio.cse.psu.edu/, along with online documentation and additional examples of annotated genomic regions.
Assuntos
Acetilcolinesterase/genética , Cromossomos Humanos Par 7/genética , Cromossomos/genética , Receptores da Transferrina/genética , Animais , Sequência de Bases , DNA/química , DNA/genética , Humanos , Internet , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Fases de Leitura Aberta , Mapeamento Físico do Cromossomo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Repetições de Trinucleotídeos , Células Tumorais CultivadasRESUMO
Apoptotic cell suicide initiated by ligation of CD95 (Fas/APO-1) occurs through recruitment, oligomerization and autocatalytic activation of the cysteine protease, caspase-8 (MACH, FLICE, Mch5). An endogenous mammalian regulator of this process, named Usurpin, has been identified (aliases for Usurpin include CASH, Casper, CLARP, FLAME-1, FLIP, I-FLICE and MRIT). This protein is ubiquitously expressed and exists as at least three isoforms arising by alternative mRNA splicing. The Usurpin gene is comprised of 13 exons and is clustered within approximately 200 Kb with the caspase-8 and -10 genes on human chromosome 2q33-34. The Usurpin polypeptide has features in common with pro-caspase-8 and -10, including tandem 'death effector domains' on the N-terminus of a large subunit/small subunit caspase-like domain, but it lacks key residues that are necessary for caspase proteolytic activity, including the His and Cys which form the catalytic substrates diad, and residues that stabilize the P1 aspartic acid in substrates. Retro-mutation of these residues to functional caspase counterparts failed to restore proteolytic activity, indicating that other determinants also ensure the absence of catalytic potential. Usurpin heterodimerized with pro-caspase-8 in vitro and precluded pro-caspase-8 recruitment by the FADD/MORT1 adapter protein. Cell death induced by CD95 (Fas/APO-1) ligation was attenuated in cells transfected with Usurpin. In vivo, a Usurpin deficit was found in cardiac infarcts where TUNEL-positive myocytes and active caspase-3 expression were prominent following ischemia/reperfusion injury. In contrast, abundant Usurpin expression (and a caspase-3 deficit) occurred in surrounding unaffected cardiac tissue, suggesting reciprocal regulation of these pro- and anti-apoptotic molecules in vivo. Usurpin thus appears to be an endogenous modulator of apoptosis sensitivity in mammalian cells, including the susceptibility of cardiac myocytes to apoptotic death following ischemia/ reperfusion injury.
Assuntos
Apoptose/fisiologia , Caspases/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Receptor fas/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Apoptose/genética , Apoptose/imunologia , Sequência de Bases , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Caspase 8 , Caspase 9 , Caspases/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 2/genética , Clonagem Molecular , Primers do DNA/genética , Ativação Enzimática , Feminino , Células HeLa , Humanos , Células Jurkat , Masculino , Modelos Biológicos , Dados de Sequência Molecular , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
In phylogenetic reconstructions by the parsimony method, utilizing 62 sequenced globin genes and pseudogenes (including 34 of the beta-globin gene family from eutherian orders Primates, Lagomorpha, Artiodactyla and Rodentia), the branch of primate psi beta pseudogenes and the goat embryonically expressed epsilon II gene group monophyletically together as orthologues of a common ancestral gene (labelled eta) distinct from orthologues of epsilon, gamma, delta and beta. This primate psi eta-goat eta branch is cladistically closer to epsilon and gamma than to delta and beta branches. In each eutherian order gene conversions replaced portions of delta by beta sequences, whereas in descent of Primates epsilon, gamma and eta mostly retained their separate ancient identities predating the radiation of Eutheria in all their exons and non-coding regions. The loci of the ancestral beta-globin gene cluster in basal eutherians and proto-primates, as deduced from beta-clusters representing the four eutherian orders, were linked 5'-epsilon-gamma-eta-delta-beta-3' with epsilon, gamma and eta being embryonically expressed genes, and delta and beta ontogenetically later expressed genes. Through deletions gamma was lost in artiodactyl evolution, eta in lagomorph and rodent evolution, and all DNA between exon 2 3' boundaries of eta and delta in prosimian lemuriform evolution (lemur having the hybrid pseudogene psi eta delta). Simian primates retained intact the five loci of the ancestral cluster. Not only did eta, after it became a pseudogene in the basal primates, persist intact in descent to present-day simians but in the line to hominoids it evolved during the last 40 million years at the decelerated rate of 1 X 10(-9) substitutions/site per year which is one-fifth the expected neutral rate. The possibility is suggested that the psi eta locus situated between fetal and adult chromosomal domains of the simian beta-globin gene cluster might play some role in a mechanism for ontogenetic switches of globin gene expression. However, not enough sequence data on genes and intergenic regions in DNA of species of primates and other mammals as yet exist to know if the slow rate of 1 X 10(-9) reflects the rate of a conserved functional gene or primarily reflects a decelerated neutral rate of hominoid DNA evolution, conceivably from enhanced DNA repair and longer generation times in hominoids. The further possibility is raised that gene correction (repair of damaged DNA that prevents emergence of new alleles) and gene conversion both more often involve strand copying of conserved than of rapidly evolving DNA.
Assuntos
Evolução Biológica , Globinas/genética , Animais , Sequência de Bases , Aves , DNA , Cabras , Humanos , Camundongos , Filogenia , Primatas , CoelhosRESUMO
Sequence analysis of epsilon and gamma genes and encoded globins and high-pressure liquid chromatography analysis of globin compositions in blood hemolysates obtained from embryos, fetuses and adults show that the prosimian primate Galago crassicaudatus expresses its epsilon and gamma genes only embryonically. Since rabbit, mouse and galago all have embryonic gamma genes but simian primates have fetal gamma genes, we conclude that gamma E evolved into gamma F in stem-simians. An elevated non-synonymous substitution rate characterizes this transition. The alignment of epsilon and gamma nucleotide sequences and the parsimoniously reconstructed evolutionary history of these sequences identify several anciently conserved cis-regulatory elements (phylogenetic footprints) important for gamma expression in primates and also cis-mutations which may have been involved in the recruitment of the gamma gene to a fetal program in simian primates.
Assuntos
Galago/genética , Globinas , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Embrião de Mamíferos/fisiologia , Feto/fisiologia , Galago/embriologia , Galago/crescimento & desenvolvimento , Genes , Globinas/metabolismo , Hemoglobinas , Dados de Sequência Molecular , Mutação , FilogeniaRESUMO
Southern blot analysis of T cell receptor (TCR) gene rearrangements has proven to be a helpful tool to establish clonality in T cell leukemias and lymphomas. To improve the detection of clonal TCR gamma (TCRG) gene rearrangements by Southern blot analysis, we designed four new Jgamma probes and determined the most optimal restriction enzymes to be used with these probes. Based on detailed analysis of the sequences as well as on hybridization experiments with the TCRGJ21 probe, the Jgamma1.2 and Jgamma2.1 downstream areas were found to be highly homologous, suggesting that during evolution the duplication of the Jgamma region was followed by deletion of the tentative Jgamma2.2 gene segment. Southern blot analysis of 51 T cell acute lymphoblastic leukemias (T-ALL) revealed that all TCRG gene rearrangements can be detected by use of the TCRGJ13 probe in EcoRI digests and the TCRGJ21 probe in PstI digests. Additional probes and digests allow a more precise identification of the exact type of TCRG gene rearrangements in the majority of cases. Almost 90% of the TCRG gene rearrangements in T-ALL involved the Jgamma2 region (16% Jgamma2.1 and 72% Jgamma2.3), whereas Jgamma1 region rearrangements were particularly found in TCRgammadelta+ T-ALL. This information has implications for design of primer sets for PCR analysis at diagnosis and for PCR target choice in detection of minimal residual disease during follow-up of T-ALL patients.