ABSTRACT Two α-actin genes of the fish Leporinus macrocephalus, referring to white and red muscle tissues, were isolated. Actin isoforms, that mainly differed by a Ser/Ala155 substitution, can have a functional significance related to actin-ATP interaction. An Ala155 residue, as observed in the α-skeletal actin from red muscle, results in a decrease in actin's affinity for ATP, which may also be associated to the slow contractile performance of this tissue. Furthermore, a Phe/Ile262 substitution at the red muscle actin leads to a hydrophobicity variation at the D-plug of the protein, which could alter its stability. Data on qRT-PCR evidenced a significant higher actin mRNA level in white muscle when compared to red muscle (T=105 Mann Whitney; p<0.001). This finding could be related to the energetic demands of the white muscle tissue, with fast contraction fibers and glycolytic metabolism for energy supply. Available data on muscle actins lead to the proposal that white and red α-skeletal actins are genetically and functionally distinguishable in fish species, a feature that is not found in other vertebrate groups.

RESUMO Dois genes de α-actina do peixe Leporinus macrocephalus, referindo-se aos tecidos musculares branco e vermelho, foram isolados. Isoformas de actinas, que principalmente diferiram por uma substituição Ser/Ala155, podem ter uma significância funcional relacionada à interação entre actina e ATP. Um resíduo de Ala155, como observado na α-actina esquelética do músculo vermelho, resulta em uma diminuição da afinidade da actina pelo ATP, o que também pode estar associado à ação contrátil lenta desse tecido. Adicionalmente, uma substituição Phe/Ile262 na actina do músculo vermelho leva a uma variação na hidrofobicidade no "plug-D" da proteína, o que pode alterar sua estabilidade. Dados de qRT-PCR evidenciaram significante maior nível de actina RNAm em músculo branco, quando comparado ao músculo vermelho (T=105 Mann Whitney; p=<0,001). Este resultado pode estar relacionado às demandas energéticas do tecido muscular branco, com fibras de contração rápida e metabolismo glicolítico para fornecimento de energia. Os dados disponíveis sobre actinas musculares levam a propor que as α-actinas esqueléticas dos músculos branco e vermelho são geneticamente e funcionalmente distinguíveis em espécies de peixes, uma característica não encontrada em outros grupos de vertebrados.

A presença de níveis mais elevados do hormônio de crescimento (GH) em animais transgênicos poderia levar a várias alterações fisiológicas. Uma linhagem transgênica de paulistinha (Danio rerio) para o GH foi comparada com amostras não transgênicas (NT) desta espécie, através de uma abordagem de DDRT-PCR, com o objetivo de identificar transcritos candidatos diferencialmente expressos em tecido cerebral que poderiam estar envolvidos na superexpressão de GH. Análises densitométricas de dois produtos de amplificação selecionados, p300 e ADCY2, apontaram uma expressão gênica significativamente menor nas amostras transgênicas de paulistinha (104.02 ± 57.71; 224.10 ± 91.73), quando comparadas com as amostras NT (249.75 ± 30.08; 342.95±65.19). Os presentes dados indicam que p300 e ADCY2 estão envolvidos em um sistema de regulação do GH, quando altos níveis circulantes desse hormônio são encontrados em paulistinha.

The presence of higher level of exogenous growth hormone (GH) in transgenic animals could lead to several physiological alterations. A GH transgenic zebrafish (Danio rerio) line was compared to nontransgenic (NT) samples of the species through a DDRT-PCR approach, with the goal of identifying candidate differentially expressed transcripts in brain tissues that could be involved in GH overexpression. Densitometric analyses of two selected amplification products, p300 and ADCY2, pointed to a significant lower gene expression in the transgenic zebrafish (104.02 ± 57.71; 224.10 ± 91.73) when compared to NT samples (249.75 ± 30.08; 342.95 ± 65.19). The present data indicate that p300 and ADCY2 are involved in a regulation system for GH when high circulating levels of this hormone are found in zebrafishes.

Actin-encoding cDNAs of Nile tilapia (Oreochromis niloticus) were isolated by RT-PCR using total RNA samples of different tissues and further characterized by nucleotide sequencing and in silico amino acid (aa) sequence analysis. Comparisons among the actin gene sequences of O. niloticus and those of other species evidenced that the isolated genes present a high similarity to other fish and other vertebrate actin genes. The highest nucleotide resemblance was observed between O. niloticus and O. mossambicus a-actin and b-actin genes. Analysis of the predicted aa sequences revealed two distinct types of cytoplasmic actins, one cardiac muscle actin type and one skeletal muscle actin type that were expressed in different tissues of Nile tilapia. The evolutionary relationships between the Nile tilapia actin genes and diverse other organisms is discussed.

In order to extend the genetic data on the Sciaenidae fish family, the present study had the purpose to characterize PCR-generated 5S rDNA repeats of twelve species of this group through PAGE (Polyacrylamide Gel Electrophoresis) analysis. The results showed the occurrence of at least two different 5S rDNA size classes in all the species. Moreover, 5S rDNA repeats of one of the studied species - Isopisthus parvipinnis - were cloned and subjected to nucleotide sequencing and Southern blot membrane hybridization analyses, which permitted to confirm the existence of two major 5S rDNA classes. Phylogenetic analysis based on the nucleotide sequences of different 5S rDNA repeats of I. parvipinnis lead to their separation into two major clusters. These results may reflect the high dynamism that rules the evolution rate of 5S rDNA repeats. The obtained data suggest that 5S rDNA can be useful in genetic analyses to identify species-specific markers and determine relationships among species of the Sciaenidae group.

Sharks are suffering from intensive exploitation by worldwide fisheries leading to a severe decline in several populations in the last decades. The lack of biological data on a species-specific basis, associated with a k-strategist life history make it difficult to correctly manage and conserve these animals. The aim of the present study was to develop a DNA-based procedure to discriminate shark species by means of a rapid, low cost and easily applicable PCR analysis based on 5S rDNA repeat units amplification, in order to contribute conservation management of these animals. The generated agarose electrophoresis band patterns allowed to unequivocally distinguish eight shark species. The data showed for the first time that a simple PCR is able to discriminate elasmobranch species. The described 5S rDNA PCR approach generated species-specific genetic markers that should find broad application in fishery management and trade of sharks and their subproducts.

An alpha actin gene segment, isolated from Nile tilapia (Oreochromis niloticus), was characterized by nucleotide sequencing, predicted amino acid sequence and Southern blot hybridization. Genomic DNA amplification resulted in a 1063-bp fragment corresponding to a partial alpha-cardiac muscle actin gene containing exons 3 to 6. Southern blot analysis of the restriction-digested DNA revealed that the Nile tilapia genome contains multiple muscle actin isoforms. Although comparison of the nucleotide sequence, amino acid residues and exon-intron organization of the isolated actin gene with those of other vertebrates showed a high level of identity, diagnostic amino acid residues can still be correlated to distinct actin genes in fish species.

Leporinus elongatus, a fish species widely distributed throughout the Paraná River basin in South America, is an important fishery resource and a valuable species in aquaculture programs. Despite its great economic importance, several wild populations have been suffering a drastic reduction. The comprehension of its population structure represents an important step for the conservation of these organisms in natural environments, and also for the selection of wild stocks to be used in hatchery programs. In order to understand the genetic-population structure of L. elongatus, D-loop mitochondrial DNA analyses were applied in six wild populations of the species. The results were used to estimate the levels of within and among population genetic variability. Although the D-loop variations could not be correlated to the geographic distribution of these organisms, it was possible to detect high levels of genetic variability within each population and the occurrence of exclusive population haplotypes, which suggests a partial genetic differentiation among them. The obtained data can be useful in selecting fish stocks that preserve a better genetic diversity of L. elongatus for use in conservation and/or hatchery programs.