南极鱼类多样性和适应性进化研究进展

南极地区是地球上唯一未被人类活动大量影响的地区, 其极端寒冷的环境为南极生物的进化提供了“温床”。过去三千万年间, 南极鱼亚目鱼类在南极海洋逐渐变冷的过程中快速进化, 从一个温暖海域的底栖祖先分化成南极海域最为多样化的鱼类类群。由于其在南极圈内和南极圈外的各种温度区间都有分布, 因而成为研究鱼类适应性进化和耐寒机制的良好生物模型。本文综述了有关南极海域鱼类区系组成与物种多样性现状, 南极鱼亚目鱼类适应低温的一系列特化的生物学性状及其关键的遗传进化机制。现有研究表明: 南极鱼类在几千万年零度以下低温环境的进化中发生了大量基因的大规模扩增和基因表达的改变, 如铁调素、卵壳蛋白和逆转座子等118个基因发生了显著的扩增。另外, 有些从南极鱼中获得的抗寒基因已经用于提高动植物低温抗性的研究并取得了良好的效果。在今后的几年中, 将会有多个南极鱼物种的全基因组得到破译, 在低温适应相关基因的功能和进化方面的研究也会更加深入, 这些研究将深入揭示低温压力下基因组的进化规律以及鱼类低温适应的分子机制。     

Radioimmunoassay of metallothionein in rabbit,rat, mouse,Chinese hamster,and human cells

We describe a competitive, solid-phase radioimmunoassay for metallothionein, which employs a rabbit antiserum directed against rat MT-2 to detect metallothionein (MT) from several different species (rabbit, mouse, rat, Chinese hamster, and human). The lower limit of detection of the assay for rat MT-2 was 0.7 ng; for rabbit MT-2 it was 2 ng. The method is capable of measuring both isoforms of MT (MT-1 and MT-2). When MT levels in rat and mouse tissues were estimated with this RIA and the silver-saturation method, both assays gave the same pattern of MT induction in control and cadmium-treated animals. Both methods measured high levels of MT in human liver samples. Chinese hamster ovary cells induced with cadmium also showed elevated MT expression. The detectability of MTs from a broad range of species is facilitated by the use of solid-phase MT, which has an avidity for the antiserum similar to that of the MT in the tested sample.     

The complete nuclear estrogen receptor family in the rainbow trout: discovery of the novel ERalpha2 and both ERbeta isoforms

Estrogen hormones interact with cellular ERs to exert their biological effects in vertebrate animals. Similar to other animals, fishes have two distinct ER subtypes, ERalpha (NR3A1) and ERbeta (NR3A2). The ERbeta subtype is found as two different isoforms in several fish species because of a gene duplication event. Although predicted, two different isoforms of ERalpha have not been demonstrated in any fish species. In the rainbow trout (Oncorhynchus mykiss), the only ER described is an isoform of the ERalpha subtype (i.e. ERalpha1, NR3A1a). The purpose of this study was to determine whether the gene for the other ERalpha isoform, ERalpha2 (i.e., NR3A1b), exists in the rainbow trout. A RT-PCR and cloning strategy, followed by screening a rainbow trout BAC library yielded a unique DNA sequence coding for 558 amino acids. The deduced amino acid sequence had a 75.4% overall similarity to ERalpha1. Both the rainbow trout ERbeta subtypes, ERbeta1 [NR3A2a] and ERbeta2, [NR3A2b] which were previously unknown in this species, were also sequenced as part of this study, and the amino acid sequences were found to be very different from the ERalphas (approximately 40% similarity). ERbeta1 and ERbeta2 had 594 and 604 amino acids, respectively, and had 57.6% sequence similarity when compared to one another. This information provides what we expect to be the first complete nuclear ER gene family in a fish. A comprehensive phylogenetic analysis with all other known fish ER gene sequences was undertaken to understand the evolution of fish ERs. The results show a single ERalpha subtype clade, with the closest relative to rainbow trout ERalpha2 being rainbow trout ERalpha1, suggesting a recent, unique duplication event to create these two isoforms. For the ERbeta subtype there are two distinct subclades, one represented by the ERbeta1 isoform and the other by the ERbeta2 isoform. The rainbow trout ERbeta1 and ERbeta2 are not closely associated with each other, but instead fall into their respective ERbeta subclades with other known fish species. Real-time RT-PCR was used to measure the mRNA levels of all four ER isoforms (ERalpha1, ERalpha2, ERbeta1, and ERbeta2) in stomach, spleen, heart, brain, pituitary, muscle, anterior kidney, posterior kidney, liver, gill, testis and ovary samples from rainbow trout. The mRNAs for each of the four ERs were detected in every tissue examined. The liver tended to have the highest ER mRNA levels along with the testes, while the lowest levels were generally found in the stomach or heart. The nuclear ERs have a significant and ubiquitous distribution in the rainbow trout providing the potential for complex interactions that involve the functioning of many organ systems.     

Mitochondrial phylogeny of notothenioids: a molecular approach to Antarctic fish evolution and biogeography

Antarctic waters represent a unique marine environment delimited by an oceanographic barrier, the Polar Front Zone, and characterized by constant subzero temperatures and presence of sea ice. A group of teleost fish, the Notothenioidei, have adapted to these challenging environmental conditions, undergoing a remarkable diversification. In the present study a total of 798 base pairs, generated from partial sequencing of 16S and 12S mitochondrial ribosomal RNA genes, were examined in 33 notothenioid species representative of all families included in the suborder Notothenioidei. Phylogenetic trees, reconstructed on the basis of sequence data by different methods, indicate that traditional hypotheses on notothenioid systematics and biogeography might be in need of reexamination. Molecular evidence suggests that vicariant speciation could be invoked to explain the early divergence of Eleginops maclovinus, a species previously included in the family Nototheniidae, which is now proposed as the closest sister group to all the rest of notothenioids apart from bovichtids. On the other hand, repeated, independent dispersal through the Polar Front is proposed for the divergence of other subantarctic notothenioid species. Likewise, multiple, independent transitions from benthic to pelagic habit are inferred from molecular data, at variance with the more conservative hypothesis based on cladograms reconstructed from morphological data.     

Characterization of the intestinal microbiota of two Antarctic notothenioid fish species

The fish fauna of the Southern Ocean is dominated by species of the perciform suborder Notothenioidei, which constitute 46% of fish species and 90% of biomass. Notothenioids have undergone rapid morphological and ecological diversification and developed physiological adaptations to a cold, highly oxygenated environment. Microbes inhabiting animal intestines include those that perform essential nutritional functions, but notothenioid gut microbial communities have not been investigated using cultivation-independent approaches. We analyzed bacterial 16S rRNA gene sequences obtained from the intestinal tract of Notothenia coriiceps and Chaenocephalus aceratus, which differ in their pelagic distribution and feeding strategies. Both samples showed dominance of Gammaproteobacteria (mostly Vibrionaceae), as has been reported for temperate teleost species. Both samples showed low diversity relative to that reported for other fish microbiota studies, with C. aceratus containing fewer OTUs than N. coriiceps. Despite the small sample size of this preliminary study, our findings suggest that Antarctic notothenioids carry a gut microbiota similar in composition to that of temperate fish, but exhibiting lower species-level diversity. The omnivorous N. coriiceps individual exhibited greater diversity than the exclusively carnivorous C. aceratus individual, which may indicate that increasing herbivory in fish leads to gut microbe diversification, as found in mammals. Lastly, we detected members of taxa containing known microbial pathogens, which have not been previously reported in Antarctic notothenioid fish.     

Positive Darwinian selection operating on the immunoglobulin heavy chain of Antarctic fishes

The cooling of the Southern Ocean to the freezing point of seawater (-1.9 degrees C) over the past 25 million years played a dominant selective role in the evolution of the Antarctic fish fauna. During this period, the perciform suborder Notothenioidei, which is largely endemic to the Antarctic, diversified and developed numerous cold-adapted characters. In this report, we provide compelling evidence that the immunoglobulin heavy chain (IgH) of the notothenioid fishes has undergone adaptive selection. Two and four IgH clones were isolated, respectively, from spleen cDNA libraries prepared from the Antarctic icefish Chaenocephalus aceratus and the yellowbelly rockcod Notothenia coriiceps. The transmembrane region of the membrane form of the rockcod IgM heavy chain was located at the end of the second constant (C(H)) domain, in contrast to other teleost IgMs in which the transmembrane region is located at the end of the third constant domain. Phylogenetic analyses of C(H) regions revealed that rates of nonsynonymous nucleotide substitution were higher than rates of synonymous nucleotide substitution. Many of the nonsynonymous substitutions introduced charge changes, consistent with positive Darwinian selection acting to adapt the structure of the notothenioid immunoglobulins. The rates of nonsynonymous nucleotide substitutions were higher than the rates of synonymous nucleotide substitutions in complementarity determining regions of variable regions, suggesting that diversity at antigen binding sites is enhanced by genomic and/or somatic selection. Results of Southern blot hybridization experiments were consistent with a translocon type of IgH gene organization reminiscent of bony fishes and tetrapods.     

The role of aquaporin 3 in teleost fish

The aquaporin isoform, AQP3 has now been identified in a number of different teleost fish species, with additional DNA sequence information on AQP3 genes in further fish species available in genome databases. In zebrafish (Danio rerio), the AQP3 gene is present as two duplicate isoforms resulting from a teleostean fish genome-wide duplication. A further splicoform/isoform has also been identified in rainbow trout (Oncorhynchus mykiss). The identification of these AQP3 isoforms in other fish species is consequently explored. The role of AQP3 in physiological/osmoregulatory processes, in various teleost organs is then described. In teleost gill, AQP3 is expressed in 'chloride' cells, and in some species, in other epithelial cell types, where it may have a number of different functions including the prevention of dehydration. In eel esophagus, immunohistochemistry shows that AQP3 is expressed in surface epithelial cells in the anterior esophagus, but in mucus cells within the epithelium of the posterior esophagus. In eel intestine, AQP3 is found in macrophage-like cells and probably plays no part in osmoregulatory processes. In the rectum, as in the posterior esophagus AQP3 is expressed in mucus cells. In eel kidney, AQP3 is expressed in a subset of renal tubules, and localizes to the apical pole of tubule cells. There is no apparent change in the location or protein abundance of renal AQP3 following the acclimation of eels from freshwater to seawater.     

Metallothionein: a multifunctional protein from toxicity to cancer

The metallothionein (MT) family is a class of low molecular weight, intracellular and cysteine-rich proteins presenting high affinity for metal ions. Although the members of this family were discovered nearly 40 years ago, their functional significance remains obscure. Four major MT isoforms, MT-1, MT-2, MT-3 and MT-4, have been identified in mammals. MTs are involved in many pathophysiological processes such as metal ion homeostasis and detoxification, protection against oxidative damage, cell proliferation and apoptosis, chemoresistance and radiotherapy resistance. MT isoforms have been shown to be involved in several aspects of the carcinogenic process, cancer development and progression. MT expression has been implicated as a transient response to any form of stress or injury providing cytoprotective action. Although MT participates in the carcinogenic process, its use as a potential marker of tumor differentiation or cell proliferation, or as a predictor of poor prognosis remains unclear. In the present review the involvement of MT in defense mechanisms to toxicity and in carcinogenicity is discussed.     

Induction,Regulation, Degradation,and Biological Significance of Mammalian Metallothioneins

MTs are small cysteine-rich metal-binding proteins found in many species and, although there are differences between them, it is of note that they have a great deal of sequence and structural homology. Mammalian MTs are 61 or 62 amino acid polypep-tides containing 20 conserved cysteine residues that underpin the binding of metals. The existence of MT across species is indicative of its biological demand, while the conservation of cysteines indicates that these are undoubtedly central to the function of this protein. Four MT isoforms have been found so far, MT-1, MT-2, MT-3, and MT-4, but these also have subtypes with 17 MT genes identified in man, of which 10 are known to be functional. Different cells express different MT isoforms with varying levels of expression perhaps as a result of the different function of each isoform. Even different metals induce and bind to MTs to different extents. Over 40 years of research into MT have yielded much information on this protein, but have failed to assign to it a definitive biological role. The fact that multiple MT isoforms exist, and the great variety of substances and agents that act as inducers, further complicates the search for the biological role of MTs. This article reviews the current knowledge on the biochemistry, induction, regulation, and degradation of this protein in mammals, with a particular emphasis on human MTs. It also considers the possible biological roles of this protein, which include participation in cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification.     

Induction, regulation, degradation, and biological significance of mammalian metallothioneins

MTs are small cysteine-rich metal-binding proteins found in many species and, although there are differences between them, it is of note that they have a great deal of sequence and structural homology. Mammalian MTs are 61 or 62 amino acid polypeptides containing 20 conserved cysteine residues that underpin the binding of metals. The existence of MT across species is indicative of its biological demand, while the conservation of cysteines indicates that these are undoubtedly central to the function of this protein. Four MT isoforms have been found so far, MT-1, MT-2, MT-3, and MT-4, but these also have subtypes with 17 MT genes identified in man, of which 10 are known to be functional. Different cells express different MT isoforms with varying levels of expression perhaps as a result of the different function of each isoform. Even different metals induce and bind to MTs to different extents. Over 40 years of research into MT have yielded much information on this protein, but have failed to assign to it a definitive biological role. The fact that multiple MT isoforms exist, and the great variety of substances and agents that act as inducers, further complicates the search for the biological role of MTs. This article reviews the current knowledge on the biochemistry, induction, regulation, and degradation of this protein in mammals, with a particular emphasis on human MTs. It also considers the possible biological roles of this protein, which include participation in cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification.     

Isolation and characterization of Mytilus edulis metallothionein genes

Metallothioneins (MTs) are crucial proteins in all organisms for the regulation of essential metals and the detoxification of heavy metals. Many studies have estimated MT levels in mussel tissues to detect marine metal pollution. In this study, we investigated the MT gene structures of the forms present in Mytilus edulis (blue mussel). One MT-10 (2413 bp) gene and one MT-20 (1906 bp) gene were obtained. These MT genes contain three exons and two long introns. The splicing signals for MT-10 and MT-20 were GTA(T/A)GT-(C/T)AG. The structural organization (length of intron, splicing signals, AT content) of MT-10 and MT-20 is compared with other MT genes.     

Karyotype and genome size of zoarcids and notothenioids (Taleostei,Perciformes) from the Ross Sea: cytotaxonomic implications

In the absence of fossils, the origin of Notothenioidei, a perciform suborder dominating the fish fauna of the Southern Ocean, remains conjectural; some morphoecological evidence suggests relationships to zoarcoids. To test this point we have compared the karyotype morphology and genome size of two species of zoarcids from the Ross Sea to those of one species each of the notothenioid families Artedidraconidae, Bathydraconidae, Channichthyidae and Nototheniidae from the same region. A karyotype of 48, mostly acrocentric, chromosomes, localization of nucleolar organizers on a pair of small dibrachial chromosomes, a genome size of about 3 pg of DNA, characterize both zoarcids; similar features can be found in the karyology of the notothenioids (especially the Nototheniidae). However, all shared characters appear as plesiomorphic in teleost karyology, which does not help in producing new data on the problem of notothenioid relationships.     

Biogeography and adaptation of Notothenioid fish: hemoglobin function and globin-gene evolution

The recognition of the important role of the polar habitats in global climate changes has awakened great interest in the evolutionary biology of polar organisms. They are exposed to strong environmental constraints, and it is important to understand how they have adapted to cope with these challenges and to what extent adaptations may be upset by current climate changes. We present an introductory overview of the evolution of the Antarctic fish fauna with emphasis on the dominant perciform sub-order Notothenioidei, as well as some specific comments on the biogeography of the three phyletically basal notothenioid families. The wealth of information on the ecology and biodiversity of the species inhabiting high-Antarctic and sub-Antarctic regions provides a necessary framework for better understanding the origin, evolution and adaptation of this unique group of fish. Notothenioidei offer opportunities for identification of the biochemical characters or the physiological traits responsible for thermal adaptation. The availability of phylogenetically related taxa in a wide range of latitudes has allowed to look into the molecular bases of environmentally driven phenotypic gain and loss of function. In the process of cold adaptation, the evolutionary trend of notothenioids has produced unique specialisations, including modification of hematological characteristics, e.g. decreased amounts and multiplicity of hemoglobins. The Antarctic family Channichthyidae (the notothenioid crown group) is devoid of hemoglobin. This loss is related to a single deletional event removing all globin genes with the exception of the inactive 3' end of adult alpha-globin. In reviewing hemoglobin structure, function and phylogeny, the evolution of the fish Root effect is analysed in detail. Adaptation of the oxygen-transport system in notothenioids seems to be based on evolutionary changes involving levels of biological organisation higher than the structure of hemoglobin.