Re-examination of the phylogeny of Rhacophoridae (Anura) based on mitochondrial and nuclear DNA

The phylogenetic relationships among rhacophorid frogs are under dispute. We use partial sequences of three mitochondrial (12S rRNA, 16S rRNA, and cytochrome b) and three nuclear protein-coding (Rag-1 rhodopsin exon 1, and tyrosinase exon 1) genes from 57

Identification of a Giardia krr1 homolog gene and the secondarily anucleolate condition of Giaridia lamblia

Giaridia lamblia was long considered to be one of the most primitive eukaryotes and to lie close to the transition between prokaryotes and eukaryotes, but several supporting features, such as lack of mitochondrion and Golgi, have been challenged recently. It was also reported previously that G. lamblia lacked nucleolus, which is the site of pre-rRNA processing and ribosomal assembling in the other eukaryotic cells. Here, we report the identification of the yeast homolog gene, krr1, in the anucleolate eukaryote, G. lamblia. The krr1 gene, encoding one of the pre-rRNA processing proteins in yeast, is actively transcribed in G. lamblia. The deduced protein sequence of G. lamblia krr1 is highly similar to yeast KRR1p that contains a single-KH domain. Our database searches indicated that krr1 genes actually present in diverse eukaryotes and also seem to present in Archaea. However, only the eukaryotic homologs, including that of G. lamblia, have the single-KH domain, which contains the conserved motif KR(K)R. Fibrillarin, another important pre-rRNA processing protein has also been identified previously in G. lamblia. Moreover, our database search shows that nearly half of the other nucleolus-localized protein genes of eukaryotic cells also have their homologs in Giardia. Therefore, we suggest that a common mechanism of pre-RNA processing may operate in the anucleolate eukaryote G. lamblia and in the other eukaryotes and that like the case of "lack of mitochondrion," "lack of nucleolus" may not be a primitive feature, but a secondarily evolutionary condition of the parasite.

Genetic diversity and origin of Chinese cattle revealed by mtDNA D-loop sequence variation

To determine the origin and genetic diversity of Chinese cattle, we analyzed the complete mtDNA D-loop sequences of 84 cattle from 14 breeds/populations from southwest and west China, together with the available cattle sequences in GenBank. Our results sh

Stability and molecular modeling of triplex DNA inhibiting DNA binding protein binding to the core promoter of hepatitis B virus.

Two three-dimensional structure models of the 21nt oligodeoxyribonucleotides, CPI (G3TG-2TGT2G5TG2TGT) and CP3 (TGTG2TGST2GTG2TG3), were constructed by InsightII (MSI) software in IRIS Indigo2 (SGI) workstation using the crystal structure of TAT tripler formation as the template. The initial structures subsequently were minimized by molecular mechanics. The final structures were believed as the dominant conformation. The results showed that the energy of CP1 is lower than that of CP3, and the former is more stable than the latter. Moreover, the results further proved that the 21nt oligodeoxyribo-nucleotide CP1 stably combines with the core promoter (Cp) fragment of hepatitis B virus (HBV) to form a tripler DNA, and CP1 specifically inhibits a specific cellular factor (DNA binding protein) binding to Cp fragment. These results indicated that specific repression of gene transcription of HBV DNA might be possible by tripler-formation DNA.

Phylogeny of east Asian bufonids inferred from mitochondrial DNA sequences (Anura : Amphibia)

We investigated the relationships of Asian bufonids using partial sequences of mitochondrial DNA genes. Twenty-six samples representing 14 species of Bufo from China and Vietnam and 2 species of Torrentophryne from China were examined. Three samples of Bufo viridis from Armenia and Georgia were also sequenced to make a comparison to its sibling tetraploid species B. danatensis. Bufo americanus, from Canada, was used as the outgroup. Sequences from the 12S ribosomal RNA, 16S ribosomal RNA, cytochrome b, and the control region were analyzed using parsimony. East Asian bufonids were grouped into two major clades. One clade included B. andrewsi, B. bankorensis, B. gargarizans, B. tibetanus, B. tuberculatus, its sister clade B. cryptotympanicus, and the 2 species of Torrentophryne. The second clade consisted of B. galeatus, B. himalayanus, B. melanostictus, and a new species from Vietnam. The placement of three taxa (B. raddei B. viridis, and its sister species, B. danatensis) was problematic. The genus Torrentophryne should be synonymized with Bufo to remove paraphyly. Because B. raddei does not belong to the clade that includes B. viridis and B. danatensis, it was removed from the viridis species group. The species status of B bankorensis from Taiwan is evaluated. (C) 2000 Academic Press.

Structural and functional characterization of the human CCR5 receptor in complex with HIV gp120 envelope glycoprotein and CD4 receptor by molecular modeling studies

The entry of human immunodeficiency virus (HIV) into cells depends on a sequential interaction of the gp120 envelope glycoprotein with the cellular receptors CD4 and members of the chemokine receptor family. The CC chemokine receptor CCR5 is such a receptor for several chemokines and a major coreceptor for the entry of R5 HIV type-1 (HIV-1) into cells. Although many studies focus on the interaction of CCR5 with HIV-1, the corresponding interaction sites in CCR5 and gp120 have not been matched. Here we used an approach combining protein structure modeling, docking and molecular dynamics simulation to build a series of structural models of the CCR5 in complexes with gp120 and CD4. Interactions such as hydrogen bonds, salt bridges and van der Waals contacts between CCR5 and gp120 were investigated. Three snapshots of CCR5-gp120-CD4 models revealed that the initial interactions of CCR5 with gp120 are involved in the negatively charged N-terminus (Nt) region of CCR5 and positively charged bridging sheet region of gp120. Further interactions occurred between extracellular loop2 (ECL2) of CCR5 and the base of V3 loop regions of gp120. These interactions may induce the conformational changes in gp120 and lead to the final entry of HIV into the cell. These results not only strongly support the two-step gp120-CCR5 binding mechanism, but also rationalize extensive biological data about the role of CCR5 in HIV-1 gp120 binding and entry, and may guide efforts to design novel inhibitors.

Comparative phylogeography of the Yellow River schizothoracine fishes (Cyprinidae): Vicariance, expansion, and recent coalescence in response to the Quaternary environmental upheaval in the Tibetan Plateau

National Natural Science Foundation of China (NSFC) [30225008, 30300036, 30530120]; Key Innovation Plan [KSCX2-SW-106]; National Basic Research Project in China [2005cb422005]; National Natural Science Foundation of China [30600062]

Distinct Evolutionary Patterns Between Two Duplicated Color Vision Genes Within Cyprinid Fishes

We investigated the molecular evolution of duplicated color vision genes (LWS-1 and SWS2) within cyprinid fish, focusing on the most cavefish-rich genus-Sinocyclocheilus. Maximum likelihood-based codon substitution approaches were used to analyze the evolution of vision genes. We found that the duplicated color vision genes had unequal evolutionary rates, which may lead to a related function divergence. Divergence of LWS-1 was strongly influenced by positive selection causing an accelerated rate of substitution in the proportion of pocket-forming residues. The SWS2 pigment experienced divergent selection between lineages, and no positively selected site was found. A duplicate copy of LWS-1 of some cyprinine species had become a pseudogene, but all SWS2 sequences remained intact in the regions examined in the cyprinid fishes examined in this study. The pseudogenization events did not occur randomly in the two copies of LWS-1 within Sinocyclocheilus species. Some cave species of Sinocyclocheilus with numerous morphological specializations that seem to be highly adapted for caves, retain both intact copies of color vision genes in their genome. We found some novel amino acid substitutions at key sites, which might represent interesting target sites for future mutagenesis experiments. Our data add to the increasing evidence that duplicate genes experience lower selective constraints and in some cases positive selection following gene duplication. Some of these observations are unexpected and may provide insights into the effect of caves on the evolution of color vision genes in fishes.

The youngest split in sympatric schizothoracine fish (Cyprinidae) is shaped by ecological adaptations in a Tibetan Plateau glacier lake

Although new empirical evidence shows that sympatric speciation has occurred in some species, there are few indisputable model organisms for this process of speciation. The two subspecies (Gymnocypris eckloni eckloni and G. e. scoliostomus) of the schizothoracine Gymnocypris fish species complex from a small glacier lake in the Tibetan Plateau, Lake Sunmcuo, fit several of the key characteristics of the sympatric speciation model. We used combined mitochondrial control region sequences and the cytochrome b gene (1894 bp) to address the phylogenetics and population genetics of 232 specimens of G. e. eckloni and G. e. scoliostomus, as well as all of its closely related sister species. We found that: (i) a total of four old lineages were uncovered in the widespread G. e. eckloni, of which only one was shown to be shared with all G. e. scoliostomus individuals and (ii) the new subspecies (G. e. scoliostomus) evolved in Lake Sunmcuo from the ancestral G. e. eckloni population within approximately 0.057 Ma. These two taxa of the species complex are morphologically distinct, and reproductive isolation is further suggested. Ecological disruptive selection based on morphological traits (e.g. mouth cleft characters) and food utilization may be a mechanism of incipient speciation of two sympatric populations within Lake Sunmcuo. This study provides the first genetic evidence for sympatric speciation in the schizothoracine fish.

Molecular characterization and expression pattern of AFPIV during embryogenesis in gibel carp(Carassiu auratus gibelio)

As a new type of AFPs, AFPIV has been firstly identified in longhorn sculpin (Myoxocephalus octodecimspinosus), and in recent years, its cDNA and amino acid sequence have been reported, and its pancreatic synthesis has been firstly reported in polar fish. However, its expression patterns during fish embryogenesis have not been elucidated yet. By differential screening, we cloned the CagAFPIV in gibel carp, Carassius auratus gibelio, demonstrated its predominant expression during embryogenesis. RT-PCR detection revealed that CagAFPIV was first transcribed from blastula stage and kept a high level during embryogenesis and declined remarkably in hatched larva. In situ hybridization revealed that CagAFPIV transcripts were firstly distributed over the margin and marginal blastomere in blastula stage embryos, at the early-gastrula stage the positive signals distributed in the marginal cells and the internalization cells, and later restricted to the cells the yolk syncytial layer (YSL) from later gastrula stage to larva stage. Consistently, the CagAFPIV protein also kept a high level during embryogenesis, and the high protein level retained some days after the larva hatched. Our work, for the first time, revealed the dynamic expression and distribution of CagAFPIV during embryogenesis.

Molecular and expression characterization of two somatostatin genes in the Chinese sturgeon, Acipenser sinensis

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species and also an important resource for the sturgeon aquaculture industry. SMART cDNA was synthesized from the hypothalamus of Chinese sturgeon, and the full-length cDNAs of two somatostatin (SS) genes were cloned and sequenced. The first cDNA (AsSS1) encodes a 116-amino acid protein that contains the SS14 sequence at its C-terminal extremity. AsSS1 shows high identity to that of human and other vertebrates. The second cDNA (AsSS2) encodes a 111-amino acid protein that contains the somatostatin variant [Pro(2)]-SS14 at its C-terminal extremity. Both the two SS mRNAs were expressed in brain and pituitary with different mRNA levels. But in peripheral tissues, AsSS2 was more widely distributed than AsSS1. High mRNA levels of AsSS2 were found in liver, kidney and heart, while low mRNA levels of AsSS2 were also detected in ovary. Throughout embryogenesis and early larval development only AsSS2 mRNAs were detected. Furthermore, in the hypothalamus of one to five year-old Chinese sturgeon, AsSS2 but not AsSS1 maintained stable expression. The mRNA distribution suggests that the Chinese sturgeon AsSS2 products play important physiological functions in adult fish as well as in cell growth and organ differentiation in embryo and larva development. (C) 2009 Elsevier Inc. All rights reserved.

Origin and evolution of the RIG-I like RNA helicase gene family

Background: The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria. Results: The DExD/H containing RNA helicases including RIG-I, MDA5 and LGP2 were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. The gene synteny of MDA5 and LGP2 is well conserved among vertebrates whilst conservation of the gene synteny of RIG-I is less apparent. Invertebrate homologues had a closer phylogenetic relationship with the vertebrate RIG-Is than the MDA5/LGP2 molecules, suggesting the RIG-I homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. Our data suggest that the RIG-I like helicases possibly originated from three distinct genes coding for the core domains including the HELICc, CARD and ATP dependent DExD/H domains through gene fusion and gene/domain duplication. Furthermore, presence of domains similar to a prokaryotic DNA restriction enzyme III domain (Res III), and a zinc finger domain of transcription factor (TF) IIS have been detected by bioinformatic analysis. Conclusion: The RIG-I/MDA5 viral surveillance system is conserved in vertebrates. The RIG-I like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. Diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral PAMPs.

Population genetic structure and geographical differentiation of the Chinese catfish Hemibagrus macropterus (Siluriformes, Bagridae): Evidence for altered drainage patterns

National Natural Science Foundation of China (NSFC) [2007CB411600, 30530120]

Molecular cloning and expression characterization of ApoC-I in the orange-spotted grouper

Endogenous yolk nutrients are crucial for embryo and larval development in fish, but developmental behavior of the genes that control yolk utilization remains unknown. Apolipoproteins have been shown to play important roles in lipid transport and uptake through the circulation system. In this study, EcApoC-I, the first cloned ApoC-I in teleosts, has been screened from pituitary cDNA library of female orange-spotted grouper (Epinephelus coioides), and the deduced amino acid sequence shows 43.5% identity to one zebrafish (Danio rerio) hypothetical protein similar to ApoC-I, and 21.2%, 21.7%, 22.5%, 20%, and 22.5% identities to Apo C-I of human (Homo sapiens), house mouse (Mus musculus), common tree shrew (Tupaia glis), dog (Canis lupus familiaris) and hamadryas baboon (Papio hamadryas), respectively. Although the sequence identity is low, amphipathic alpha-helices with the potential to bind to lipid were predicted to exist in the EcApoC-I. RT-PCR analysis revealed that it was first transcribed in gastrula embryos and maintained a relatively stable expression level during the following embryogenesis. During embryonic and early larval development, a very high level of EcApoC-I expression was in the yolk syncytial layer, indicating that it plays a significant role in yolk degradation and transfers nutrition to the embryo and early larva. By the day 7 after hatching, EcApoC-I transcripts were observed in brain. In adult, EcApoC-I mRNA was detected abundantly in brain and gonad. In transitional gonads, the EcApoC-I expression is restricted to the germ cells. The data suggested that EcApoC-I might play an important role in brain and gonad morphogenesis and growth.

Phylogeography of the freshwater catfish Hemibagrus guttatus (Siluriformes, Bagridae): Implications for South China biogeography and influence of sea-level changes

National Natural Science Foundation of China (NSFC) ; [2007CB411600]; [30530120]