Molecular phylogenetic systematics of twelve species of acipenseriformes based on mtDNA ND4L-ND4 gene sequence analysis

Acipenseriformes is an endangered primitive fish group, which occupies a special place in the history of ideas concerning fish evolution, even in vertebrate evolution. However, the classification and evolution of the fishes have been debated. The mitochondrial DNA (mtDNA) ND4L and partial ND4 genes were first sequenced in twelve species of the order Acipenseriformes, including endemic Chinese species. The following points were drawn from DNA sequences analysis: (i) the two species of Huso can be ascribed to Acipenser; (ii) A. dabryanus is the mostly closely related to A. sinensis, and most likely the landlocked form of A. sinensis; (iii) genus Acipenser in trans-Pacific region might have a common origin; (iv) mtDNA ND4L and ND4 genes are the ideal genetic markers for phylogenetic analysis of the order Acipenseriformes.

Evolution of the DRD2 gene haplotype and its association with alcoholism in Mexican Americans

The human D2 dopamine receptor gene (DRD2) plays a central role in the neuromodulation of appetitive behaviors and is implicated in having a possible role in susceptibility to alcoholism. We genotyped an SNP in DRD2 Exon 8 in 251 nonalcoholic, unrelated, healthy controls and 200 alcoholic Mexican Americans. The DRD2 haplotypes were analyzed using the Exon 8 genotype in combination with five other SNP genotypes, which were obtained from our previous study. The ancestral origins of the DRD2 polymorphisms have been determined by sequencing the homologous region in other higher primates. Twenty DRD2 haplotypes, defined as H1 to H20 based on their frequency from high to low, were obtained in this major minority population. The ancestral haplotype "I-132-G-C-G-A1" and two one-step mutation haplotypes were absent in our study population. The haplotype H1, "I-B1-T-C-A-A1", with the highest frequency in the population, is a three-step mutation from the ancestral form. The first five or eight major haplotypes make up 87% or 95% of the entire population, respectively. The prevalence of the haplotype H1+ (H1/H1 and H1/Hn genotypes) is significantly higher in alcoholics and alcoholic subgroups, including early onset drinkers and benders, than in their respective control groups. The Promoter -141C allele is in linkage disequilibrium (LD) with five other loci in the nonalcoholic group, but not in the alcoholic group. All of the other five loci are in LD in both the alcoholic and control groups. The DRD2 TaqI B allele is in complete LD with the allele located in intron 6. Five SNPs, Promoter -141C, TaqI B (or Intron 6), Exon 7, Exon 8, and TaqI A, are sufficient to define the DRD2 haplotypes in Mexican Americans. Our data indicate that the DRD2 haplotypes are associated with alcoholism in Mexican Americans. (c) 2005 Elsevier Inc. All rights reserved.

Episodic evolution of prolactin gene in primates

In the present study, we obtained exon 2-5 of prolactin (PRL) gene from four primate species by PCR and sequencing. Adding other genes available in GenBank, we calculate amino acid substitution rates for prolactin gene in primate. Comparison of nonsynonymous substitution rate to synonymous substitution rate ratios shows no evidence of positive selection for any lineage of primate prolactin gene. According to this and the facts that (i) no sites under positive selection are inferred by using maximum-likelihood method; (ii) among 32 amino acid replacement that occurred along the rapid evolutionary phase, only two are included in the 40 functionally important residues, indicating that amino acid replacement tends to occur in those functionally unimportant residues; (iii) partial of prolactin function is replaced by placental lactogen in primate at the rapid evolutionary phase of prolactin gene, we thus deem that it is relaxation of purifying selection to some extent rather than positive selection that enforces the rapid evolution of primate prolactin gene.

Parallel and Convergent Evolution of the Dim-Light Vision Gene RH1 in Bats (Order: Chiroptera)

Rhodopsin, encoded by the gene Rhodopsin (RH1), is extremely sensitive to light, and is responsible for dim-light vision. Bats are nocturnal mammals that inhabit poor light environments. Megabats (Old-World fruit bats) generally have well-developed eyes, while microbats (insectivorous bats) have developed echolocation and in general their eyes were degraded, however, dramatic differences in the eyes, and their reliance on vision, exist in this group. In this study, we examined the rod opsin gene (RH1), and compared its evolution to that of two cone opsin genes (SWS1 and M/LWS). While phylogenetic reconstruction with the cone opsin genes SWS1 and M/LWS generated a species tree in accord with expectations, the RH1 gene tree united Pteropodidae (Old-World fruit bats) and Yangochiroptera, with very high bootstrap values, suggesting the possibility of convergent evolution. The hypothesis of convergent evolution was further supported when nonsynonymous sites or amino acid sequences were used to construct phylogenies. Reconstructed RH1 sequences at internal nodes of the bat species phylogeny showed that: (1) Old-World fruit bats share an amino acid change (S270G) with the tomb bat; (2) Miniopterus share two amino acid changes (V104I, M183L) with Rhinolophoidea; (3) the amino acid replacement I123V occurred independently on four branches, and the replacements L99M, L266V and I286V occurred each on two branches. The multiple parallel amino acid replacements that occurred in the evolution of bat RH1 suggest the possibility of multiple convergences of their ecological specialization (i.e., various photic environments) during adaptation for the nocturnal lifestyle, and suggest that further attention is needed on the study of the ecology and behavior of bats.

Ribosomal DNA gene and phylogenetic relationships of Diplura and lower Hexapods

The monophyly of Diplura and its phylogenetic relationship with other hexapods are important for understanding the phylogeny of Hexapoda. The complete 18SrRNA gene and partial 28SrRNA gene (D3-D5 region) from 2 dipluran species (Campodeidae and Japygidae), 2 proturan species, 3 collembolan species, and 1 locust species were sequenced. Combining related sequences in GenBank, phylogenetic trees of Hexapoda were constructed by MP method using a crustacean Artemia salina as an outgroup. The results indicated that: (i) the integrated data of 18SrDNA and 28SrDNA could provide better phylogenetic information, which well supported the monophyly of Diplura; (ii) Diplura had a close phylogenetic relationship to Protura with high bootstrap support.

Different protein tyrosine phosphatase superfamilies resulting from different gene reading frames

Protein tyrosine phosphatases (PTPs) are comprised of two superfamilies, the phosphatase I superfamily containing a single low-molecular-weight PTP (lmwPTP) family and the phosphatase II superfamily including both the higher-molecular-weight PTP (hmwPTP) and the dual-specificity phosphatase (DSP) families. The phosphatase I and H superfamilies are often considered to be the result of convergent evolution. The PTP sequence and structure analyses indicate that lmwPTPs, hmwPTPs, and DSPs share similar structures, functions, and a common signature motif, although they have low sequence identities and a different order of active sites in sequence or a circular permutation. The results of this work suggest that lmwPTPs and hmwPTPs/DSPs are remotely related in evolution. The earliest ancestral gene of PTPs could be from a short fragment containing about 90similar to120 nucleotides or 30similar to40 residues; however, a probable full PTP ancestral gene contained one transcript unit with two lmwPTP genes. All three PTP families may have resulted from a common ancestral gene by a series of duplications, fusions, and circular permutations. The circular permutation in PTPs is caused by a reading frame difference, which is similar to that in DNA methyltransferases. Nevertheless, the evolutionary mechanism of circular permutation in PTP genes seems to be more complicated than that in DNA methyltransferase genes. Both mechanisms in PTPs and DNA methyltransferases can be used to explain how some protein families and superfamilies came to be formed by circular permutations during molecular evolution.

Expression regulation and functional characterization of a novel interferon inducible gene Gig2 and its promoter

Grass carp hemorrhagic virus (GCHV)-induced gene 2 (Gig2) is a novel gene previously identified from UV-inactivated GCHV-treated Carassius auratus blastulae embryonic (CAB) cells, suggesting that it should play a pivotal role in the interferon (IFN) antiviral response. In this study, a polyclonal anti-Gig2 antiserum was generated and used to study the inductive expression pattern by Western blot analysis, showing no basal expression in normal CAB cells but a significant up-regulation upon UV-inactivated GCHV, polyinosinic:polycytidylic acid (Poly I:Q and recombinant IFN (rIFN). However, constitutive expression of Gig2 is observed in all tested tissues from grass carp (Ctenopharyngodon idellus), and Poly I:C injection increases the relative amount of Gig2 protein in skin, spleen, trunk kidney, gill, hindgut and thymus. Moreover, the genomic sequence covering the whole Gig2 ORF and the upstream promoter region were amplified by genomic walking. Significantly, the Gig2 promoter contains three IFN-stimulated response elements (ISREs), nine GAAA/TfTC motifs and five gamma-IFN activating sites (GAS), which are the characteristics of genes responsive to both type I IFN and type 11 IFN. Subsequently, the complete Gig2 promoter sequence was cloned into pGL3-Basic vector, and its activity was measured by luciferase assays in the transfected CAB cells. The Gig2 promoter-driven construct is highly induced in CAB cells after treatment with Poly I:C or rIFN, and the functional capability is dependent on IFN regulatory factor 7 (IRF7), because its activity can be stimulated by IRF7. Collectively, the data provide strong evidence that Gig2 is indeed a novel IFN inducible gene and its expression is likely dependent on IRF7 upon Poly I:C or IFN. (C) 2009 Elsevier Ltd. All rights reserved.

Cloning, expression and subcellular distribution of a Rana grylio virus late gene encoding ERV1 homologue

An essential for respiration and viability (ERV1) homologue, 88R, was cloned and characterized from Rana grylio virus (RGV). Database searches found its homologues in all sequenced iridoviruses, and sequence alignment revealed a highly conserved motif shared by all ERV1 family proteins: Cys-X-X-Cys. RT-PCR and western blot analysis revealed that 88R begins to transcribe and translate at 6 h postinfection (p.i.) and remains detectable at 48 h p.i. during RGV infection course. Furthermore, using drug inhibition analysis by a de novo protein synthesis inhibitor and a viral DNA replication inhibitor, RGV 88R was classified as a late (L) viral gene during the in vitro infection. 88R-EGFP fusion protein was observed in both the cytoplasm and nucleus of pEGFP-N3-88R transfected EPC cells. Although result of immunofluorescence is similar, 88R protein was not detected in viromatrix. Moreover, function of RGV 88R on virus replication were evaluated by RNAi assay. Nevertheless, effect of knockdown of RGV 88R expression on virus replication was not detected in cultured fish cell lines. Collectively, current data indicate that RGV 88R was a late gene of iridovirus encoding protein that distributed both the cytoplasm and nucleus.

In vivo study on the effects of microcystin extracts on the expression profiles of proto-oncogenes (c-fos, c-jun and c-myc) in liver, kidney and testis of male Wistar rats injected i.v. with toxins

Microcystins (MCs) are a potent liver tumor promoter, possessing potent tumor-promoting activity and weak initiating activity. Proto-oncogenes are known to be involved in the tumor-promoting mechanisms of microcystin-LR. However, few data are available on the effects of MCs oil proto-oncogenes in the whole animal. To investigate the effects of MCs on the expression profile of the proto-oncogenes in different organs, male Wistar rats were injected intravenously with microcystin extracts at a dose of 86.7 mu g MC-LR eq/kg bw (MC-LR eq, MC-LR equivalents). mRNA levels of three proto-oncogenes c-fos, c-jun and c-myc in liver, kidney and testis were analyzed using quantitative real-time PCR at several time points post-injection. Significant induction of these genes at transcriptional level was observed in the three organs. In addition, the increase of mRNA expression of all three genes was much higher in liver than in kidney and testis. Meanwhile, the protein levels of c-Fos and c-Jun were investigated by western blotting. Both proteins were induced in the three organs. However, elevations of protein levels were Much lower than those of mRNA levels. These findings suggest that the expression of c-fos, c-jun and c-myc might be one possible mechanism for the tumor-promoting activity and initiating activity of microcystins. (c) 2008 Published by Elsevier Ltd.

Comparative study and expression analysis of the interferon gamma gene locus cytokines in Xenopus tropicalis

Using bioinformatics approach, the genome locus containing interleukin (IL)-22, IL-26, and interferon gamma (IFN-gamma) genes has been identified in the amphibian, Xenopus tropicalis. Like that in other vertebrates such as fish, birds, and mammals, the Xenopus IL-22, IL-26, and IFN-gamma are clustered in the same chromosome and the adjacent genes are conserved. The genomic structures of the Xenopus IL-22, IL-26, and IFN-gamma gene were identical to that of their mammalian counterparts. The Xenopus IL-22 and IL-26 genes contained five exons and four introns while the Xenopus IFN-gamma gene consisted of four exons and three introns. The Xenopus IL-22, IL-26, and IFN-gamma share 14.1-41.6%, 14.6-31.2%, and 23.7-36.5% identity to their counterparts in other species, respectively. Reverse-transcription polymerase chain reaction (PCR) and real-time quantitative PCR analyses revealed that the expression of IL-22, IL-26, and IFN-gamma genes was significantly upregulated after simulation with bacterial polyliposaccharide and/or synthetic double-stranded poly(I:C), suggesting these cytokines like those in other vertebrates play an important role in regulating immune response in Xenopus.

Developmental toxicity and alteration of gene expression in zebrafish embryos exposed to PFOS

Perfluorooetanesulfonate (PFOS) is a persistent organic pollutant, the potential toxicity of which is causing great concern. In the present study, we employed zebrafish embryos to investigate the developmental toxicity of this compound. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to 0.1, 0.5, 1, 3 and 5 mg/L PFOS. Hatching was delayed and hatching rates as well as larval survivorship, were significantly reduced after the embryos were exposed to 1, 3 and 5 mg/L PFOS until 132 hpf. The fry displayed gross developmental malformations, including epiboly deformities, hypopigmentation, yolk sac edema, tail and heart malformations and spinal curvature upon exposure to PFOS concentrations of I mg/L or greater. Growth (body length) was significantly reduced in the 3 and 5 mg/L PFOS-treated groups. To test whether developmental malformation was mediated via apoptosis, flow cytometry analysis of DNA content, acridine orange staining and TUNEL assay was used. These techniques indicated that more apoptotic cells were present in the PFOS-treated embryos than in the control embryos. Certain genes related to cell apoptosis, p53 and Bax, were both significantly up-regulated upon exposure to all the concentrations tested. In addition, we investigated the effects of PFOS on marker genes related to early thyroid development (hhex and pax8) and genes regulating the balance of androgens and estrogens (cyp19a and cyp19b). For thyroid development, the expression of hhex was significantly up-regulated at all concentrations tested, whereas pax8 expression was significantly up-regulated only upon exposure to lower concentrations of PFOS (0.1, 0.5, 1 mg/L). The expression of cyp19a and of cyp19b was significantly down-regulated at all exposure concentrations. The overall results indicated that zebrafish embryos constitute a reliable model for testing the developmental toxicity of PFOS, and the gene expression patterns in the embryos were able to reveal some potential mechanisms of developmental toxicity. (C) 2008 Elsevier Inc. All rights reserved.

Variation patterns of the mitochondrial 16S rRNA gene with secondary structure constraints and their application to phylogeny of cyprinine fishes (Teleostei : Cypriniformes)

The mitochondrial 16S ribosomal RNA (rRNA) gene sequences from 93 cyprinid fishes were examined to reconstruct the phylogenetic relationships within the diverse and economically important subfamily Cyprininae. Within the subfamily a biased nucleotide composition (A > T, C > G) was observed in the loop regions of the gene, and in stem regions apparent selective pressures of base pairing showed a bias in favor of G over C and T over A. The bias may be associated with transition-transversion bias. Rates of nucleotide substitution were lower in stems than in loops. Analysis of compensatory substitutions across these taxa demonstrates 68% covariation in the gene and a logical weighting factor to account for dependence in mutations for phylogenetic inference should be 0.66. Comparisons of varied stem-loop weighting schemes indicate that the down-weightings for stem regions could improve the phylogenetic analysis and the degree of non-independence of stem substitutions was not as important as expected. Bayesian inference under four models of nucleotide substitution indicated that likelihood-based phylogenetic analyses were more effective in improving the phylogenetic performance than was weighted parsimony analysis. In Bayesian analyses, the resolution of phylogenies under the 16-state models for paired regions, incorporating GTR + G + I models for unpaired regions was better than those under other models. The subfamily Cyprininae was resolved as a monophyletic group, as well as tribe Labein and several genera. However, the monophyly of the currently recognized tribes, such as Schizothoracin, Barbin, Cyprinion + Onychostoma lineages, and some genera was rejected. Furthermore, comparisons of the parsimony and Bayesian analyses and results of variable length bootstrap analysis indicates that the mitochondrial 16S rRNA gene should contain important character variation to recover well-supported phylogeny of cyprinid taxa whose divergences occurred within the recent 8 MY, but could not provide resolution power for deep phylogenies spanning 10-19 MYA. (c) 2008 Published by Elsevier Inc.

Gene structures and promoter characteristics of interferon regulatory factor 1 (IRF-1), IRF-2 and IRF-7 from snakehead Channa argus

Three interferon regulatory factor (IRF) genes, CaIRF-1, CaIRF-2 and CaIRF-7, and their promoters of snakehead (Channa argus) were cloned and characterized. The CaIRF-1 gene consists of ten exons, spans 4.3 kb and encodes a putative peptide of 299 aa. The CaIRF-2 gene consists of nine exons, spans 8 kb and encodes a putative peptide of 328 aa. The gene organizations of CaIRF-1 and CaIRF-2 are very similar to that of human IRF-1 and IRF-2 except more compact. Comparison of exon-intron organization of the two genes indicated a common evolutionary structure, notably within the exons encoding the DNA binding domain (DBD) of the two factors. The CaIRF-7 gene spans 4.1 kb and encodes a putative peptide of 437 aa. However, the gene organization of CaIRF-7 consisting of ten exons is different to human IRF-7a gene which has an intron in 5' UTR. Three CaIRFs share homology in N-terminal encompassing the DBD that contains a characteristic repeat of tryptophan residues. The promoters of CaIRF-1 and CaIRF-2 genes contain the conserved sites for NF-kappa B and Sp1. The gamma-IFN activation sites (GAS) were found in the promoters of CaIRF-1 and CaIRF-7. The promoter of CaIRF-7 contains conserved interferon stimulating response element (ISRE) which is characteristic of IFN-induced gene promoter, and suggests that there also exist intracellular amplifier circuit in fish IFN signal pathway. Moreover, the element GAAANN oriented in both directions is repeated in CaIRF promoter regions, which confers to further inducibility by IFN. The constitutive expression of CaIRF genes were found to increase obviously in response to induction by the known IFN-inducer poly I:C. (c) 2008 Published by Elsevier Ltd.

Rapid growth cost in “all-fish” growth hormone gene transgenic carp: Reduced critical swimming speed

Evidence has accumulated that there is a trade-off between benefits and costs associated with rapid growth. A trade-off between growth rates and critical. swimming speed (U-crit) had been also reported to be common in teleost fish. We hypothesize that growth acceleration in the F-3 generation of "all-fish" growth hormone gene (GH) transgenic common carp (Cyprinus carpio L.) would reduce the swimming abilities. Growth and swimming performance between transgenic fish and non-transgenic controls were) compared. The results showed that transgenic fish had a mean body weight 1.4-1.9-fold heavier, and a mean specific growth rate (SGR) value 6%-10% higher than the controls. Transgenic fish, however, had a mean absolute U-crit (cm/s) value 22% or mean relative Ucrit (BL/s) value 24% lower than the controls. It suggested that fast-growing "all-fish" GH-transgenic carp were inferior swimmers. It is also supported that there was a trade-off between growth rates and swimming performance, i.e. faster-growing individuals had lower critical swimming speed.

Identification and characterization of two homologues of interferon-stimulated gene ISG15 in crucian carp

ISG15 is one of the most strongly induced genes upon viral infection, interferon (IFN) stimulation, and lipopolysaccharide, (LPS) stimulation, and only one copy has been found in mammals so far. Here two fish ISG15 genes, termed CaISG15-1 and CaISG15-2, have been cloned and sequenced from UV-inactivated GCHV (grass carp haemorrhagic virus)-infected and IFN-produced CAB cells (crucian carp Carassius auratus blastulae embryonic cells) by suppression subtractive hybridization. The full-length cDNA sequences of two crucian carp ISG15 encode a 155-amino-acid protein and a 161-amino-acid protein, both of which show 78.9% identity overall and possess the characteristic structures of mammalian ISG15 proteins including two tandem ubiquitin-like domains and the C-terminal canonical LRLRGG motif. In CAB cells treated with different stimuli including active virus, UV-inactivated GCHV and IFN containing supernatant (ICS), the expression of both CaISG15-1 and CaISG15-2 was up-regulated but displayed different kinetics. Poly I:C and LPS were also able to induce an increase in mRNA for both genes. In CAB cells responsive to active GCHV, UV-inactivated GCHV, CAB ICS, Poly 1:12 and LPS, CaISG15-1 was upregulated more significantly than CaISG15-2. These results suggest that there are two ISG15 homologues in crucian carp, both of which might play distinct roles in innate immunity against viral and bacterial infection. (c) 2006 Elsevier Ltd. All rights reserved.