Differential expression profiling of orange-spotted grouper larvae, Epinephelus coioides (Hamilton), that survived a betanodavirus outbreak.

Nervous necrosis virus (NNV), a piscine nodavirus, has caused serious viral nervous necrosis and viral encephalopathy and retinopathy in hatchery-reared larvae and juveniles of a wide range of marine teleost species worldwide in the last two decades. Although the mortality of NNV-infected larvae is nearly 100%, there are still some larvae that survive this catastrophe. To comprehensively understand the variations of these survivors at the molecular level, we collected orange-spotted grouper larvae that survived an NNV outbreak in an indoor hatchery in southern Taiwan to study differential gene expression. Healthy larvae with high, medium and low levels of detected NNV were compared with morbid larvae using a 9600-clone-containing grouper larva cDNA microarray, and differential gene expression was further confirmed by a quantitative real-time polymerase chain reaction. Significant variation exists in healthy larvae. The following genes were upregulated: adenylate kinase 1-2, myosin binding protein H-like, myosin light chain 2, myosin light chain 3, tropomyosin, fast/white muscle troponin T embryonic isoform, and parvalbumin 1 and 2 genes. The following genes were downregulated: apolipoprotein A-I, trypsinogen, pyruvate kinase and astacin-like metalloprotease. Moreover, immunoglobulin M heavy chain gene transcription was significantly higher in healthy larvae that had high virus levels, indicating that humoral immunity might protect organisms from viral infection. These results suggest that some non-immune-related genes may have played important roles in survival during the larval metamorphosis stage, after betanodavirus infection.

Identifications, classification, and evolution of the vertebrate alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit genes.

The AMPA receptor (AMPAR), a pharmacologically defined ionotropic glutamate receptor, mediates fast excitatory synaptic transmission in the vertebrate central nervous system. Mammalian and avian AMPARs are assembled from the products of four genes (GRIA1-GRIA4) conserved in their translated sequences and gene organizations. Teleost fish also express AMPAR subunits; however, the AMPAR genes have not been extensively investigated in lower vertebrates. To elucidate the evolution of vertebrate AMPAR genes, reverse-transcriptase PCR-based surveys of subunits expressed in the brains of eight nonmammalian vertebrates were performed. The newly cloned vertebrate AMPAR subunits were classified by their sequence identities to the mammalian AMPAR subunits. The results of molecular and phylogenetic analyses indicated that the members of the AMPAR gene family increased from two in the jawless hagfish to four in the tetrapods and the shark and to more than four in the teleost fish. The sizes of AMPAR gene families correlate well with those of many multigene families observed in various vertebrates. Moreover, all vertebrates expressed at least one AMPAR subunit bearing an arginine (R) at the Q/R site, at which no invertebrate glutamate receptor subunit has been found to have an R residue, suggesting that the low calcium-permeable AMPARs appeared at early evolutionary stages of vertebrate central nervous systems. Uniquely, the loop 1 (L1) regions between hydrophobic domain 1 and hydrophobic domain 2 of the hagfish putative GRIA2 and all the teleost GRIA1 subunits were much longer than those of the remaining known ionotropic glutamate receptor subunits. The length and sequence of the L1 of teleost GRIA1 subunits were heterogeneous, suggesting that the amino acid residues in L1 were not highly selected.

A phylogeny of freshwater eels inferred from mitochondrial genes.

The genus Anguilla Shaw of Family Anguillidae consists entirely of freshwater eels, including 15 species and 2 subspecies. Conventionally, variegated markings and the length of the dorsal fin are the major morphological features used for reconstruction of phylogenetic relationships. The evolutionary history of these species remains unclear, especially for the Atlantic eels, whose habitats are far from the Metropolis in the Indo-Pacific region. This study reexamined the phylogenetic relationships of 12 Anguilla species by sequencing of the cytochrome b and 12S rRNA genes. In our analysis, species bearing similar coloration patterns or dorsal fin morphology are not necessarily clustered in the same clade, indicating that these morphological features might be unstable or might have occurred independently in different lineages during evolution. Combining our molecular data and geographical evidence, we speculate that (1) Anguilla first radiated about 20 million years ago, (2) the ancestors of Atlantic eels did not migrate by drifting through the Tethys Seaway at the leptocephali stage but instead trekked across the Central American Isthmus to the Sargasso Sea for spawning at the adult stage, and (3) multiple radiation events had occurred at the Metropolis during Anguilla evolution.

Genomic divergence between human and chimpanzee estimated from large-scale alignments of genomic sequences.

To study the genomic divergence between human and chimpanzee, large-scale genomic sequence alignments were performed. The genomic sequences of human and chimpanzee were first masked with the RepeatMasker and the repeats were excluded before alignments. The repeats were then reinserted into the alignments of nonrepetitive segments and entire sequences were aligned again. A total of 2.3 million base pairs (Mb) of genomic sequences, including repeats, were aligned and the average nucleotide divergence was estimated to be 1.22%. The Jukes-Cantor (JC) distances (nucleotide divergences) in nonrepetitive (1.44 Mb) and repetitive sequences (0.86 Mb) are 1.14% and 1.34%, respectively, suggesting a slightly higher average rate in repetitive sequences. Annotated coding and noncoding regions of homologous chimpanzee genes were also retrieved from GenBank and compared. The average synonymous and nonsynonymous divergences in 88 coding genes are 1.48% and 0.55%, respectively. The JC distances in intron, 5' flanking, 3' flanking, promoter, and pseudogene regions are 1.47%, 1.41%, 1.68%, 0.75%, and 1.39%, respectively. It is not clear why the genetic distances in most of these regions are somewhat higher than those in genomic sequences. One possible explanation is that some of the genes may be located in regions with higher mutation rates.

Characterization of the C-terminal domain of Helicobacter pylori vacuolating toxin and its relationship with extracellular toxin production.

Helicobacter pylori vacuolating cytotoxin (VacA) induces gastric epithelial necrosis. Its C-terminal domain is hypothesized to be responsible for extracellular translocation of the mature cytotoxin. In this study, genetic-structural properties of VacA C-terminal domain and the level of cytotoxin secretion were investigated. Sau3AI-HaeIII restriction fragment length polymorphism (RFLP) analysis of the 1.1-kb PCR-amplified vacA fragment revealed 14 distinct combined patterns among 87 clinical isolates. Of the 4 popular groups (A-a, A-b, A-f, and B-a), A-a strains produced a higher level of the VacA protein than A-b strains and than A-f strains (P < 0.05). Sequence analysis and secondary structure prediction supported a beta-barrel structure that might act as a selective export channel like Iga beta-core of IgA proteases. Sequence differences in the predicted beta-barrel were present among strains of different RFLPs.

The complete nucleotide sequence of the Crossostoma lacustre mitochondrial genome: conservation and variations among vertebrates.

The complete mitochondrial (mt) genome of Crossostoma lacustre, a freshwater loach from mountain stream of Taiwan, has been cloned and sequenced. This fish mt genome, consisting of 16558 base-pairs, encodes genes for 13 proteins, two rRNAs, and 22 tRNAs, in addition to a regulatory sequence for replication and transcription (D-loop), is similar to those of the other vertebrates in both the order and orientation of these genes. The protein-coding and ribosomal RNA genes are highly homologous both in size and composition, to their counterparts in mammals, birds, amphibians, and invertebrates, and using essentially the same set of codons, including both the initiation and termination signals, and the tRNAs. Differences do exist, however, in the lengths and sequences of the D-loop regions, and in space between genes, which account for the variations in total lengths of the genomes. Our observations provide evidence for the first time for the conservation of genetic information in the fish mitochondrial genome, especially among the vertebrates.

Maternal transmission of mitochondrial DNA in ducks.

Maternal transmission of mitochondrial DNA (mtDNA) has been studied in amphibians, insects and mammals, but little is known about mtDNA inheritance in the ovaripirous avian species. In this study, we have constructed the physical maps of mitochondrial genomes from two different genera of ducks (Cairina and Anas) and taken advantage of the availability of their hybrids to demonstrate that mtDNA is maternally inherited.