Molecular cloning and expression analyses of a novel swine gene-ARF4

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus muscle tissues from Meishan and Large White pigs. One novel gene that was differentially expressed was identified through semi-quantitative RT-PCR and the cDNA complete sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. The nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 180 amino acids that contains the putative conserved domain of ADP-ribosylation factor (ARF) which has high homology with the ADP-ribosylation factor 4 (ARF4) of six species-bovine (98%), human and orangutan (96%), African clawed frog (96%), mouse and rat (98%)-so that it can be defined as swine ADP-ribosylation factor 4 (ARF4). This novel porcine gene was finally assigned to GeneID:595108. The phylogenetic tree analysis revealed that the swine ARF4 has a closer genetic relationship with the rat and mouse ARF4 than with those of human and African clawed frog. The tissue expression analysis indicated that the swine ARF4 gene is over expressed in muscle, fat, heart, spleen, liver, and ovary and moderately expressed in lung and kidney but weakly expressed in small intestine. Our experiment is the first to establish the primary foundation for further research on the swine ARF4 gene.     

Molecular evidence for a clade of turtles.

Although turtles have been generally grouped with the most primitive reptile species, the origin and phylogenetic relationships of turtles have remained unresolved to date. To confirm the phylogenetic position of turtles in amniotes, we have cloned and determined the cDNA sequences encoding for skink lactate dehydrogenase (LDH)-A and LDH-B, snake LDH-A, and African clawed frog LDH-A; four alpha-enolase cDNA sequences from turtle, alligator, skink, and snake were also cloned and determined. All of these eight cDNA sequences, as well as the previously published LDH-A, LDH-B, and alpha-enolase of mammals, birds, reptiles, and African clawed frog, were analyzed by the phylogenetic tree reconstruction methods of neighbor-joining, maximum parsimony, and maximum likelihood. In the phylogenetic analyses, the turtle was found to be closely related to the alligator. Also, we found that the turtle had diverged after the divergence of squamates and birds. This departs from previous hypotheses of turtle evolution and further suggests that turtles are the latest of divergent reptiles, having been derived from an ancestor of crocodilian lineage within the last 200 million years.     

The amphibian globin gene repertoire as revealed by the Xenopus genome

The draft genome sequence of the Western clawed frog Xenopus (Silurana) tropicalis facilitates the identification, expression analysis and phylogenetic classification of the amphibian globin gene repertoire. Frog and mammalian neuroglobin display about 67% protein sequence identity, with the expected predominant expression in frog brain and eye. Frog and mammalian cytoglobins share about 69% of their amino acids, but the frog protein lacks the mammalian-type extension at the C-terminus. Like in mammals, X. tropicalis cytoglobin is expressed in many organs including neural tissue. Neuroglobin and cytoglobin genomic regions are syntenically conserved in all vertebrate classes. Frog and fish globin X show only 57% amino acid identity, but gene synteny analysis confirms orthology. The expression pattern of X. laevis globin X differs from that in fish, with a prominent expression in the eye and weak expression in most other examined tissues. Globin X is possibly present as two paralogous copies in X. tropicalis, with one copy showing transition stages of non-functionalization. The amphibian genome contains a previously unknown globin type (tentatively named 'globin Y') which is expressed in a broad range of tissues and is distantly related to the cytoglobin lineage. The globin Y gene is linked to a cluster of larval and adult hemoglobin alpha and beta genes which contains substantially more paralogous hemoglobin gene copies than previously published. Database and gene synteny analyses confirm the absence of a myoglobin gene in X. tropicalis.     

Genomic structure and expression of the soluble guanylyl cyclase alpha2 subunit gene in the medaka fish Oryzias latipes

A cDNA clone encoding the soluble guanylyl cyclase alpha2 subunit was isolated from medaka fish (Oryzias latipes) and designated as OlGCS-alpha2. The OlGCS-alpha2 cDNA was 3,192 bp in length and the open reading frame (ORF) encodes a protein of 805 amino acids. The deduced amino acid sequence has high similarity to that of the mammalian alpha2 subunit gene except for the N-terminal regulatory domain. The C-terminal 5 amino acids, "RETSL", which have been reported to interact with the post synaptic density protein (PSD)-95 were conserved. An RNase protection assay with adult fish organs showed that OlGCS-alpha2 was expressed mainly in the brain and testis. The complete nucleotide sequence (about 41 kbp) of the OlGCS-alpha2 genomic DNA clone isolated from a medaka fish BAC library indicated that the OlGCS-alpha2 gene consisted of 9 exons and 8 introns. The 5'-flanking region and larger introns, such as introns 1, 4, and 7, contained the several fragments conserved in the nucleotide sequences of Rex6 (non-long terminal repeat retrotransposon), MHC class I genomic region, and OlGC1, the medaka fish homolog of the mammalian guanylyl cyclase B gene. Linkage analysis on the medaka fish chromosome demonstrated that the OlGCS-alpha2 gene was mapped to LG13; this mapping position was different from those for the OlGCS-alpha1 and OlGCS-beta1 genes (LG1).     

Structure and diversity of T-lymphocyte antigen receptors alpha and gamma in Xenopus

Short primer PCR directed at conserved regions of amino acid sequence within the T-cell antigen receptor (TCR) and immunoglobulin (Ig) light chain variable ( V) regions was used to amplify putative TCRgamma V region amplicons from stage 45 Xenopus laevis (African clawed frog) mRNA (cDNA). An adult Xenopus spleen cDNA library was screened using the Vgamma and a putative TCRValpha amplicon. Full copy length cDNAs containing the specific PCR-derived Valpha and Vgamma amplicons were recovered at relatively low frequency. Probes complementing the TCRalpha and TCRgamma constant ( C) regions were employed to isolate equivalent numbers of additional TCR alpha and TCR gamma cDNAs in an unbiased (non- V-based) manner. Few Vgamma genes appear to be expressed relative to the highly diverse expression of V alpha genes in equivalent numbers of cDNAs that were analyzed. Two TCRgamma C regions differ at only two positions; whereas two TCRalpha C regions differ at 33 coding positions as well as in their respective 3' untranslated regions, consistent with two independent loci. However, genomic Southern blots revealed considerably higher numbers of hybridizing bands when probed with C gamma than with C alpha. A potential novel mechanism of diversification is suggested by an unusual TCR alpha cDNA in which the V region can be translated in two frames through utilization of two closely linked V genes and an alternative splicing process. This process produces a translatable cDNA that is not readily predictable from the genomic locus utilizing normal recombination and splicing mechanisms.     

PCR detection of excision suggests mobility of the medaka fish Tol1 transposable element in the frog Xenopus laevis

Tol1 is a DNA-based transposable element identified in the medaka fish Oryzias latipes and a member of the hAT (hobo/Activator/Tam3) transposable element family. Its mobility has already been demonstrated in the human and mouse, in addition to its original host species. This element is thus expected to be useful in a wide range of vertebrates as a genomic manipulation tool. Herein, we show that the Tol1 element can undergo excision in the African clawed frog Xenopus laevis, a major model organism for vertebrate genetics and developmental biology. An indicator plasmid carrying a Tol1 element was injected into 2- or 4-cell-stage embryos together with either a helper plasmid coding for the full-length Tol1 transposase or a modified helper plasmid yielding a truncated protein, and recovered from tailbud-stage embryos. Deletion of the Tol1 region of the indicator plasmid was observed in the experiment with the full-length transposase, and not in the other case. The deletion was associated with various footprint sequences at breakpoints, as frequently observed with many DNA-based transposable elements. These results indicate that the Tol1 element was excised from the indicator plasmid by catalysis of the transposase, and suggest that the Tol1 element is mobile in this frog species.     

Structure and developmental expression of hatching enzyme genes of the Japanese eel<Emphasis Type="Italic"> Anguilla japonica</Emphasis>: an aspect of the evolution of fish hatching enzyme gene

We isolated seven cDNA clones from embryos of the Japanese eel Anguilla japonica. Each deduced amino acid sequence consisted of a signal peptide, a propeptide and a mature enzyme portion belonging to the astacin protease family. A phylogenetic analysis showed that the eel enzymes resembled the high choriolytic enzyme (HCE) of medaka Oryzias latipes, and the hatching enzymes of the zebra fish Danio rerio and masu salmon Oncorhynchus masou. Hatching enzymes of these teleosts belonged to the group of the medaka HCE, and not the medaka low choriolytic enzyme (LCE), another hatching enzyme of medaka. Southern blot analysis showed that the genes of the eel hatching enzymes were multicopy genes like the medaka HCE genes. However, one of the eel hatching enzyme genes comprised eight exons and seven introns, and the exon-intron organization was similar to the medaka LCE gene, which is a single-copy gene. The molecular evolution of the fish hatching enzyme genes is discussed. In addition, whole-mount in situ hybridization and immunocytochemistry showed that the eel hatching enzyme was first expressed in the pillow anterior to the forebrain of early neurula, and finally in the cell mass on the yolk sac of later stage embryos. The early differentiation profile of eel hatching gland cells was similar to that of medaka, masu salmon and zebrafish, whereas the final location of the gland cells was different among fishes.Edited by N. Satoh     

Characterization of the mitochondrial respiratory pathways in Candida albicans

cDNA and genomic clones encoding guanylate cyclase activating proteins (GCAP1 and GCAP2) in the Japanese puffer fish (Fugu rubripes) were identified by probing, respectively, a retinal cDNA library and a whole genomic cosmid library with human GCAP1 and GCAP2 cDNA probes. Clones were identified as GCAP1 and GCAP2 on the basis of amino acid identity with the equivalent frog sequences and their placement into GCAP1 and GCAP2 clades within a GCAP phylogenetic tree. The Fugu genes have an identical four exon/three intron structure to GCAP1 and GCAP2 genes from other vertebrates but the introns are smaller, with the result that the four exons spread over approximately 1 kb of DNA in each case. The two genes are separated on to separate cosmids. However, the results of Southern analysis of the cosmids and of genomic DNA are consistent with a tail-to-tail gene arrangement, as in other species, but with a surprisingly large intergenic separation of around 18.7 kb. Recombinant Fugu GCAP1 failed to activate human retinal guanylate cyclase (retGC) in vitro although CD spectroscopy shows that the protein is folded with a similar secondary structure to that of human GCAP1. The failure to activate may be due therefore to a lack of molecular compatibility in this heterologous assay system.     

Cloning,sequencing and phylogenetic/phenetic classification of an estrogen receptor alpha (alpha) subtype of sheepshead minnow (Cyprinodon variegatus)

The estrogen receptor (ER) is a key component of the reproductive system of both teleosts and tetrapods. In this study, the sequence and evolutionary relationship of sheepshead minnow (Cyprinodon variegatus) ER were examined. Total RNA from livers of adult laboratory-reared gravid female C. variegatus was reverse-transcribed to prepare cDNA. Nested pairs of gene-specific degenerative primers derived from conserved amino acid sequences of the ER DNA binding domain were used to amplify an internal fragment of the ER cDNA of C. variegatus using polymerase chain reaction (PCR) followed by rapid amplification of cDNA ends (RACE). The amplified cDNA products were inserted into pGEM T-Easy Vector for cloning and sequencing. The cloned ER cDNA segments gave a 524-amino-acid ER sequence, which represents approximately 80% of the sequence. The use of PHYLIP for phylogenetic analysis with the maximum parsimony method and for phenetic analysis with the neighbor-joining method, along with bootstrap resampling, using 24 known sequences of alpha and beta ER subtypes (both teleosts and tetrapods) indicated that the ER cDNA sequence of C. variegatus has strong homology to the alpha-subtype (ER alpha) of other teleostean fish, especially the closely related killifish species, Japanese medaka (Oryzias latipes). Because ER alpha was the only subtype found, it appears that the alpha-subtype is predominant in C. variegatus liver.     

鳜鱼胰蛋白酶和淀粉酶与胃蛋白酶原基因的克隆与序列分析

采用RT-PCR及RACE法,克隆得到鳜鱼(Siniperca chuatsi)肝胰脏胰蛋白酶(trypsin, Try)、淀粉酶(amylase, Amy)基因 cDNA全序列.结果表明,鳜鱼Try基因cDNA全长为896 bp,其中开放阅读框 (open reading frame,ORF)为744 bp,编码247个氨基酸. 序列同源性分析发现,鳜鱼Try与 斑马鱼(Danio rerio)、非洲爪蟾(Xenopus laevis)、 小鼠Try和人TRY氨基酸序列同源性分别为81.4%、75.3%、74.5%和71.4%.鳜鱼Amy 基因cDNA全长为1 647 bp,其中ORF为1 539 bp,编码512个氨基酸.鳜鱼Amy与斑马鱼 、非洲爪蟾、小鼠Amy和人AMY氨基酸序列同源性分别为79.7%、75.4%、71.9%和70.9%. 同时对鳜鱼基因组进行PCR,获得鳜鱼Try、Amy与胃蛋白酶原(pepsinogen, Pep)全基因组DNA序列.序列分析表明,鳜鱼Try基因由4个内含子和5个外显子组成,全长1 362 bp;鳜鱼Amy基因由8个内含子和9个外显子组成,全长4 267 bp;鳜鱼Pep基因由8个内含子和9个外显子组成,全长 4 032 bp,与其它脊椎动物基因结构相似.应用Genome walker方法在鳜鱼克隆得到长度分别为1 189 bp、413 bp和527 bp的Try、Amy和Pep基因的5′侧翼区序列以及1段长为704 bp的Pep 基因3′侧翼区序列,并利用相关软件预测其中具有多个可调节其表达的调控元件.鳜鱼Try、Am y和Pep基因组全序列的克隆及其序列、结构分析和分子系统进化等的研究,为鱼类消化代谢相关基因的生理功能及表达调控机理进一步研究提供依据.     

Gene expression of leucocytes in vaccinated Japanese flounder (Paralichthys olivaceus) during the course of experimental infection with Edwardsiella tarda

In this paper, we focused on the detection of differentially expressed genes in peripheral blood leucocytes (PBL) during the course of Edwardsiella tarda infection in vaccinated and non-vaccinated Japanese flounder (Paralichthys olivaceus). cDNA microarray analysis was performed to compare the gene expression patterns of the PBL between the vaccinated and non-vaccinated fish in response to E. tarda inoculation. Fish were vaccinated twice, at a two-week interval and experimentally challenged with E. tarda two weeks after the second vaccination. Among the 1187 analyzed genes, 42 genes were up-regulated during the course of infection either in vaccinated or non-vaccinated fish. These genes included immune-related genes, such as MMP-9, MMP-13, CXC chemokine, CD20 receptor and hepcidin. Some immune-related genes were down-regulated after the E. tarda challenge, i.e. interferon inducible Mx protein, MHC class II-associated invariant chain, MHC class II alpha and MHC class II beta encoding genes, immunoglobulin light chain precursor, immunoglobulin light chain and IgM. These responses are thought to be a common reaction of Japanese flounder PBL in the course of edwardsiellosis, irrespective of immunized condition. Ten genes were significantly up-regulated only in vaccinated fish, and 11 genes were significantly up-regulated only in non-vaccinated fish. These genes may have a correlation with the efficacy of vaccination, although we have no evidence to link the different gene expression patterns and the efficacy of vaccination at present.     

Evolutionary analysis of putative olfactory receptor genes of medaka fish, Oryzias latipes

To obtain an understanding of the origin, diversification and genomic organization of vertebrate olfactory receptor genes, we have newly cloned and characterized putative olfactory receptor genes, mfOR1, mfOR2, mfOR3 and mfOR4 from the genomic DNA of medaka fish (Oryzias latipes). The four sequences contained features commonly seen in known olfactory receptor genes and were phylogenetically most closely related to those of catfish and zebrafish.Among them, mfOR1 and mfOR2 showed the highest amino acid (aa) similarity (93%) and defined a novel olfactory receptor gene family that is most divergent among all other vertebrate olfactory receptor genes. Southern hybridization analyses suggested that mfOR1 and mfOR2 are tightly linked to each other (within 24kb), although suitable marker genes were not available to locate their linkage group. Unlike observation in catfish olfactory receptor sequences, nucleotide (nt) substitutions between the two sequences did not show any evidence of positive natural selection. mfOR3 and mfOR4, however, showed a much lower aa similarity (26%) and were both mapped to a region in the medaka linkage group XX.After including these medaka fish sequences, olfactory receptors of terrestrial and aquatic animals formed significantly different clusters in the phylogenetic tree. Although the member genes of each olfactory receptor gene subfamily are less in fish than that in mammals, fish seem to have maintained more diverse olfactory receptor gene families. Our finding of a novel olfactory receptor gene family in medaka fish may provide a step towards understanding the emergence of the olfactory receptor gene in vertebrates.