Expression of the dnmt3 genes in zebrafish development: similarity to Dnmt3a and Dnmt3b

The zebrafish differs from mammals in that they have six dnmt3 genes as opposed to the two that can produce a catalytically active protein in mammals. Zebrafish also do not show evidence of genomic imprinting and lack the Dnmt3l gene necessary to that process in mammals. As such, they offer a unique opportunity to compare the two genetic situations in order to define the roles of the multiple genes in developmental gene methylation. To this end, we have analyzed the developmental expression of the six dnmt3 genes in zebrafish and find that they fall into two distinct patterns. The expression patterns of the dnmt6 and dnmt8 genes, which more closely resemble the mammalian Dnmt3a gene in sequence, also show an expression pattern that is more similar to the expression of Dnmt3a rather than Dnmt3b. Conversely, the other four dnmt3 genes in zebrafish (dnmt3, dnmt4, dnmt5, and dnmt7) show an expression pattern that is more similar to Dnmt3b. The dnmt6 and dnmt8 genes are also expressed in the adult zebrafish and in the brain in particular. In situ expression analyses show that the dnmt6 and/or dnmt8 genes also show tissue-specific differences in expression with those two genes being more ubiquitously expressed in the developing zebrafish than the other dnmt3 genes. Although differences in dnmt3 function may exist between mammals and fish, our results showing similar expression patterns between the genes in fish and mammals suggest that the six dnmt3 genes in the zebrafish may be analogous to the two Dnmt3 genes in mammals.     

Analysis of genes expressed in head kidney of common carp Cyprinus carpio L treated with cortisol

We analyzed genes expressed from head kidney of common carp Cyprinus carpio L. treated with cortisol. The results of single-pass sequencing of expressed sequence tags (ESTs) from 188 clones (AU240288–AU240367, AU301120–AU301227) from kidney cDNA are presented. One-hundred-twenty-seven clones (67.6%) were completely unknown and are likely to represent newly described genes, whereas 61 clones (32.4%) were identified based on matches to sequences in the database. The putative genes contain several ribosomal proteins, cytochrome oxidase subunits. Immune related cDNA clones identified from kidney were immunoglobulin light chain (n=4), FK506/rapamycin-binding protein (FKBP), CXC chemokine receptor type 4, complement factor B/C2-A3, peptidylprolyl isomerase (cyclophilin; Cyp)-like1, cyclophilin S1, heat shock-70 kDa protein-4, stress-activated protein kinase-3 (n=2). FKBP and cyclophilin genes expressed in normal tissues (head kidney, spleen, liver, brain and heart). Expression of FKBP and cyclophilin genes were not detected in liver, brain and heart when treated with cortisol for 16 h.     

Expression patterns of dnmt3aa,dnmt3ab,and dnmt4 during development and fin regeneration in zebrafish

Epigenetic modifications such as DNA methylation and chromatin modifications are critical for regulation of spatiotemporal gene expression during development. In mammals, the de novo-type DNA methyltransferases (Dnmts), Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns during development. In addition to developmental processes, we recently showed that DNA methylation levels are dynamically changed during zebrafish fin regeneration, suggesting that the de novo-type Dnmts might play roles in the regulation of gene expression during regeneration processes. Here, we showed the detailed expression profiles of three zebrafish dnmt genes (dnmt3aa, dnmt3ab, and dnmt4), which were identified as the orthologues of mammalian dnmt3a and dnmt3b, during embryonic and larval development, as well as fin regeneration processes. dnmt3aa and dnmt3ab are expressed in the brain, pharyngeal arches, pectoral fin buds, intestine, and swim bladder; the specific expression of dnmt3aa is observed in the pronephric duct during larval development. dnmt4 expression is observed in the zona limitans intrathalamica, midbrain–hindbrain boundary, ciliary marginal zone, pharyngeal arches, auditory capsule, pectoral fin buds, intestine, pancreas, liver, and hematopoietic cells in the aorta–gonad–mesonephros and caudal hematopoietic tissue from 48 to 72 h post-fertilization. Furthermore, during fin regeneration, strong dnmt3aa expression, and faint dnmt3ab and dnmt4 expression are detected in blastema cells at 72 h post-amputation. Taken together, our results suggest that zebrafish Dnmt3aa, Dnmt3ab, and Dnmt4 may play roles in the formation of various organs, such as the brain, kidney, digestive organs, and/or hematopoietic cells, as well as in the differentiation of blastema cells.     

半滑舌鳎miR-200a和miR-200b前体克隆及其免疫应答分析

为了探究半滑舌鳎（Cynoglossus semilaevis）miR-200a和miR-200b在免疫应答中的作用,采用PCR方法克隆了半滑舌鳎miR-200家族的miR-200a和miR-200b的前体序列,长度分别为82和88 bp;用The mfold Web Server和Clustalx1.83软件对其前体序列进行了二级结构和同源性分析,miR-200a和miR-200b都具有典型的颈环结构,与其他物种具有较高的同源性。qRT-PCR分析结果显示,miR-200a和miR-200b在健康半滑舌鳎13种组织（肝脏、肠、脾脏、头肾、后肾、鳃、血液、脑、皮肤、肌肉、胃、心脏和卵巢）中均有表达,miR-200a在头肾中表达量最高,在血液中表达量最低,miR-200b在肝脏中表达量最高,在肌肉中表达量最低;miR-200a和miR-200b在鳗弧菌（Vibrio anguillarum）感染半滑舌鳎后不同时间点的4种免疫相关组织（肝脏、肠、脾脏和头肾）中的表达呈现出先上调后下降的规律,但表达达到峰值的时间点有所不同。miR-200a在肝脏和脾中的表达峰值出现在鳗弧菌感染后6h,在肠和头肾中则是鳗弧菌感染后12h,miR-200b在肠、脾和头肾中均在鳗弧菌感染后12h达到表达高峰;miR-200a和miR-200b在脂多糖（LPS）、肽聚糖（PGN）、葡聚糖（WGP）、聚肌胞苷酸（poly I:C）4种病原模拟物刺激后的半滑舌鳎肝脏细胞系中呈现出上调表达趋势,其中Poly I:C刺激半滑舌鳎肝脏细胞系后miR-200a上调表达趋势明显,6h的表达量为0h的9倍,在WGP刺激半滑舌鳎肝脏细胞后miR-200b上调表达趋势明显,2h的表达量为0的9倍。研究结果为揭示miRNA在半滑舌鳎免疫应答中的作用提供了科学依据。     

Immunoreactivity of subunits of the (Na + K)-ATPase Cross-reactivity of the α,α+ and β forms in different organs and species

The immunologic cross-reactivity of the α and α+ forms of the large subunit and the β subunit of the (Na⁺ + K⁺)-ATPase from brain and kidney preparations was examined using rabbit antiserum prepared against the purified holo lamb kidney enzyme. As previously reported by Sweadner ((1979) J. Biol. Chem. 254, 6060–6067) phosphorylation of the large subunit of the (Na⁺ + K⁺)-ATPase in the presence of Na⁺, Mg²⁺, and [γ-³²P]ATP revealed that dog and, very likely, rat brain contain two forms of the large subunit (designated α and α+) while dog, rat, and lamb kidney contain only one form (α). The cross-reactivity of the α and α+ forms in these preparations was investigated by resolving the subunits by SDS-polyacrylamide gel electrophoresis. The separated polypeptides were transferred to unmodified nitrocellulose paper, and reacted with rabbit anti-lamb kidney serum, followed by detection of the antigen-antibody complex with ¹²⁵I-labeled protein A and autoradiography. By this method, the α and α+ forms of rat and dog brain, as well as the α form found in kidney, were shown to cross-react. In addition, membranes from human cerebral cortex were shown to contain two immunoreactive bands corresponding to the α and α+ forms of dog brain. In contrast, the brain of the insect Manduca sexta contains only one immunoreactive polypeptide with a molecular weight intermediate to the α and α+ forms of dog brain. The β subunit from lamb, dog and rat kidney and from dog and rat brain cross-reacts with anti-lamb kidney (Na⁺ + K⁺)-ATPase serum. The mobility of the β subunit from dog and rat brain on SDS-polyacrylamide electrophoresis gels is greater than the mobility of the β subunit from lamb, rat or dog kidney.     

Cloning of cDNAs coding for the Gαil and Gαi2 G-proteins from chick brain

We have cloned and characterized several cDNAs coding for G-protein inhibitory α subunits (Gai) from a chick brain cDNA library. Based on homology to Ga subunits from other eukaryotes, these clones were designated chick Gαil and Gαi2. On the deduced amino-acid level, Gαi1 and Gαi2 were found to be 98 and 95% identical to rat Goal and Gαi2, respectively. Using RNase protection analysis, the Gαi1 and Gαi2 mRNAs were found to be expressed in chick atria, ventricle, lung, liver, brain and kidney.     

Novel splice variants associated with one of the zebrafish <Emphasis Type="Italic">dnmt3</Emphasis>genes

Background  

DNA methylation and the methyltransferases are known to be important in vertebrate development and this may be particularly true for the Dnmt3 family of enzymes because they are thought to be the de novo methyltransferases. Mammals have three Dnmt3 genes; Dnmt3a, Dnmt3b, and Dnmt3L, two of which encode active enzymes and one of which produces an inactive but necessary cofactor. However, due to multiple promoter use and alternative splicing there are actually a number of dnmt3 isoforms present. Six different dnmt3 genes have recently been identified in zebrafish.     

Epigenetic regulation of left–right asymmetry by DNA methylation

DNA methylation is a major epigenetic modification; however, the precise role of DNA methylation in vertebrate development is still not fully understood. Here, we show that DNA methylation is essential for the establishment of the left–right (LR) asymmetric body plan during vertebrate embryogenesis. Perturbation of DNA methylation by depletion of DNA methyltransferase 1 (dnmt1) or dnmt3bb.1 in zebrafish embryos leads to defects in dorsal forerunner cell (DFC) specification or collective migration, laterality organ malformation, and disruption of LR patterning. Knockdown of dnmt1 in Xenopus embryos also causes similar defects. Mechanistically, loss of dnmt1 function induces hypomethylation of the lefty2 gene enhancer and promotes lefty2 expression, which consequently represses Nodal signaling in zebrafish embryos. We also show that Dnmt3bb.1 regulates collective DFC migration through cadherin 1 (Cdh1). Taken together, our data uncover dynamic DNA methylation as an epigenetic mechanism to control LR determination during early embryogenesis in vertebrates.     

Identification and expression analysis of irak1 gene in common carp Cyprinus carpio L.: indications for a role of antibacterial and antiviral immunity

In this study, the full‐length complementary (c)DNA of interleukin‐1 receptor‐associated kinase 1 gene (irak1) was cloned from common carp Cyprinus carpio. The complete open reading frame of irak1 contained 2109 bp encoding a protein of 702 amino acid residues that comprised a death domain, a ProST region, a serine–threonine‐specific protein kinase catalytic domain and a C‐terminal domain. The amino‐acid sequence of C. carpio Irak1 protein shared sequence homology with grass carp Ctenopharyngodon idellus (84·5%). The phylogenetic tree of IRAKs separated the polypeptides into four clades, comprising IRAK1s, IRAK2s, IRAK3s and IRAK4s. Cyprinus carpio Irak1 fell into the cluster with previously reported IRAK1s including teleost Irak1s. The irak1 gene was highly expressed in gills, followed by brain, skin, hindgut, buccal epithelium, spleen, foregut, head kidney and liver, and was expressed at lowest levels in gonad and muscle. The irak1 messenger (m)RNA expression was up‐regulated in liver, spleen, head kidney, foregut, hindgut, gills and skin after stimulation with Vibrio anguillarum and poly(I:C), and significantly high up‐regulated expression was observed in liver and spleen. These results implied that irak1 might participate in antibacterial and antiviral innate immunity. These findings gave the indications that irak1 may participate in antibacterial and antiviral immunity.     

日本鳗鲡的C-型和G-型溶菌酶研究

研究以日本鳗鲡(Anguilla japonica Temminck et Schlegel)为研究对象, 根据其基因组数据库, 预测并扩增出2类, 共5个溶菌酶基因, 包括1个C-型溶菌酶和4个G-型溶菌酶, 分别命名为AJLysC、AJLysG1、AJLysG2、AJLysG3和AJLysG4。它们的cDNA全长分别为811、749、1352、1175和733 bp, 编码143、193、185、185和187个氨基酸。SignalP预测表明, AJLysC和AJLysG1的N-端分别包括15和19氨基酸的信号肽, 另外3种溶菌酶没有信号肽。基因组分析显示, AJLysC、AJLysG2、AJLysG3和AJLysG4的基因结构与其他鱼类的同类溶菌酶的基因结构相似, C-型溶菌酶具有4个外显子, G-型则具有5个。但是, AJLysG1的基因结构与其他鱼类G-型溶菌酶不同, 具有6个外显子, 与其他鱼类溶菌酶的蛋白序列比较, 发现AJLysG1缺失其他G-型溶菌酶存在的第2个酶活性位点氨基酸, 即天冬氨酸Asp。AJLysC与其他很多物种的C-型溶菌酶具有较高的同一性, 如与牙鲆的同一性为72.7%。G-型溶菌酶中AJLysG2、AJLysG3、AJLysG4彼此之间以及与其他物种G-型溶菌酶的同一性相对较高; 而AJLysG1与其他物种以及与其他3种G-型溶菌酶的同一性均不高, 且都在50%以下。组织表达分析显示, 所有5个溶菌酶基因在12种检测的组织中均有表达。C-型溶菌酶在胃及免疫相关组织的表达量较高; G-型溶菌酶在各组织/器官中的表达则差异较大, AJLysG1在皮肤和肌肉中的表达量最高, AJLysG2在免疫组织/器官如血液、头肾、体肾和鳃中表达量较高。经迟缓爱德华氏菌(Edwardsiella tarda)刺激48h后, 这5个溶菌酶基因在组织/器官中的表达量均有上调, 其中在血液、肠道和头肾等的上调较为显著。此外, 研究尝试重组表达这些抗菌肽, 获得了AJLysG2、AJLysG3和AJLysG4基因在鲤上皮瘤细胞(Epithelioma papulosum cyprinid, EPC)细胞中的表达, 重组蛋白表现出对溶壁微球菌(Micrococcus lyso-deikticus)生长的明显抑制作用。文章较全面地研究了日本鳗鲡溶菌酶基因的组成和类型及其表达变化, 并重组表达了部分基因, 这为进一步研究这些溶菌酶的功能, 特别是对病原微生物的作用奠定了基础。