Modulatory effect of environmental endocrine disruptors on N-ras oncogene expression in the hermaphroditic fish, Kryptolebias marmoratus

Kryptolebias marmoratus is the only known internally self-fertilizing vertebrate. It shows high susceptibility to many chemical carcinogens and has been proposed as a potential cancer model species alternative to mammals. Since use of this fish species is expected to rise in cancer research, regulation of oncogenes from K. marmoratus needs proper understanding. We cloned and deduced full-length sequence of cDNA of N-ras oncogene from K. marmoratus. Study of expression profile of N-ras by using quantitative real-time RT-PCR revealed that brain had the highest level of expression compared to other tissues. Some embryonic stages showed more N-ras expression than juveniles and adults. Exposure to two environmental endocrine disrupting chemicals (EDCs), bisphenol A (BPA) and 4-nonylphenyl (NP) caused up-regulation of N-ras in gonad, intestine and liver of hermaphrodite K. marmoratus. It is suggested that K. marmoratus may be a suitable model species for oncogene expression studies. The observed EDC-induced expression of N-ras supports the assumption that EDC exposure may predispose the host to the risk of environmental carcinogenesis.     

Nonylphenol modulates expression of androgen receptor and estrogen receptor genes differently in gender types of the hermaphroditic fish Rivulus marmoratus

To uncover the effect of estrogenic chemicals [4-nonylphenol (NP) and bisphenol A (BisA)] on the expression of androgen receptor (AR) and estrogen receptors (ERalpha and ERbeta) in the hermaphroditic fish Rivulus marmoratus, we cloned the full length of the cDNAs encoding AR, ERalpha, and ERbeta from gonadal tissue of R. marmoratus and analyzed the modulation of expression of these genes following exposure to estrogenic chemicals using real-time RT-PCR. R. marmoratus AR, ERalpha, and ERbeta genes showed a high similarity to the relevant fish species on amino acid residues, respectively. Rm-ERalpha and Rm-ERbeta cDNAs included a serine-rich region when compared to other teleost fish ER genes. Tissue-specific expression of Rm-AR and Rm-ERbeta mRNAs in adult hermaphrodite R. marmoratus was high in the gonad, while Rm-ERalpha mRNA was high in the liver based on real-time RT-PCR. In addition, Rm-AR and Rm-ERalpha mRNAs increased along with developmental stage from stage 3 (5 dpf) to hatching, while Rm-ERbeta mRNA increased from stage 2 (2 dpf). To uncover the effect of estrogenic chemicals on R. marmoratus, we exposed the fish to NP (300 microg/l) and BisA (600 microg/l) for 96 h. Significant down-regulation of Rm-AR, Rm-ERalpha, and Rm-ERbeta mRNA was observed in gonadal tissue after exposure to NP but not BisA. In the liver, there were gender differences in gene expression after EDC exposure. These results demonstrate that expression patterns of the Rm-AR, Rm-ERalpha, and Rm-ERbeta genes in the hermaphroditic fish, R. marmoratus, vary according to tissue and developmental stage as well as the specificity of environmental estrogenic chemicals. These genes can be useful as molecular biomarkers in assessing the potential impact of estrogenic compounds using this species as a model system.     

The structure and expression of the murine wildtype p53-induced phosphatase 1 (Wip1) gene

The human wildtype p53-induced phosphatase 1 (Wip1; GenBank symbol Ppm1d) gene encodes a type 2C protein phosphatase (PP2C) that is induced by ionizing radiation in a p53-dependent manner. We have cloned and sequenced the mouse Wip1 gene and its encoded mRNA. The mouse Wip1 gene is composed of six exons and spans over 36 kb of DNA. The mouse cDNA sequence predicts a 598-amino-acid protein with a molecular mass of roughly 66 kDa. Comparison of human and mouse Wip1 sequences revealed 83% overall identity at the amino acid level. The 5'-flanking region of exon 1 had promoter elements characteristic of a housekeeping gene. The Wip1 coding sequences share conserved functional regions with other PP2Cs from a diverse array of species. Expression of Wip1 mRNA was detected ubiquitously in adult and embryonic tissues, though expression in the testis was much higher than in other tissues. Wip1 has been mapped near the p53 gene on mouse chromosome 11.     

Cloning and comparative sequence analysis of TP53 in Xiphophorus fish hybrid melanoma models

We have cloned and sequenced the p53-encoding cDNA of green swordtail (X. helleri) and southern platyfish (X. maculatus). These two fish species are often used to produce hybrids that develop melanomas after genetic crossing. Computer translation of derived cDNA sequences revealed that p53 polypeptides from these two species are virtually identical, exhibiting only two conservative amino acid substitutions. TP53 mRNA expression was detected in virtually all tissues tested. Comparison of these fish p53 polypeptide sequences with those of other vertebrates, including other fishes, amphibians, and mammals, revealed that conservation is especially high in several previously defined protein domains. In addition, sequencing of the 3′ TP53 genomic region of X. maculatus reveals similarity to the human TP53 locus in overall organization. Knowledge of the Xiphophorus TP53 sequences will allow assessment of mutational alterations within tumors generated from numerous fish genetic crosses.     

The Structure and Expression of the Murine Wildtype p53-Induced Phosphatase 1 (Wip1) Gene

The human wildtype p53-induced phosphatase 1 (Wip1; GenBank symbol Ppm1d) gene encodes a type 2C protein phosphatase (PP2C) that is induced by ionizing radiation in a p53-dependent manner. We have cloned and sequenced the mouse Wip1 gene and its encoded mRNA. The mouse Wip1 gene is composed of six exons and spans over 36 kb of DNA. The mouse cDNA sequence predicts a 598-amino-acid protein with a molecular mass of roughly 66 kDa. Comparison of human and mouse Wip1 sequences revealed 83% overall identity at the amino acid level. The 5′-flanking region of exon 1 had promoter elements characteristic of a housekeeping gene. The Wip1 coding sequences share conserved functional regions with other PP2Cs from a diverse array of species. Expression of Wip1 mRNA was detected ubiquitously in adult and embryonic tissues, though expression in the testis was much higher than in other tissues. Wip1 has been mapped near the p53 gene on mouse chromosome 11.     

黄颡鱼HSC70基因及其组织表达分析

热休克蛋白70（HSP70）与生物体的抗胁迫能力密切相关。本文采用RACE (Rapid amplification of cDNA ends) 技术,从黄颡鱼Pelteobagrus fulvidraco克隆到一种组成型热休克蛋白（HSC70）基因及其cDNA。该cDNA全长2245bp,包括5′非编码区82bp,3′非编码区225bp,开放阅读框(ORF) 1938bp,编码645个氨基酸组成的蛋白质。黄颡鱼HSC70基因含有8个内含子,与人、鼠、虹鳟和花斑溪鳉的HSC70基因内含子数目相同,位置相似。其中,最长内含子（873bp）位于5′端非编码区,其余内含子（长度在80－251bp之间不等）均在编码区以内。黄颡鱼HSC70基因编码的氨基酸序列与南方鲶的相似度最高,达96.13%,与欧洲银鲫和团头鲂的相似度分别为94.45%和94.14%。RT-PCR检测显示,正常情况下黄颡鱼HSC70在血细胞、心脏、肝、头肾、脾、鳃、肌肉和脑中均有表达,但表达量在鳃中最高,肌肉中最低;统计结果显示,热激后HSC70在血细胞、肝、头肾和脑中的表达量显著上升(p<0.05),而在其余组织中热激前后的表达差异不显著(p>0.05)。     

草鱼和翘嘴鲌fgfrhl-1基因的克隆和表达分析

成纤维细胞生长因子受体同源类似物1(fgfrhl-1)基因是目前仅在鱼类基因组中检测到的fgfr基因家族成员, 该序列在鱼类进化过程中高度保守。为研究fgfrhl-1基因的表达情况和具体的功能, 在亲缘关系较远的草鱼(Ctenopharyngodon idellus)和翘嘴鲌(Culter alburnus Basilewsky)中克隆了fgfrhl-1的cDNA序列, 并通过半定量RT-PCR和冰冻切片原位杂交分析了该基因在成体不同组织中的表达情况。克隆结果的序列分析表明: 草鱼fgfrhl-1 cDNA序列全长为1472 bp, 5′-UTR长213 bp, 3′-UTR长56 bp, 开放阅读框长1203 bp; 翘嘴鲌fgfrhl-1 cDNA序列全长为1886 bp, 5′-UTR长298 bp, 3′-UTR长385 bp, 开放阅读框长1203 bp。在两种鱼类中该基因都编码400个氨基酸, 其预测的氨基酸序列同源性高达95.5%。蛋白二级结构预测表明Fgfrhl-1具有FGFRs家族蛋白的胞内酪氨酸激酶区, 跨膜的螺旋区和胞外配体识别结合区, 但其胞外区比FGFRs缺少了3个免疫球蛋白样结构域。通过RT-PCR方法在两种鱼类的心脏、鳃、肝、脾、尾鳍以及肌肉组织的肌间隔中均检测到了fgfrhl-1表达, 但在肌纤维中均没有检测到其表达。对这两种鱼类的肌肉组织、肝脏和脾脏进行的组织切片原位杂交表明fgfrhl-1只在这些组织和器官的结缔组织及导管中表达, 不在间质细胞结构中表达。这些结果说明: fgfrhl-1的成体组织特异性表达模式在不同鱼类中基本一致, fgfrhl-1在鱼类各组织和器官的结缔组织和导管的细胞中表达, 不在间质细胞中表达。因此, fgfrhl-1可能在鱼类结缔组织及导管分化调控或功能维持中有独特作用。     

虾夷扇贝脂多糖诱导的肿瘤坏死因子LITAF基因的克隆及表达分析

脂多糖诱导的肿瘤坏死因子(Lipopolysaccharide-induced TNF-alpha factor,LITAF)是一类重要的炎症细胞因子,在先天性免疫系统中发挥重要的介质作用。文章根据虾夷扇贝LITAF基因EST序列,应用RACE技术克隆了虾夷扇贝LITAF全长cDNA,对序列及编码的氨基酸进行生物信息学分析。结果显示,该基因cDNA全长1 551 bp,其5′非编码区包含76 bp,3′非编码区包含1 001 bp;开放阅读框(ORF)为474 bp,编码157个氨基酸,氨基酸序列中存在一个保守的LITAF结构域;理论分子量16.99 kDa,等电点为6.24。LITAF基因序列为3 698 bp,由3个外显子和两个内含子组成。利用实时荧光定量PCR技术分析LITAF在虾夷扇贝不同组织、不同胚胎发育阶段以及鳗弧菌(Vibrio anguillarum)刺激后各时间段的表达情况。结果表明:LITAF基因在所检测的6个成体组织中均有表达,其中肾脏的表达量最高;胚胎发育的7个时期中,担轮幼体时期表达量最高;菌刺激36 h实验组与对照组的表达量差异大。LITAF基因是LITAF家族的一员,推测LITAF基因参与虾夷扇贝的先天性免疫反应。     

IGF—ImRNA在不同年龄鲤表达的差异和LHRH—A对IGF—ImRNA表达的影响

利用RNA酶保护法对7月龄性未成熟幼鲤和2龄性成熟鲤组织胰岛素样生长因子-I（IGF-I）mRNA的表达水平进行测定,结果表明成鱼肝和肾脏组织IGF-ImRNA的丰度显著高于幼鱼,对鲤成鱼和幼鱼腹腔注射促性腺激素释放激素类似物（LHRH-A,D-Ala^6-Pro^9-NEt-LHRH)使血清生长激素（GH）水平和肝组织IGF-ImRNA水平都显著升高,而成鱼生殖腺IGF-ImRNA的丰度比对照组显著增加,研究结果提示鲤在不同发育阶段肝组织IGF-ImRNA的丰度比对照组显著增加,研究结果提示鲤在不同发育阶段肝组织IGF-ImRNA的表达存在差别,其中2龄成鱼大于7月龄幼鱼;LHRH-A可能通过刺激垂体GH的释放间接促进肝组织IGF-ImRNA的表达,亦可能通过某种未知途径刺激生殖腺IGF-ImRNA的表达。