Cloning and tissue expression of chicken heart fatty acid-binding protein and intestine fatty acid-binding protein genes

Fatty acid-binding proteins (FABPs) are members of a superfamily of lipid-binding proteins, occurring intracellularly in invertebrates and vertebrates. This study was designed to clone and characterize the genes of heart fatty acid-binding protein and intestine fatty acid-binding protein in the chicken. PCR primers were designed according to the chicken EST sequences to amplify cDNA of H-FABP and I-FABP genes from chicken heart and intestinal tissues. Analysis of sequence showed that the cDNA of the chicken H-FABP gene is 75 to 77% homologues to human, mouse, and pig H-FABP genes, and the chicken I-FABP gene is 71 to 72% homologues to human, mouse, and pig I-FABP genes. In addition, Northern blot analysis indicated that of the two genes, similar to the copartner of the mammal, H-FABP gene was expressed in a wide variety of tissues, and I-FABP gene was expressed only in intestinal tissues. The expression levels of the chicken H-FABP mRNA in heart and I-FABP mRNA in intestine had significant differences between the broilers from fat line and Bai'er layers at six weeks of age. The results of this study provided basic molecular information for studying the role of two FABPs in the regulation of fatty acid metabolism in avian species.     

Cloning and Tissue Expression of Chicken Heart Fatty Acid-Binding Protein and Intestine Fatty Acid-Binding Protein Genes

Fatty acid-binding proteins (FABPs) are members of a superfamily of lipid-binding proteins, occurring intracellularly in invertebrates and vertebrates. This study was designed to clone and characterize the genes of heart fatty acid-binding protein and intestine fatty acid-binding protein in the chicken. PCR primers were designed according to the chicken EST sequences to amplify cDNA of H-FABP and I-FABP genes from chicken heart and intestinal tissues. Analysis of sequence showed that the cDNA of the chicken H-FABP gene is 75 to 77% homologues to human, mouse, and pig H-FABP genes, and the chicken I-FABP gene is 71 to 72% homologues to human, mouse, and pig I-FABP genes. In addition, Northern blot analysis indicated that of the two genes, similar to the copartner of the mammal, H-FABP gene was expressed in a wide variety of tissues, and I-FABP gene was expressed only in intestinal tissues. The expression levels of the chicken H-FABP mRNA in heart and I-FABP mRNA in intestine had significant differences between the broilers from fat line and Bai'er layers at six weeks of age. The results of this study provided basic molecular information for studying the role of two FABPs in the regulation of fatty acid metabolism in avian species.     

鸡脂肪酸结合蛋白基因的克隆和测序分析

根据哺乳动物脂肪酸结合蛋白基因序列设计一对引物对鸡基因组进行PCR扩增,将163bp扩增片段进行克隆和测序,并与猪的脂肪酸结合蛋白（fatty acid binding protein,FABP）基因序列进行同源性比较。该基因因片段与猪的心脏脂肪酸结合蛋白（heart fatty acid binding protein,H-FABP）基因有68%的同源性,与猪的脂肪型脂肪酸结合蛋白（adipocyte fatty acid binding protein,A-FABP）基因有75%的同源性,演绎成氨基酸之后与猪的脂肪型脂肪酸结合蛋白相应的氨基酸有75%的同源性。Northern结果表明该基因只在脂肪组织中表达。     

Isolation and characterization of a fatty acid binding protein in adipose tissue of Gallus domesticus.

1. Fatty acid binding protein (A-FABP) was isolated from chicken adipose cytosol. 2. Relative mol. wt of chicken A-FABP was determined to be 14,400 from SDS-polyacrylamide electrophoresis; the pI was 5.1; and amino acid composition data indicated structural homology with mammalian heart and adipose FABPs. 3. Polyclonal antisera prepared against A-FABP exhibited monospecificity for chicken A-FABP and no cross-reactivity with chicken liver proteins was observed. 4. Determination of relative ligand binding characteristics indicated A-FABP exhibited greatest binding activity in response to linoleate, followed by oleate, palmityl CoA and palmitate; no binding affinity for cholesterol was detected.     

Identification in chickens of an evolutionarily conserved cellular ets-2 gene (c-ets-2) encoding nuclear proteins related to the products of the c-ets proto-oncogene.

In chicken cells, we previously identified a set of proteins (p58-64) structurally related to, but distinct from, the products encoded by the c-ets proto-oncogene. We report here the isolation and nucleotide sequence of a cDNA encoding nuclear products of mol. wt 58, 60, 62 and 64 kd, indistinguishable from those detected in chicken cells. The p60 and p64 species appear to represent phosphorylated versions on serine and threonine residues of p58 and p62. The homology of p58-64 to other ets-related proteins, including the v-ets encoded domain of the transforming protein of avian leukemia virus E26 and p54c-ets, the translation product of the chicken (Ck) c-ets gene, is confined to two regions of 175 and 96 amino acid residues localized respectively at the carboxy-terminal domain and close to the amino-terminal domain of these molecules. This cDNA corresponds to a gene localized in a locus distinct from that of c-ets which is transcribed as a 4.0-kb RNA species in most chicken tissues. We also identified the human (Hu) c-ets-2-encoded products as two proteins of 60 and 62 kd, highly related to chicken p58-64. This, together with the fact that the amino acid sequence of the cDNA encoding p58-64 is 95% identical to the reported partial sequence of a Hu-c-ets-2 cDNA, indicates that p58-64 are the translation products of the Ck-c-ets-2 gene.     

人柠檬酸合成酶cDNA的克隆及其表达谱分析

在动物、植物、微生物细胞中普遍存在的三羧酸循环(TCA循环)是产生ATP的主要途径,它不仅参与糖的分解代谢,也参与蛋白质和脂肪的分解代谢。柠檬酸合成酶在TCA循环中起着关键的调节作用,它通过催化乙酰辅酶A与草酰乙酸缩合成柠檬酸。本文用基因组信息学方法获得的一个长1636bp的EST重叠群序列,它与猪柠檬酸合成酶cDNA序列高度同源。从这一序列出发,又用PCR方法从人的睾丸组织和骨胳肌组织的cDNA分子库中,分别克隆到一个1492bp的cDNA片段,在其序列中含有一个长1401bp的可读框,该可读框推导的编码蛋白由466个氨基酸组成,它与猪柠檬酸合成酶、鸡柠檬酸合成酶及酵母柠檬酸合成酶的同源度分别达95．9％,92％和60．9％,故认为该cDNA序列可能就是来自人类柠檬酸合成酶基因的转录本。Northern分析表明人类柠檬酸合成酶在心脏和骨胳肌中呈高表达,在脑、肾和胰腺组织中度表达,在小肠和胸腺中未能检出表达,而在其他9种被检测的组织中仅有低表达。     

Molecular Cloning and Expression of Aven Gene in Chicken

Aven was originally identified as a protein that regulates apoptosis by binding to apoptotic regulators, Bcl-xL and Apaf-1. Recently was found that Aven protein is a potent activator of ATM, critical for its DNA damage-induced activation. An Aven cDNA clone was isolated from chicken (Gallus gallus) after screening of a cerebellum cDNA library. The full-length cDNA is 1,430 nt in size, encoding for a polypeptide of 352 amino acid residues. The predicted amino acid sequence of the chicken Aven is 69, 46, 45 and 37% identical to those of zebra finch, human, xenopus and zebrafish orthologs, respectively. Expression analysis reveals that the chicken Aven gene is expressed in the adult brain, heart, intestine, kidney, lung, stomach and spleen, as well as in the whole embryos of 4- and 6-days old. Phylogenetic analysis of the Aven ortholog proteins from various organisms clusters the chicken Aven in the same group with other bird Avens.     

Human thymidine kinase gene: molecular cloning and nucleotide sequence of a cDNA expressible in mammalian cells.

A cDNA containing the entire coding region of the human thymidine kinase gene has been molecularly cloned. The cDNA is under the control of a simian virus 40 promoter and is expressible in mammalian cells. The complete nucleotide sequence of the human thymidine kinase cDNA has been determined. The cDNA is 1,421 base pairs in length and has a large open reading frame of 702 base pairs capable of specifying a protein with a molecular weight of 25,504. Genomic Southern blotting experiments show that sequences homologous to the human thymidine kinase cDNA are conserved among many vertebrates, including prosimians (lemur), tree shrews, rats, mice, and chickens. Direct comparison of the nucleotide sequences of the human thymidine kinase cDNA and the chicken thymidine kinase gene reveals ca. 70% overall homology. This homology is extended further at the amino acid sequence level, with greater than 74% amino acid residues matched between the human and chicken thymidine kinase proteins.     

Isolation and characterization of a cDNA encoding a chicken beta thyroid hormone receptor.

We have isolated and characterized a cDNA encoding a chicken beta homolog of c-erbA, or thyroid hormone receptor (TR). Chicken liver cDNA libraries were screened with a rat TR beta-1 cDNA probe, and several cDNA inserts were isolated and characterized. The sequence of one cDNA predicts a 369-amino-acid open reading frame (ORF), with a protein sequence that possesses 96% identity with that of rat TR beta-1, but only 88% identity with chicken TR alpha. These data indicate that the cDNA likely encodes a beta form of TR that has the expected putative DNA and T3 binding domains. The chicken TR beta (chTR beta) in vitro translated protein binds T3 with high affinity, and binds both the thyroid hormone response element (TRE) from the rat growth hormone gene and the Xenopus vitellogenin A2 gene estrogen response element (ERE), similarly to that of the rat TR beta-1. Northern blot analysis revealed the expression of a 7.0-kb RNA in several tissues including cerebellum, pituitary, kidney, and liver. This chicken liver TR beta cDNA sequence varies in both the 5' and 3' untranslated regions from the chicken kidney TR beta cDNA sequence recently reported (Forrest et al., 1990). The 5' untranslated cDNA sequence divergence occurs near a potential splice site junction of the human TR beta gene, suggesting that this chicken liver cDNA may represent an alternatively spliced RNA product of the chicken TR beta gene.     

红树林植物木榄水通道基因的克隆和表达

根据植物水通道基因保守区设计简并引物,采用RT-PCR方法,从木榄树叶中分离出水通道基因的cDNA片段;3′RACE获得3′端cDNA序列;再经5′RACE获得5′端部分cDNA序列,命名为PIP2,GenBank登录号为EF126757。该基因全长843个碱基,编码281个氨基酸,具有典型的植物水通道基因结构。该基因编码的蛋白质与含羞草(PIP2;5)、欧洲葡萄(PIP)、拟南芥(PIP3)等水通道蛋白的同源性分别为90%、91%、88%。Northern杂交分析表明,该基因在木榄树不同器官中的表达差异明显:根部有较高的表达水平,茎部较弱,而在叶中只能检测到微弱的信号。     

Molecular cloning, expression and evolutionary analysis of the avian tyrosine kinase JAK1

The Janus protein tyrosine kinases (JAK) constitute a protein family that plays a pivotal role in signalling of a large number of cytokine receptors. The cDNA of the chicken homologue of JAK1 was cloned and its nucleotide sequence determined. Chicken JAK1 protein comprises 1150 amino acids as deduced from its cDNA sequence with a calculated molecular mass of 133 kDa. The overall structure of JAK proteins exemplified by the JAK homology domains JH1–JH7 is also preserved in chicken JAK1. Additionally, phylogenetic analysis demonstrates that chicken JAK1 is more closely related to mammalian JAK1 than to those of fish, exhibiting 80%, 79% and 63% identity in amino acid sequence to human, mouse and zebrafish JAK1, respectively. JAK1 proteins were found to be most conserved in the kinase (JH1) and pseudokinase (JH2) domains. This data is supported by Southern hybridization studies of ZOO blots. Chicken JAK1 shows a ubiquitous expression pattern and is transcribed as a 5.5 kb mRNA in various tissues and cell types. JAK1 expression was particularly high in lymphoid cells.     

Molecular cloning of a cold-shock domain protein, zfY1, in zebrafish embryo(1).

Cold-shock domain proteins in vertebrates contain a highly conserved domain which is related to the Escherichia coli cold-shock proteins. Here we report the cloning of a cold-shock domain protein from zebrafish embryo. Using the combination of PCR techniques with degenerate primers, 5'RACE and 3'RACE, the full length cDNA of a cold-shock domain protein in the zebrafish embryo was successfully cloned without constructing and screening a library. Determined from the deduced amino acid sequence, this protein is most similar to Xenopus, FRGY1, and this newly cloned zebrafish gene was therefore designated as zfY1.     

A novel early estrogen-regulated gene gec1 encodes a protein related to GABARAP

We have isolated, in guinea-pig endometrial cells, an estrogen-induced 1.8 kb RNA called gec1. Screening of a guinea-pig genomic library led to identification of gec1 gene consisting of 4 exons and 3 introns. Exon 1 contains the 5'UTR and the ATG initiation codon. A guinea-pig gec1 cDNA was obtained by 5'-RACE. The 351 bp coding sequence shares 76.8% identity with that of the human GABARAP 924 bp cDNA while UTRs of the two cDNAs differ. A gec1 probe from the 3'UTR revealed a 1.9 kb mRNA in human tissues and a human GEC1 cDNA was isolated from placenta. Its coding sequence shares 93 and 79% identity with that of guinea-pig gec1 and human GABARAP, respectively. The human and guinea-pig GEC1 proteins have 100% identity. GEC1 and GABARAP proteins have 87% identity and N terminus featuring a tubulin binding motif. Thus, estrogen-regulated gec1 is a new gene which could encode a microtubule-associated protein.     

Characterization and expression of chicken selenoprotein W

As an essential trace element, selenium (Se) deficiency results in White Muscle Disease in livestock and Keshan disease in humans. The main objectives of this study were to clone and characterize the chicken selenoprotein W (SeW) gene and investigate SeW mRNA expression in chicken tissues. The deduced amino acid (AA) sequence of chicken SeW contains 85 AAs with UAG as the stop codon. Like all SeW genes identified in different species, chicken SeW contains one well-conserved selenocysteine (Sec) at the 13th position encoded by the UGA codon. The proposed glutathione (GSH)-binding site at the Cys₃₇ of SeW is not conserved in the chicken, but Cys₉ and Sec₁₃, with possible GSH binding, are conserved in SeWs identified from all species. There are 23–59% and 50–61% homology in cDNA and deduced AA sequences of SeW, respectively, between the chicken and other species. The predicted secondary structure of chicken SeW mRNA indicates that the selenocysteine insertion sequence element is type II with invariant adenosines within the apical bulge. The SeW mRNA expression is high in skeletal muscle followed by brain, but extremely low in other tissues from chickens fed a commercial maize-based diet. The SeW gene is ubiquitously expressed in heart, skeletal muscle, brain, testis, spleen, kidney, lung, liver, stomach and pancreas in chickens fed a commercial diet supplemented with sodium selenite. These results indicate that dietary selenium supplementation regulates SeW gene expression in the chicken and skeletal muscle is the most responsive tissue when dietary Se content is low.