Cloning and tissue expression characterization of the chicken APOB gene

Apolipoprotein B (APOB) serves an essential role in the assembly and secretion of triglyceride-rich lipoproteins and lipids transport. This study was designed to clone the full-length cDNA of the chicken APOB gene, to characterize the expression profile, and investigate the differential expression between layer and broiler of the chicken APOB gene. The full-length cDNA sequence (14,150-bp) that contained a 13,896-bp ORF encoding 4,631 amino acids was obtained by RT-PCR, RACE, and bioinformatics analysis. qReal-Time PCR analysis showed that the chicken APOB gene was highly expressed in kidney, liver, and intestine. The results of differential expression showed that the APOB gene was more highly expressed in intestine and kidney in Bai'er layer than in broiler, but there was no significant difference in liver between the two breeds. The results of this study provided basic molecular information for studying the role of APOB in the energy transportation in avian species.     

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.     

A pore-forming protein,perforin, from a non-mammalian organism,Japanese flounder,<Emphasis Type="Italic"> Paralichthys olivaceus</Emphasis>

A perforin cDNA of Japanese flounder, Paralichthys olivaceus, was cloned from a cDNA library of kidney stimulated with ConA/PMA. The full-length cDNA is 2,157 bp, which encodes 587 amino acids. The Japanese flounder perforin gene consists of five exons and four introns, with a length of approximately 3 kb. The amino acid sequence of the Japanese flounder perforin is 36% identical to that of rat perforin and 37% identical to amino acid sequences of mouse and human perforin. The Japanese flounder perforin also showed low homology to human and mouse complement components (C6, C7, C8 and C9), ranging from 19% to 24%. However, the membrane attack complex/perforin domain is conserved. A phylogenetic analysis placed the Japanese flounder perforin in the same cluster with other known mammalian perforins. RT-PCR analysis revealed that the perforin gene was expressed in the peripheral blood leukocytes, head kidney, trunk kidney, spleen, heart, gill and intestine of healthy fish. Recombinant perforin produced in insect cells using the baculovirus expression system showed calcium-dependent hemolytic activity.     

Antisense oligonucleotide-induced alternative splicing of the <Emphasis Type="Italic">APOB</Emphasis> mRNA generates a novel isoform of APOB

Background  

Apolipoprotein B (APOB) is an integral part of the LDL, VLDL, IDL, Lp(a) and chylomicron lipoprotein particles. The APOB pre-mRNA consists of 29 constitutively-spliced exons. APOB exists as two natural isoforms: the full-length APOB100 isoform, assembled into LDL, VLDL, IDL and Lp(a) and secreted by the liver in humans; and the C-terminally truncated APOB48, assembled into chylomicrons and secreted by the intestine in humans. Down-regulation of APOB100 is a potential therapy to lower circulating LDL and cholesterol levels.     

Cloning, identification and accurate normalization expression analysis of PPARα gene by GeNorm in Megalobrama amblycephala

Megalobrama amblycephala suffers from serious liver diseases recently and PPARα gene has been reported to play an important role in the immune system of animal liver. On the basis of these facts, we have cloned and identified full-length cDNA of PPARα and examined its expression patterns at different embryo developmental stages and in different tissues of adult and young fish in order to improve liver disease immunity of M. amblycephala. We also accurately normalized seven reference genes by GeNorm and calculated their gene expression normalization factors. The total length of PPARα cDNA was 2021 bp, comprising of 214-bp 5'-untranslated region; 1404-bp open reading frame (encoding 467-amino acids); and 403-bp 3'-untranslated region. PPARα peptide was predicted to consist of 4 domains, i.e. A/B, C, D, and E/F. PPARα mRNAs were detected in different tissues of adult and young fish including adipose tissue, gill, heart, liver, spleen, kidney, white muscle, intestine, brain and gonad. In adult fish, the expression of PPARα in white muscles was highest followed by liver and it was lowest in gonads. Its expression in male gonads was significantly higher than female gonads. In young fish, the expression of PPARα was highest in brain, followed by intestines and it was lowest in spleen. At different embryo developmental stages, the expression of PPARα was highest at 2 cells stage and it was lowest at gastrula stage, but it increased on first day after hatching. In unfertilized spermatozoa, the expression of PPARα was higher than unfertilized ovum.     

军曹鱼催乳素受体PRLR1基因的克隆及其在不同盐度条件下mRNA表达差异

催乳素受体通过结合催乳素,能调节鱼体渗透压。为研究催乳素受体1(PRLR1)在高盐水体和低盐水体中对军曹鱼(Rachycentron canadum)的渗透调节作用,利用cDNA末端快速扩增(RACE-PCR)技术,获得了军曹鱼PRLR1全长cDNA序列。该基因全长为2629 bp,包含1953 bp的开放阅读框ORF,可编码650个氨基酸。氨基酸序列包含了2个纤维连接蛋白3型结构域(FN3)、保守的WS区和box1。采用qRT-PCR技术,检测不同盐度(10‰、30‰和35‰)条件下鳃、肠、体肾中PRLR1基因mRNA表达情况。结果显示,PRLR1基因在军曹鱼的各个组织中均有表达,其中鳃表达量最高,其次是肌肉、体肾和肠,而在胃、脾、脑和心脏中则微量表达。低盐组、正常组和高盐组中,PRLR1基因的表达量均为鳃最高;肠次之;体肾最低。随着盐度提高,PRLR1基因的鳃、肠和体肾组织表达量变化规律均呈逐步下降趋势。以上结果反映了军曹鱼PRLR1在渗透压器官中的功能差异性,说明PRLR1在军曹鱼渗透压调节上具有重要作用。     

Molecular cloning, sequence characterization, SNP detection, and tissue expression analysis of duck FMO3 gene

Flavin-containing monooxygenase 3 (FMO3) is an important monooxygenase for catalytic oxygenation of many harmful xenobiotics. Mutations in the FMO3 gene have been identified as causing trimethylaminuria in human and fishy off-flavor in cow milk and chicken eggs. In this study, the full-length cDNA sequence of Pekin duck FMO3 gene was cloned, sequenced, and characterized. The full-length cDNA sequence consisted of 1,846 bp and contained a 1,599 bp open-reading frame encoding 532 amino acids. Duck FMO3 gene shared a similar nine exon–eight intron structure with chicken and human. The duck FMO3 putative protein sequence showed high identity with that of chicken (82 %), and relative low identity with those of mammals (61–66 %). We also found that the duck FMO3 gene was dramatically expressed in liver, lung, and kidney compared to that in other tissues in the ducks, indicating the possible roles the FMO3 gene could play in the three tissues. By bidirectional sequencing, we also found one nonsense mutation, 5 nonsynonymous, and 21 synonymous mutations in the coding region of the FMO3 gene in 11 duck breeds and some of them were predicted to be potentially associated with the activities of FMO3 protein.     

Molecular cloning, gene expression, and identification of a splicing variant of the mouse parkin gene

We have isolated mouse cDNA clones that are homologous to human Parkin gene, which was recently found to be responsible for the pathogenesis of autosomal recessive juvenile parkinsonism (AR-JP). One of these cDNA clones had the 1,392-bp open reading frame encoding a protein of 464 amino acids with presumed molecular weight of 51,615. The amino acid sequence of mouse parkin protein exhibits 83.2% identity to human Parkin protein, including the ubiquitin-like domain at the N-terminus (identity = 89.5%) and the RING finger-like domain at the C-terminus (identity = 90.6%). Two other clones had the 783-bp open reading frame encoding a truncated protein of 261 amino acids without RING finger-like domain. It was proved to be a novel splicing variant by 3′-RACE method. Northern blot analysis revealed that mouse parkin gene is expressed in various tissues including brain, heart, liver, skeletal muscle, kidney, and testis. It is notable that mouse parkin gene expression appears evident in 15th day mouse embryo and increases toward the later stage of development. These mouse parkin cDNA clones will be useful for elucidating the essential physiological function of parkin protein in mammals. Received: 5 May 1999 / Accepted: 11 February 2000     

A novel sheep gene,MMP7, differentially expressed in muscles from black-boned sheep and local common sheep

Differences in gene expression in muscles from Chinese black-boned sheep and local common sheep were investigated using mRNA differential display. One differentially expressed novel gene was identified through semi-quantitative RT-PCR, and the full-length cDNA sequence was then obtained using the rapid amplification of cDNA ends (RACE). The nucleotide sequence of this gene is not homologous to any of the known sheep genes, but it contains an open reading frame encoding a protein of 416 amino acids, which has high homology with matrix metallopeptidase 7 (matrilysin, uterine) (MMP7) of 10 species: bovine (93%), rhesus monkey (75%), human (74%), pig (73%), chimpanzee (73%), dog (73%), horse (72%), mouse (66%), rat (65%), and chicken (53%). Thus the novel gene can be defined as the sheepMMP7 gene. It was finally assigned to GeneID: 100192317. The phylogenetic tree analysis revealed that the sheepMMP7 gene is closely related to the bovineMMP7. Our experiment is the first one to establish the primary foundation for further research on the sheepMMP7 gene.     

鸡PPARγ基因的表达特性及其对脂肪细胞增殖分化的影响

为分析鸡PPARγ基因的组织表达特性及其在脂肪细胞增殖和分化过程中的功能,文章以东北农业大学高、低腹脂双向选择品系肉鸡为实验材料,利用Western blotting方法,检测PPARγ基因的组织表达特性及其在高、低脂系肉鸡腹部脂肪组织间的表达差异;采用RNAi技术,在鸡原代脂肪细胞中抑制PPARγ基因的表达后,通过MTT和油红O提取比色的方法,研究鸡PPARγ基因对脂肪细胞增殖和分化的调控作用;利用Real-timePCR和Western blotting技术,分析PPARγ基因表达下调后,其他脂肪细胞分化转录因子以及与脂肪细胞分化相关的重要基因的表达变化情况。结果表明,PPARγ基因在7周龄高脂系肉鸡腹部脂肪组织、肌胃、脾脏、肾脏组织中表达量较高,在心脏中表达量较低,在肝脏、胸肌、腿肌、十二指肠中未检测到表达信号;与高脂系相比,PPARγ基因在5和7周龄低脂系肉鸡腹部脂肪组织中的表达量较低(P<0.05);PPARγ基因的表达量下降后,鸡脂肪细胞的增殖能力增强,分化能力减弱;同时,C/EBPα、SREBP1、A-FABP、Perilipin1、LPL、IGFBP-2基因的表达量均下降(P<0.05)。由此可见,PPARγ基因的表达可能与肉鸡腹部脂肪的沉积有一定的关系,该基因可能是调控鸡脂肪细胞增殖与分化的关键因子。     

Molecular characterization and expression profile of a novel porcine gene differentially expressed in the muscle and backfat tissues from Chinese Meishan and Russian Large White pigs

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus dorsi muscle and backfat tissues from Chinese Meishan and Russian Large White pigs. One novel gene that was differentially expressed was identified through semiquantitative RT-PCR, and the cDNA complete sequence was then obtained using the rapid amplification of the cDNA ends (RACE) method. The cDNA sequence of this 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 402 amino acids that contains the putative conserved transposase DDE domain, and further Blast analysis revealed that this protein has 100% homology with the Tn10 transposase from Oryza sativa, Serratia marcescens, and Salmonella, and, therefore, this gene can be defined as the swine Tn10 transposase gene. This novel porcine gene was finally assigned to Gene ID: 100049649. The RT-PCR analysis of the tissue expression profile was carried out using the tissue cDNAs of one Meishan pig as the templates, and the result indicated that this novel swine gene is moderately expressed in fat and weakly expressed in small intestine, liver, kidney, and spleen but almost not expressed in heart, ovary, muscle, and lung. Our experiment established the primary foundation for further research into the biological significance of swine Tn10 transposase gene.     

Isolation,characterization, and chromosomal mapping of a novel cDNA clone encoding human selenium binding protein

We have isolated the full-length human 56 kDa selenium binding protein (hSP56) cDNA clone, which is the human homolog of mouse 56 kDa selenium binding protein. The cDNA is 1,668 bp long and has an open reading frame encoding 472 amino acids. The calculated molecular weight is 52.25 kDa and the estimated isoelectric point is 6.13. Using Northern blot hybridization, we found that this 56 kDa selenium binding protein is expressed in mouse heart with an intermediate level between those found in liver/lung/kidney and intestine. We have also successfully expressed hSP56 in Escherichia coli using the expression vector-pAED4. The hSP56 gene is located at human chromosome 1q21–22. J. Cell. Biochem. 64:217–224. © 1997 Wiley-Liss, Inc.