The major low molecular weight apolipoprotein from normal and hyperlipidemia atherosclerosis-prone (LAP) Japanese quail

The low molecular weight (LMW) apolipoprotein of apo C plays an important role in the metabolism of triglyceride-rich lipoproteins. This study aimed at a characterization of the major LMW apolipoproteins from normal quail strain, and also from LAP (hyperlipidemia atherosclerosis-prone) strain to identify its genetic disorder. The major LMW apoprotein cDNA clone from normal quail comprised of approximately 500 bp, and encoded polypeptide of 78 amino acid residues containing 57 amino acids as a mature apolipoprotein. Although the quail LMW apoprotein showed a low homology to either apo C-I, C-II, or C-III of other animals, it retained a well-developed amphipathic alpha-helix structure. There was no difference in the deduced primary structure of the quail LMW apoprotein between LAP and normal strain. An analysis of the mRNA expression showed that the quail LMW apoprotein was only expressed in the liver of both LAP and normal Japanese quail. No difference was noted in the hepatic expression of the quail LMW apoprotein mRNA between normal and LAP strains with neither normal nor atherogenic dietary conditions. The structure and expression of the major LMW apoprotein thus had no relevance to higher susceptibility of LAP strain to the experimental atherosclerosis.     

Rabbit apolipoprotein A-I mRNA and gene. Evidence that rabbit apolipoprotein A-I is synthesized in the intestine but not in the liver

In order to study the tissue-specific expression of rabbit apolipoprotein (apo) A-I, a 923-base-pair clone, pRBA-502, complementary to rabbit apo A-I mRNA was identified from a rabbit intestinal cDNA library by hybrid-select translation and immunoprecipitation methods. Northern blot and dot-blot hybridization, utilizing 32P-labeled pRBA-502, revealed that the rabbit apo A-I gene is expressed in the intestine, not in the liver and that rabbit apo A-I mRNA is about 950 nucleotides in length. The entire nucleotide sequence of pRBA-502 has been determined and the complete amino acid sequence of the corresponding apo A-I has been deduced. The mRNA codes for a protein comprising 265 amino acids. Amino acids 1-18 and 19-24 of the primary translation product represent the presegment and prosegment, respectively, of apo A-I. Matured rabbit apo A-I contains 241 amino acids and has a molecular mass of 27612 Da. Using pRBA-502 as a probe, a 15.5-kb genomic fragment, which contains the entire apo A-I gene, was isolated from a rabbit liver genomic library. Sequence analysis of the gene shows that the 200 base pairs of the 5' upstream flanking region of the rabbit and human apo A-I genes showed 78% sequence homology. Like the human apo A-I gene, the rabbit apo A-I gene is interrupted by three intervening sequences. Except for two nucleotides in the fourth exon, the coding sequence of the rabbit liver apo A-I gene is identical to that of pRBA-502. Our data showed that the lack of expression of apo A-I gene in rabbit liver is not due to the alternation of rabbit liver apo A-I gene sequence and suggest that the expression of apo A-I gene in rabbit liver is regulated by a trans-acting regulating element(s).     

Distribution of apolipoprotein A-I, C-II, C-III, and E mRNA in fetal human tissues. Time-dependent induction of apolipoprotein E mRNA by cultures of human monocyte-macrophages

Recently developed molecular probes for human apolipoprotein (apo) genes have been used to study the specificity of human tissue expression of the apo A-I, apo C-II, apo C-III, and apo E genes. We have found that apo E mRNA was present in all tissues examined. On the basis of total RNA concentration the relative abundance of apo E mRNA expressed as a percentage of the liver value is as follows: adrenal gland and macrophages, 74-100%; gonads and kidney, 12-15%; spleen, brain, thymus, ovaries, intestine, and pancreas, 3-9%; heart, 1.5%; stomach, striated muscle, and lung, less than 1%. The relative concentration of apo E mRNA in cultures of human peripheral blood monocyte-macrophages increases dramatically as a function of time in culture, and after 5 days, it compares to that of liver. The human tissues shown to synthesize apo E mRNA were also examined for their ability to synthesize apo A-I, apo C-II, and apo C-III mRNA. The relative abundance of apo A-I, apo C-III, and apo C-II mRNA expressed as a percentage of the liver value is as follows: apo A-I, intestine, 50%; apo A-I, pancreas and gonads, 12%; apo A-I, kidney, 4%; apo A-I, adrenal, 2.5%; apo A-I, ovaries and heart, 1%; apo A-I, stomach and thymus, less than 1%; apo C-III, intestine, 62%; apo C-III, pancreas, 7%; apo C-II, intestine, 3%; apo C-II, pancreas, less than 1%. The knowledge of tissue specificities in the synthesis of apolipoproteins is important for our understanding of the regulation of apolipoproteins and lipoprotein metabolism.     

Structure, evolution, and regulation of chicken apolipoprotein A-I

A full-length cDNA clone for the precursor form of chicken liver apolipoprotein A-I (apoA-I) was isolated by antibody screening of a chicken liver cDNA library in the expression vector lambda gt11. The complete nucleotide sequence and predicted amino acid sequence of this clone is presented. The identity of the clone was confirmed by comparison with partial amino acid sequences for chicken apolipoprotein A-I. Chicken preproapolipoprotein A-1 consists of an 18-amino acid prepeptide, a 6-amino acid propeptide, and 240 amino acids of mature protein. The sequence of the protein is homologous to mammalian apoA-I and is highly internally repetitive, consisting largely of 11-amino acid repeats predicted to have an amphipathic alpha-helical structure. The sequence of the propeptide (Arg-Ser-Phe-Trp-Gln-His) differs in two positions from that of mammalian apoA-I. The mRNA for chicken apoA-I is about 1 kilobase in length and is expressed in a variety of tissues including liver, intestine, brain, adrenals, kidneys, heart, and muscle. This quantitative tissue distribution has been determined and is similar to that observed for mammalian apoE and different from that of mammalian apoA-I mRNA. This reinforces the concept that avian apoA-I performs functions analogous to those of mammalian apoE. Moreover, comparisons revealed sequences of chicken apoA-I similar to the region of mammalian apoE responsible for interaction with cellular receptors. Previous studies have demonstrated striking changes in the rates of synthesis of apoA-I in breast muscle during development and in optic nerve after retinal ablation. We now demonstrate that these changes are paralleled by changes in mRNA levels. ApoA-I mRNA levels increase approximately 50-fold in breast muscle between 14 days postconception and hatching and then decrease about 15-fold to adult levels. The levels of apoA-I mRNA increase about 3-fold in optic nerve following retinal ablation. ApoA-I mRNA is also found in the brain in the absence of nerve injury. This may indicate that locally synthesized apoA-I has a routine or housekeeping function in lipid metabolism in the central nervous system.     

Chicken apolipoprotein A-I: cDNA sequence,tissue expression and evolution

Using an antibody against chicken apolipoprotein (apo) A-I, we identified multiple cDNA clones for the protein in two intestinal cDNA libraries in λgtll. The complete nucleotide sequence of chicken apoA-I cDNA was determined. The sequence predicts a mature protein of 240 amino acids, a 6-amino acid propeptide and an 18-amino acid signal peptide. Using a ³²P-cDNA probe, we detected the presence of apoA-I mRNA in 21 day old chicken intestine, liver, kidney, spleen, breast muscle and brain. The primary sequence of apoA-I contains numerous tandem repeats of 11 and 22 residues in a manner similar to the mammalian proteins. Our analysis of apoA-I sequences from human, rabbit, dog, rat, and chicken indicates that the rate of amino acid substitution is considerably faster in the rat lineage than in other mammalian lineages.     

Structure and expression of dog apolipoprotein A-I, E, and C-I mRNAs: implications for the evolution and functional constraints of apolipoprotein structure

Dog apolipoprotein (apo) C-I, A-I, and E cDNA clones were identified in a dog liver cDNA library in lambda gt10 by hybridization to synthetic oligonucleotide probes with the corresponding human DNA sequences. The longest clone for each apolipoprotein was completely sequenced. The apoC-I cDNA sequence predicts a protein of 62 residue mature peptide preceded by a 26 amino acid signal peptide. The apoA-I cDNA sequence predicts a 242 residue mature peptide, a 6 residue pro-segment, and an 18 residue signal peptide. The apoE cDNA, which lacks the signal peptide region, predicts a mature peptide of 291 amino acid residues. Slot blot hybridization of total RNA isolated from various dog tissues to dog apoC-I, A-I, and E cDNA probes indicates that apoC-I mRNA is detectable in liver only, apoA-I mRNA is present in liver and small intestine, though the concentration in the latter tissue is only approximately 15% of that in the liver, and apoE mRNA is present in multiple tissues including liver, jejunum, urinary bladder, ileum, colon, brain, kidney, spleen, pancreas, and testis with relative concentrations (%) of 100, 17.5, 7.5, 6.9, 5.9, 5.5, 5.0, 3.3, 1.0, and 1.0, respectively. These tissue distributions indicate that nascent lipoprotein particles produced in the dog small intestine would contain apoA-I and apoE but not apoC-I. The widespread tissue distribution of apoE mRNA indicates that like other mammals, peripheral synthesis of apoE contributes significantly to the total apoE pool in dog. We next compared the cDNA sequences among different vertebrate species for apoC-I (human and dog), A-I (human, rat, dog, rabbit and chicken), and E (human, rat, dog and rabbit) and calculated the rate of nucleotide substitution for each gene. Our results indicate that apoC-I has evolved rather rapidly and that on the whole, apoA-I is more conservative than apoE, contradictory to an earlier suggestion. ApoA-I is also more conservative than a region (residues 4204-4536) at the carboxyl-terminal portion, but less conservative than a region (residues 595-979) at the amino-terminal portion of apoB-100. Some regions in each of the apolipoproteins studied are better conserved than others and the rate of evolution of individual regions seems to be related to the stringency of functional requirements. Finally, we estimate that the human apoC-I pseudogene arose more than 35 million years ago, becoming nonfunctional soon after its formation.     

版纳微型猪近交系 GHR 基因克隆及生物信息学分析简

目的 获得版纳微型猪近交系（BMI）生长激素受体基因（GHR）序列,通过生物信息学分析预测GHR功能并进行GHR mRNA多组织表达谱分析.方法 以版纳微型猪近交系的肝脏组织为材料提取RNA,RT-PCR方法扩增GHR基因编码区序列,将序列连接至pMD18-T载体进行克隆、测序和生物信息学分析;半定量PCR检测GHR mRNA在BMI不同组织中表达量的差异.结果克隆出了BMI GHR 编码区序列,提交GenBank获得登录号KC999114.该基因CDS长1917 bp,编码638个氨基酸.生物信息学分析表明,与长白猪的GHR序列相比BMI存在4处氨基酸替换,分别为p.E381D、p.A409S、p.L556V和p.A580G,均发生在胞内域.GHR基因多组织表达谱分析显示：GHR mRNA几乎在各组织中均有表达,在肌肉中表达量最高,在小肠、心、肝、神经纤维、脾、卵巢中表达量较高,在肺、胃、大脑、胰和肾中的表达量较低.结论 成功克隆了版纳微型猪近交系GHR全长编码区序列,进行了生物信息学功能分析和组织表达谱分析,为进一步阐明版纳微型猪近交系生长矮小机理奠定了基础.     

Cloning and expression analysis of the sheep ceruloplasmin cDNA.

The cDNA encoding sheep ceruloplasmin (sCP) was isolated from a sheep liver cDNA library. The cDNA contig was 3530 nucleotides in length and encoded a protein of 1048 amino acids. The deduced amino acid sequence showed a high degree of conservation (87%) when compared to the human ceruloplasmin (hCP) sequence. Northern blot analysis of sheep tissue revealed that the sheep ceruloplasmin gene (sCP) was expressed primarily in the liver, but low levels of mRNA were detected in the hypothalamus, spleen and uterus. No sCP mRNA was detected in the cortex, heart, intestine or kidney. Expression was not significantly affected by hepatic copper content. Northern blot analysis of sheep liver during development demonstrated little sCP expression during fetal life, but significant levels of mRNA were observed after birth. Significantly, the developmental expression pattern of sCP was closely correlated with that of the sheep Wilson disease gene (sATP7B), suggesting that the expression of the two genes may be coordinated to ensure that copper is supplied to apoceruloplasmin. Overall, the structure and expression of sCP appeared similar to other mammals, suggesting that abnormalities in CP were not responsible for the unusual sheep copper phenotype.     

Molecular cloning and characterization of beta-defensin cDNA expressed in distal ileum of buffalo (Bubalus bubalis).

Defensins play a prominent role in protection of various epithelial surfaces. In this study, we have cloned and characterized the mRNA from the distal ileum of Bubalus bubalis. Total RNA after isolation from ileal epithelium was reverse transcribed to synthesize cDNA using primers designed by taking conserved region of cattle enteric beta-defensin (EBD) mRNA, goat beta-defensin 2 (BD 2) and cattle lingual antimicrobial peptide (LAP) mRNA sequences. The PCR amplified cDNA of 254 bp was ligated to pDrive cloning vector and transformed into XL-blue strain of E coli. The sequence analysis indicated 29 nucleotide substitutions with reported cattle EBD mRNA sequence sharing 86.2% homology, 92.1% with cattle LAP, 81.6% with cattle tracheal antimicrobial peptide and 84.6% with goat BD 2. The deduced amino acid sequence encodes for a 64 amino acid precursor peptide. Both nucleotide and amino acid sequence homology shows that the cloned sequence is closer to cattle LAP.     

大黄鱼PPAR β全长cDNA的克隆和组织表达

利用RT-PCR和SMART RACE的方法从大黄鱼肝脏中克隆了PPARβ全长cDNA。该序列长3390bp,5′端非翻译区146bp,3′端非翻译区1711bp,开放阅读框1533bp,可编码一个由510个氨基酸组成的蛋白质,该蛋白质分子量为57.2kDa、等电点为6.46。氨基酸序列分析表明,PPARβ基因具有较高的保守性,大黄鱼PPARβ与花鲈、金头鲷、欧鲽、大西洋鲑、红鳍东方鲀、人、黑猩猩、牛、兔及小鼠等10个物种的同源性为72%~91%,其中与花鲈同源性最高,为91%。用RT-PCR法检测PPARβ基因的组织表达,结果表明该基因在大黄鱼肌肉、眼、脾、肾、肝、鳃、心、肠和脑等9个组织中均有表达,其中肝脏组织表达量最大,肌肉最少。     

锦鲤Hepcidin基因的克隆及其组织特异性表达分析

【目的】克隆锦鲤hepcidin全长cDNA序列(k-hepc),并获得此基因在鱼体内的表达模式。【方法】利用RT-PCR和RACE PCR的方法,从锦鲤肝脏中克隆锦鲤hepcidin的全长cDNA,进行序列测定和分析;锦鲤经肌肉注射维氏气单胞菌0、4、8、12、24和48 h后,分别取其肝、脾、肾、肠、脑、心、肌肉和鳃组织,采用实时荧光定量PCR的方法,以β-actin为内参基因,检测k-hepc基因的表达量。【结果】锦鲤抗菌肽(GenBank登录号KC795559)全长755 bp,编码序列276 bp,编码91个氨基酸,包括信号肽、原肽和成熟肽,成熟肽C端含有8个半胱氨酸,可形成4个分子内二硫键。与已报道的普通鲤鱼hepcidin氨基酸序列的一致性为93%,与其他鱼类hepcidin氨基酸序列的一致性为29%?93%。在本研究所检测的正常锦鲤的组织中,k-hepc均有表达,其中在肝组织中表达量最高,鳃组织中表达量最低。经维氏气单胞菌感染后,k-hepc在肝和心组织中的表达量明显增加,在其余组织中变化不显著。【结论】k-hepc编码的蛋白是Hepcidin家族的成员之一。锦鲤Hepcidin的表达主要受内在调节因素影响。     

Molecular cloning and nucleotide sequence of cDNA encoding the rat kidney S-adenosylmethionine synthetase.

We previously reported the isolation of a cDNA encoding the liver-specific isozyme of rat S-adenosylmethionine synthetase from a lambda gt11 rat liver cDNA library. Using this cDNA as a probe, we have isolated and sequenced cDNA clones for the rat kidney S-adenosylmethionine synthetase (extrahepatic isoenzyme) from a lambda gt11 rat kidney cDNA library. The complete coding sequence of this enzyme mRNA was obtained from two overlapping cDNA clones. The amino acid sequence deduced from the cDNAs indicates that this enzyme contains 395 amino acids and has a molecular mass of 43,715 Da. The predicted amino acid sequence of this protein shares 85% similarity with that of rat liver S-adenosylmethionine synthetase. This result suggests that kidney and liver isoenzymes may have originated from a common ancestral gene. In addition, comparison of known S-adenosylmethionine synthetase sequences among different species also shows that these proteins have a high degree of similarity. The distribution of kidney- and liver-type S-adenosylmethionine synthetase mRNAs in kidney, liver, brain, and testis were examined by RNA blot hybridization analysis with probes specific for the respective mRNAs. A 3.4-kilobase (kb) mRNA species hybridizable with a probe for kidney S-adenosylmethionine synthetase was found in all tissues examined except for liver, while a 3.4-kb mRNA species hybridizable with a probe for liver S-adenosylmethionine synthetase was only present in the liver. The 3.4-kb kidney-type isozyme mRNA showed the same molecular size as the liver-type isozyme mRNA. Thus, kidney- and liver-type S-adenosylmethionine synthetase isozyme mRNAs were expressed in various tissues with different tissue specificities.     

Human brain glycogen phosphorylase. Cloning, sequence analysis, chromosomal mapping, tissue expression, and comparison with the human liver and muscle isozymes

We have cloned the cDNA encoding a new isozyme of glycogen phosphorylase (1,4-D-glucan:orthosphosphate D-glucosyltransferase, EC 2.4.1.1) from a cDNA library prepared from a human brain astrocytoma cell line. Blot-hybridization analysis reveals that this message is preferentially expressed in human brain, but is also found at a low level in human fetal liver and adult liver and muscle tissues. Although previous studies have suggested that the major isozyme of phosphorylase found in all fetal tissues is the brain type, our data show that the predominant mRNA in fetal liver (24-week gestation) is the adult liver form. The protein sequence deduced from the nucleotide sequence of the brain phosphorylase cDNA is 862 amino acids long compared with 846 and 841 amino acids for the liver and muscle isozymes, respectively; the greater length of brain phosphorylase is entirely due to an extension at the far C-terminal portion of the protein. The muscle and brain isozymes share greater identity with regard to nucleotide and deduced amino acid sequences, codon usage, and nucleotide composition than either do with the liver sequence, suggesting a closer evolutionary relationship between them. Spot blot hybridization of the brain phosphorylase cDNA to laser-sorted human chromosome fractions, and Southern blot analysis of hamster/human hybrid cell line DNA reveals that the exact homolog of the newly cloned cDNA maps to chromosome 20, but that a slightly less homologous gene is found on chromosome 10 as well. The liver and muscle genes have previously been localized to chromosomes 14 and 11, respectively. This suggests that the phosphorylase genes evolved by duplication and translocation of a common ancestral gene, leading to divergence of elements controlling gene expression and of structural features of the phosphorylase proteins that confer tissue-specific functional properties.     

Cloning and regulation of rat apolipoprotein B mRNA

Recombinant cDNA clones that code for apolipoprotein B(apoB) were isolated from a rat liver cDNA library, using synthetic oligonucleotide probe derived from the sequence of human apoB cDNA. The nucleotide and deduced amino acid sequences of the rat apoB clone pRB5, 1.2 kb in length, showed 83% and 84% homology to those of human apoB. Northern blot analysis revealed that rat apoB cDNA probe cross-reacts with human and rabbit apoB mRNA sequences and the size of those mRNAs, approximately 15 kb long, were not discernibly different. In addition, apoB mRNA was abundant only in the liver and intestine. Finally, cholesterol feeding to rats for six weeks resulted in a several-fold increase in the level of apoB mRNA in the liver.