1,25-dihydroxyvitamin D-responsive element and glucocorticoid repression in the osteocalcin gene

The active hormonal form of vitamin D3, 1,25-dihydroxyvitamin D3[1,25(OH), which regulates cellular replication and function in many tissues and has a role in bone and calcium homeostasis, acts through a hormone receptor homologous with other steroid and thyroid hormone receptors. A 1,25(OH)2D3-responsive element (VDRE), which is within the promoter for osteocalcin [a bone protein induced by 1,25(OH)2D3] is unresponsive to other steroid hormones, can function in a heterologous promoter, and contains a doubly palindromic DNA sequence (TTGGTGACTCACCGGGTGAAC; -513 to -493 bp), with nucleotide sequence homology to other hormone responsive elements. The potent glucocorticoid repression of 1,25(OH)2D3 induction and of basal activity of this promoter acts through a region between -196 and +34 bp, distinct from the VDRE.     

1,25-dihydroxyvitamin D3: a novel immunoregulatory hormone

The hormonal form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], at picomolar concentrations, inhibited the growth-promoting lymphokine interleukin-2, which is produced by human T lymphocytes activated in vitro by the mitogen phytohemagglutinin. Other metabolites of vitamin D3 were less effective than 1,25(OH)2D3 in suppressing interleukin-2; their order of potency corresponded to their respective affinity for the 1,25(OH)2D3 receptor, suggesting that the effect on interleukin-2 was mediated by this specific receptor. The proliferation of mitogen-activated lymphocytes was also inhibited by 1,25(OH)2D3. This effect of the hormone became more pronounced at later stages of the culture. These findings demonstrate that 1,25(OH)2D3 is an immunoregulatory hormone.     

Molecular cloning of the chicken progesterone receptor

To define the functional domains of the progesterone receptor required for gene regulation, complementary DNA (cDNA) clones encoding the chicken progesterone receptor have been isolated from a chicken oviduct lambda gt11 cDNA expression library. Positive clones expressed antigenic determinants that cross-reacted with six monospecific antibodies derived from two independent sources. A 36-amino acid peptide sequence obtained by microsequencing of purified progesterone receptor was encoded by nucleotide sequences in the longest cDNA clone. Analysis of the amino acid sequence of the progesterone receptor deduced from the cDNA clones revealed a cysteine-rich region that was homologous to a region found in the estrogen and glucocorticoid receptors and to the avian erythroblastosis virus gag-erb-A fusion protein. Northern blot analysis with chicken progesterone receptor cDNA's indicated the existence of at least three messenger RNA species. These messages were found only in oviduct and could be induced by estrogens.     

维生素D对动物免疫细胞的调节作用

维生素D3能调节和控制免疫细胞的增殖和分化,产生免疫调节作用。1,25-二羟维生素D3是维生素D3的活性形式,其功能研究的进展和免疫调节机制的新认识,提示它们在畜禽免疫营养上增强动物抵抗力具有广泛的应用潜力。1,25-二羟维生素D3除了直接作用于T细胞外,还通过各种机制调节单核细胞、巨噬细胞等免疫细胞的表型和功能。该文综述了维生素D3及其活性形式1,25-二羟维生素D3的细胞免疫调节功能的新认识。     

鸡瘦蛋白基因克隆中的若干问题研究

根据GenBank中鸡的LeptinmRNA序列(AccessionNo.AF012727)设计12对引物,用RT-PCR方法从不同品种(系)、不同时期及不同处理鸡的脂肪、肝脏和卵巢组织总RNA中没有扩增出鸡的Leptin基因片段;根据鸡的EST数据库中查到的一条鸡Leptin基因前体序列设计4对引物,用RT-PCR方法在包括卵巢在内的多个组织的cDNA中没能扩增出正确序列。为提高鸡Leptin基因的表达水平,通过给鸡注射胰岛素,利用RT-PCR方法对肝脏、卵巢和脂肪组织的总RNA进行扩增,没有得到目的序列。根据已发表的哺乳动物的Leptin基因序列设计兼并引物对鸡基因组DNA和脂肪、肝脏组织的cDNA进行PCR,结果没有特异性扩增条带,但在小鼠的基因组中可以获得稳定的扩增条带。用扩增长片段LATaq酶从鸡基因组DNA中也没有扩增出Leptin基因片段,而从小鼠的基因组DNA中,可扩增出小鼠Leptin基因片段;以小鼠LeptincDNA片段为探针,对鸡脂肪组织和肝脏组织来源的总RNA进行NorthernBlot分析,并未获得杂交信号;以猪的LeptincDNA片段为探针,对鸡基因组DNA进行SouthernBlot分析,并未获得特异性的结果。研究结果表明,在鸡的脂肪、肝脏和卵巢组织中不存在与小鼠Leptin基因同源性如此高的mRNA序列,在鸡的基因组中也不存在与小鼠、猪等哺乳动物Leptin基因序列同源性如此高的基因?     

Beta-adrenergic receptor kinase: primary structure delineates a multigene family

The beta-adrenergic receptor kinase (beta-ARK), which specifically phosphorylates only the agonist-occupied form of the beta-adrenergic and closely related receptors, appears to be important in mediating rapid agonist-specific (homologous) desensitization. The structure of this enzyme was elucidated by isolating clones from a bovine brain complementary DNA library through the use of oligonucleotide probes derived from partial amino acid sequence. The beta-ARK cDNA codes for a protein of 689 amino acids (79.7 kilodaltons) with a protein kinase catalytic domain that bears greatest sequence similarity to protein kinase C and the cyclic adenosine monophosphate (cyclic AMP)--dependent protein kinase. When this clone was inserted into a mammalian expression vector and transfected into COS-7 cells, a protein that specifically phosphorylated the agonist-occupied form of the beta 2-adrenergic receptor and phosphorylated, much more weakly, the light-bleached form of rhodopsin was expressed. RNA blot analysis revealed a messenger RNA of four kilobases with highest amounts in brain and spleen. Genomic DNA blot analysis also suggests that beta-ARK may be the first sequenced member of a multigene family of receptor kinases.     

Vitamin D receptor as an intestinal bile acid sensor

The vitamin D receptor (VDR) mediates the effects of the calcemic hormone 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We show that VDR also functions as a receptor for the secondary bile acid lithocholic acid (LCA), which is hepatotoxic and a potential enteric carcinogen. VDR is an order of magnitude more sensitive to LCA and its metabolites than are other nuclear receptors. Activation of VDR by LCA or vitamin D induced expression in vivo of CYP3A, a cytochrome P450 enzyme that detoxifies LCA in the liver and intestine. These studies offer a mechanism that may explain the proposed protective effects of vitamin D and its receptor against colon cancer.     

低磷条件下VD_3对大鼠小肠NaPi-Ⅱb mRNA表达及磷吸收的调控

【目的】本试验旨在研究低磷条件下VD3对磷吸收的调节作用。【方法】选择40只断奶后大白鼠(雄),随机分为0.2%(低磷组)和0.6%(正常磷组)两磷(总磷)水平组,每组5个重复,每重复4只大白鼠,试验期7d。试验前6d,每组每只大白鼠肌注VD3代谢抗干忧药物EHDP(二磷酸盐);宰前的12h,每组2只老鼠以600ng/kg.wt剂量注射VD3,为试验组;另2只不注射,为对照组。第7天早晨屠宰、取样。测定血清和骨组织中钙磷及小肠NaPi-Ⅱb载体蛋白mRNA表达量和磷的吸收等指标。【结果】(1)低磷水平对照组骨钙、骨磷分别比正常磷水平低3.33倍(P0.01)和3.03倍(P0.01)。两磷水平试验组VD3含量比对照组高3.08倍和2.32倍,差异极显著;(2)0.2%磷水平小肠各段和肾脏Na+/PiⅡb mRNA表达量试验组皆极显著高于对照组;(3)两磷水平试验组4个部位磷的吸收皆显著或极显著高于对照组,0.2%磷水平试验组较对照组磷吸收4个部位分别提高:51.76%、62.68%、57.79%、47.37%。0.6%磷水平组分别提高18.19%、39.22%、54.72%、39.83%。【结论】(1)低磷情况下,VD3是调节磷吸收的一个重要因素,补充VD3可提高NaPi-Ⅱb转运蛋白mRNA的表达和磷的吸收;(2)VD3对磷吸收的提高作用随着磷水平的增加而减弱。     

鸡生长激素cDNA及其5'端调控区的克隆分析

从肉用品种鸡构建的垂体文库中克隆分离了鸡生长激素ｃＤＮＡ,并作了核苷酸序列测定。克隆的鸡生长激素ｃＤＮＡ序列全长为７８９个碱基对,其中５＇非转译区为４０个碱基对,３’非转译区为１０１个碱基对,编码区为６４８个碱基对。通过序列比较分析,克隆的鸡生长激素ｃＤＮＡ序列与已报道的蛋用品种鸡生长激素ｃＤＮＡ序列的同源性为９６％,与鸭生长激素序列的同源性为８７％,但与火鸡生长激素序列的同源性只有５２％。通过ＰＣＲ技术,本研究还扩增了６个鸡品种的生长激素基因５’端调控区。尽管这些鸡品种的生产性能差异显著,但序列分析表明鸡生长激素基因５’端调控区十分保守。因而,鸡生长激素基因表达的差异可能受到内含子或３’端侧翼序列的影响。另外,鸡生长激素对多人数量性状的影响也在本文中进行了讨论。     

Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family

A complementary DNA (cDNA) for the rat luteal lutropin-choriogonadotropin receptor (LH-CG-R) was isolated with the use of a DNA probe generated in a polymerase chain reaction with oligonucleotide primers based on peptide sequences of purified receptor protein. As would be predicted from the cDNA sequence, the LH-CG-R consists of a 26-residue signal peptide, a 341-residue extracellular domain displaying an internal repeat structure characteristic of members of the leucine-rich glycoprotein (LRG) family, and a 333-residue region containing seven transmembrane segments. This membrane-spanning region displays sequence similarity with all members of the G protein-coupled receptor family. Hence, the LH-CG-R gene may have evolved by recombination of LRG and G protein-coupled receptor genes. Cells engineered to express LH-CG-R cDNA bind human choriogonadotropin with high affinity and show an increase in cyclic adenosine monophosphate when exposed to hormone. As revealed by RNA blot analysis and in situ hybridization, the 4.4-kilobase cognate messenger RNA is prominently localized in the rat ovary.     

Molecular cloning of a complementary DNA encoding human macrophage-specific colony-stimulating factor (CSF-1)

Complementary DNA (cDNA) clones encoding human macrophage-specific specific colony-stimulating factor (CSF-1) were isolated. One cDNA clone codes for a mature polypeptide of 224 amino acids and a putative leader of 32 amino acids. This cDNA, which was cloned in the Okayama-Berg expression vector, specifies the synthesis of biologically active CSF-1 in COS cells, as determined by a specific radioreceptor assay, macrophage bone marrow colony formation, and antibody neutralization. Most of the cDNA isolates contain part of an intron sequence that changes the reading frame, resulting in an abrupt termination of translation; these cDNA's were inactive in COS cells. The CSF-1 appears to be encoded by a single-copy gene, but its expression results in the synthesis of several messenger RNA species, ranging in size from about 1.5 to 4.5 kilobases.     

稀有鮈鲫雄激素受体基因的克隆和内分泌干扰物对其表达的影响

采用RT-PCR和RACE法分离了稀有鮈鲫(Gobiocypris rarus)精巢雄激素受体基因(AR)的cDNA,其核苷酸序列3 130 bp,编码844个氨基酸。它的氨基酸序列与鲤科鱼类AR的同源性较高。AR基因在稀有鮈鲫的性腺、肝、脑、肠和肌肉等组织中均有表达,在雄性个体中精巢和肝脏的表达量最高,其他组织较低,而在雌性个体除肌肉中表达量较低外,其他组织均为中等水平的表达。0.01和0.1 nmol/L的乙炔基雌二醇暴露3 d后,能够分别非显著和显著地提高稀有鮈鲫幼鱼AR的mRNA表达,而1 nmol/L的乙炔基雌二醇则对其表达有下调的趋势,0.1~10 nmol/L的双酚A对其表达均有显著下调,0.01μmol/L壬基酚对其表达有显著下调,而0.1和1μmol/L壬基酚对其表达均有下调的趋势,因此不同种类内分泌干扰物及其不同暴露浓度对稀有鮈鲫AR的mRNA表达有不同影响。     

Cloning of human androgen receptor complementary DNA and localization to the X chromosome

The androgen receptor (AR) mediates the actions of male sex steroids. Human AR genomic DNA was cloned from a flow-sorted human X chromosome library by using a consensus nucleotide sequence from the DNA-binding domain of the family of nuclear receptors. The AR gene was localized on the human X chromosome between the centromere and q13. Cloned complementary DNA, selected with an AR-specific oligonucleotide probe, was expressed in monkey kidney (COS) cells and yielded a high-affinity androgen-binding protein with steroid-binding specificity corresponding to that of native AR. A predominant messenger RNA species of 9.6 kilobases was identified in human, rat, and mouse tissues known to contain AR and was undetectable in tissues lacking AR androgen-binding activity, including kidney and liver from androgen-insensitive mice. The deduced amino acid sequence of AR within the DNA-binding domain has highest sequence identity with the progesterone receptor.     

A chemoattractant receptor controls development in Dictyostelium discoideum

During the early stages of its developmental program, Dictyostelium discoideum expresses cell surface cyclic adenosine monophosphate (cyclic AMP) receptors. It has been suggested that these receptors coordinate the aggregation of individual cells into a multicellular organism and regulate the expression of a large number of developmentally regulated genes. The complementary DNA (cDNA) for the cyclic AMP receptor has now been cloned from lambda gt-11 libraries by screening with specific antiserum. The 2-kilobase messenger RNA (mRNA) that encodes the receptor is undetectable in growing cells, rises to a maximum at 3 to 4 hours of development, and then declines. In vitro transcribed complementary RNA, when hybridized to cellular mRNA, specifically arrests in vitro translation of the receptor polypeptide. When the cDNA is expressed in Dictyostelium cells, the undifferentiated cells specifically bind cyclic AMP. Cell lines transformed with a vector that expresses complementary mRNA (antisense) do not express the cyclic AMP receptor protein. These cells fail to enter the aggregation stage of development during starvation, whereas control and wild-type cells aggregate and complete the developmental program within 24 hours. The phenotype of the antisense transformants suggests that the cyclic AMP receptor is essential for development. The deduced amino acid sequence of the receptor reveals a high percentage of hydrophobic residues grouped in seven domains, similar to the rhodopsins and other receptors believed to interact with G proteins. It shares amino acid sequence identity and is immunologically cross-reactive with bovine rhodopsin. A model is proposed in which the cyclic AMP receptor crosses the bilayer seven times with a serine-rich cytoplasmic carboxyl terminus, the proposed site of ligand-induced receptor phosphorylation.     

Norwalk virus genome cloning and characterization

Major epidemic outbreaks of acute gastroenteritis result from infections with Norwalk or Norwalk-like viruses. Virus purified from stool specimens of volunteers experimentally infected with Norwalk virus was used to construct recombinant complementary DNA (cDNA) and derive clones representing most of the viral genome. The specificity of the clones was shown by their hybridization with post- (but not pre-) infection stool samples from volunteers infected with Norwalk virus and with purified Norwalk virus. A correlation was observed between the appearance of hybridization signals in stool samples and clinical symptoms of acute gastroenteritis in volunteers. Hybridization assays between overlapping clones, restriction enzyme analyses, and partial nucleotide sequence information of the clones indicated that Norwalk virus contains a single-stranded RNA genome of positive sense, with a polyadenylated tail at the 3' end and a size of at least 7.5 kilobases. A consensus amino acid sequence motif typical of viral RNA-dependent RNA polymerases was identified in one of the Norwalk virus clones. The availability of Norwalk-specific cDNA and the new sequence information of the viral genome should permit the development of sensitive diagnostic assays and studies of the molecular biology of the virus.     

鸭肝脏基础型脂肪酸结合蛋白Lb-FABP基因的克隆、组织表达谱和序列特征分析

 【目的】克隆仙湖肉鸭肝脏基础型脂肪酸结合蛋白Lb-FABP基因的cDNA,并进行组织表达谱和蛋白结构分析,为肉鸭的分子育种提供基础资料。【方法】通过比较基因组学,采用RT-PCR和RACE技术,获得了Lb-FABP基因的cDNA序列全长序列;并采用半定量PCR分析了Lb-FABP基因在16个组织的表达;通过生物信息学方法预测了Lb-FABP基因的蛋白结构。【结果】仙湖肉鸭Lb-FABP基因的cDNA全长548bp,包括97bp长的5′非翻译区(5′UTR)和70 bp长的 3′非翻译区(3′UTR)以及381 bp开放阅读框(ORF,含终止密码子)。Lb-FABP基因组序列包括4个外显子和3个内含子,3个内含子分别长为991 bp、292 bp和713 bp。在所检测的16个组织中Lb-FABP基因mRNA均有表达,尤其在肝脏组织中的表达明显高于其它组织。Lb-FABP理化性质表明该蛋白为一偏碱性蛋白,无明显的信号肽和跨膜区域;蛋白二级结构主要由β折叠和少量的α螺旋、loop环构成,预测发现在第5—22氨基酸残基处存在一个细胞溶质脂肪酸结合蛋白活性功能区;其三维结构由反向平行的10条β链及2条短α链组成的有一开口的“蛋白桶”构成。Lb-FABP基因氨基酸序列与鸡的该基因氨基酸的相似性为97.0%,与其它非哺乳脊椎动物的同源性达80%以上,比对尚未发现哺乳动物存在该基因。【结论】成功克隆肉鸭Lb-FABP基因cDNA序列以及基因组序列,该基因在肝脏组织的表达高于其它组织,并获知在5—22氨基酸处存在其蛋白活性功能区。