一种新的五步蛇蛇毒类凝血酶Cdna的克隆和序列分析

目的：克隆出五步蛇的类凝血酶cDNA，为研究其结构和功能的关系。并为下一步基因表达开发抗血栓药物提供依据和基础。方法：从五步蛇蛇腺中提取了总RNA，通过反转录合成第一链cDNA，经PCR扩增出五步蛇毒腺中类凝血酶TLE1的cDNA片段，并将其连接到质粒载体扩增，然后进行DNA测序。结果：成功克隆出一种新的五步蛇类凝血酶的cDNA片段。此片段全长794bp，包括编码部分的全长为777bp。推导其编码的蛋白质序列为258个氨基酸，其中包括18个氨基酸的信号肽，6个氨基权的前肽和234个氨基酸的成熟氨基酸顺序。TLE1成熟肽与其它蛇毒类凝血酶相比，蛋白质一级结构具有较高的同源性。结论：从五步蛇中成功克隆出一 种新的类凝血酶cDNA，并确定了它的DNA顺序。     

人酪氨酸羟化酶基因真核表达载体的构建及在哺乳动物细胞中的表达

用限制性内切酶EcoRI从pKS(-)HTH_1切下全长为1.9 kb的人酪氨酸羟化酶基因,在T_4DNA连接酶的作用下连接在真核表达载体pCDNA_3的EcoR Ⅰ位点,构建成重组质粒pcD-NA_3HTH_1,该质粒转染COS-7细胞,免疫荧光组织化学染色证实酪氨酸羟化酶在其中的表达。     

利用cDNA微阵列技术筛选成年期与胚胎期睾丸差异表达基因——钙联接蛋白基因

目的 :用cDNA微阵列技术克隆和筛选成年期与胚胎期睾丸差异表达基因 ,以进行睾丸发育及精子发生的调控研究。　方法 :构建人睾丸cDNA芯片 ,分别用正常成人及胚胎睾丸的mRNA探针进行杂交 ,对成人和胚胎睾丸中差异表达的基因进行高流量的比较。　结果 :发现 1条成人睾丸高表达、胚胎低表达的基因———钙联接蛋白 (CLGN)基因。　结论 :运用cDNA微阵列方法可筛选到成年期与胚胎期睾丸差异表达基因     

Gene structure and genetic localization of the PCLO gene encoding the presynaptic active zone protein Piccolo

Piccolo belongs to a family of presynaptic cytoskeletal proteins likely to be involved in the assembly and function of presynaptic active zones as sites of neurotransmitter release. Given that abnormalities in the formation of synaptic junctions are thought to contribute to cognitive dysfunction during brain development, we have analyzed and compared the gene structure of the Piccolo gene, PCLO, from humans and mice and determined their chromosomal localization. A comparison of the deduced amino acid sequence of cDNA clones encoding Piccolo from human, mouse, rat and chicken reveals the presence of distinct homology domains. Only subsets of these are also present in the structurally related active zone protein Bassoon indicating that Piccolo and Bassoon perform related but distinct functions at active zones. Characterization of the PCLO gene reveals the presence of 25 coding exons spread over 380kb of genomic DNA. The human PCLO gene maps to 7q11.23-q21.3, a region of chromosome 7 implicated as a linkage site for autism and Williams Syndrome suggesting that alterations in the expression of Piccolo or the PCLO gene could contribute to developmental disabilities and mental retardation.     

核酸杂交反应中影响因素分析

以光敏生物素标记的R3 cDNA克隆为模型,采用正交设计的方法,对影响核酸杂交反应的主要因素:杂交反应温度,反应时间,探针浓度及甲酰胺浓度作了综合性分析。当探针浓度为800μg/L,甲酰胺浓度为10mol/L,50℃杂交反应6h,可获得最高灵敏度(10pg)。     

Pharmacologic preconditioning effects: Prostaglandin E1 induces heat-shock proteins immediately after ischemia/reperfusion of the mouse liver

Prostaglandin E1 (PGE1) has several potential therapeutic effects, including cytoprotection, vasodilation, and inhibition of platelet aggregation. This study investigates the protective action of PGE1 against hepatic ischemia/reperfusion injury in vivo using a complementary DNA microarray. PGE1 or saline was continuously administered intravenously to mice in which the left lobe of the liver was made ischemic for 30 minutes and then reperfused. Livers were harvested 0, 10, and 30 minutes postreperfusion. Messenger RNA was extracted, and the samples were labeled with two different fluorescent dyes and hybridized to the RIKEN set of 18,816 full-length enriched mouse complementary DNA microarrays. Serum alanine aminotransferase and aspartate aminotransferase levels at 180 minutes postreperfusion were significantly lower in the PGE1-treated group than in the saline-treated group. The cDNA microarray analysis revealed that the genes encoding heat-shock protein (HSP) 70, glucose-regulated protein 78, HSP86, and glutathione S-transferase were upregulated at the end of the ischemic period (0 minutes postreperfusion) in the PGE1 group. Our results suggested that PGE1 induces HSPs immediately after ischemia reperfusion. HSPs might therefore play an important role in the protective effects of PGE1 against ischemia/reperfusion injury of the liver.     

神经节细胞胶质瘤恶变及其差异表达基因分析

目的探讨神经节细胞胶质瘤恶性进展的分子变化,为进一步研究分子病因奠定基础。方法取同一患者不断恶化的3次手术标本,行cDNA微阵列检测,分析在不同阶段持续存在的差异表达基因。结果初发(WHO II级)、复发(WHO III级)和再发(WHO IV级)的标本与正常脑组织相比,差异表达基因共19条,其中下调者16条,上调者3条。在下调的基因中,功能明确者有抑制RAF1/MEK/ERKs激酶通路激活的磷酸酰乙醇胺结合蛋白,抑制肿瘤血管增生、侵袭,调控细胞凋亡的碳酰还原酶;抑制c-myc基因表达的肺库否样因子;参与人DNA切除修复的多聚酶ε。结论神经节细胞胶质瘤恶变过程中持续存在的分子变化,以抑制肿瘤增殖基因表达下调为主,其中RAF激酶抑制蛋白、DNA多聚酶ε和碳酰还原酶是参与细胞信号传导和DNA损伤修复等具重要功能的基因,值得进一步研究。     

HIGH-LEVEL EXPRESSION OF RATD1A DOPAMINE RECEPTOR cDNA IN MOUSE FIBROBLASTLTK- CELLS BY n-BUTYRATE

1. In orefer to develop a simple, efficient system for the highlvel expression of dopamine recetors in eukaryotic cells, we have studied the effects of n-butyrate on the expression of rat D_1A dopamine receptor cDNA in mouse fibroblast LTK- cells as compared with those of n-butyrate on endogenous D₁ receptor levles in opossum kidney cells.
2. In the transfected LTK- cell mebranes with pRc/CMVD_1A receptor cDNA, a selective D₁ dopamine antagonist, [³H]-SCH 23390, exhibited a K_d of 0.9 ± 0.1 nmol/L and a B_max of 0.35 ± 0.05 pmol/mg protien (n = 5).
3. Addtion of n-butrate (2–10 mmol/L) to the culture medium for 48h dose-dependently increased the D_1A receptor level up to 1.5 ± 0.3 pmol/mg protien (n = 7), although the K_d values were not affected. The increase in receptor level was accompanined by an elevation of selective D₁ agoinist-induced adenyly cyclase activity.
4. In contrast, n-butyrate treatment (2–10 mmol/L) did not affect either endogenous D₁ receptor levels or fendoldopmainduced adenylyl cyclase activity in possum kidney cells.
5. These results sugges n-butyrate is a sueful tool for obtaining high-level expression of D_1A dopamine receptor cDNA in mouse fibroblast LTK- cells.