人类β-1,4-半乳糖苷转移酶Ⅲ基因的分离与克隆

以人、牛、鼠、鸡、蜗牛的β-1,4-半乳糖苷转移酶催化区的编码核苷酸序列为探针,在NCBI GenBank EST数据库中进行同源搜寻,获得若干有高度同源的EST.在拼接序列的两端设计引物,以从人胎盘cDNA文库中PCR扩增获得的片段为探针,在人胎盘cDNA分子库中步移获得一个长1,907bp的cDNA片段,包含一个长1,179bp的开放阅读框(ORF,open reading frame),编码393个氨基酸残基。该基因与已知的人类β1,4-GalTI的氨基酸同源性为43.8%,在蛋白质催化区的同源性更高达60.9%。表达谱分析发现该基因在人体16种组织中均有不同程度的表达,转录本大小约为2.4kb.通过该cDNA人/啮齿类杂种细胞株DNA Southern杂交将该基因定位在1号染色体。     

多发性骨髓瘤细胞中一个新的Alu超基因家族成员的克隆与分析

根据Genebank收录的多发性骨髓瘤细胞株（ARH－77）表达上调EST AF497797设计引物,采用半定量RT－PCR证实了多发性骨髓瘤患者及正常人骨髓细胞中该expressed sequence tages(EST)存在表达差异。用EST AF497797作探针筛选人胚肾cDNA文库,获得cDNA克隆经测序并用生物信息学方法对该序列进行了初步分析。AF497797在多发性骨髓瘤患者骨髓中确有较高的表达,而在正常人骨髓细胞中低表达。获得的全长cDNA克隆序列长1248bp(Genebank登录号：AY094612）。生物信息学分析显示该片段全长cDNA编码44个氨基酸的蛋白产物且可能属于Alu家族成员。基因AY094612为一个在多发性骨髓瘤中表达上调的新基因,其改变可能与多发性骨髓瘤的发生与发展有关。     

大白菜碳酸酐酶基因的克隆与序列分析

以大白菜雄性不育系和保持系花蕾差异表达片段EST H9为信息探针,在GenBank数据库中进行同源EST序列检索,并对亲缘关系近的同源EST序列进行拼接,得到大白菜EST H9的5'-cDNA序列.根据拼接组装所得的5'-cDNA序列,进行3'-RACE引物的设计,经RACE扩增、测序,获得了大白菜α-碳酸酐酶3基因的cDNA全长序列并将其登录到GenBank(登录号为GU143061),命名为BrACA3.该cDNA全长998 bp,编码270个氨基酸.同源分析显示该cDNA序列推导的氨基酸序列与拟南芥α-碳酸酐酶3一致性达78%.氨基酸序列分析表明,该蛋白具备跨膜功能,在第19和20个氨基酸之间存在一个信号肽序列,存在丝氨酸、苏氨酸和酪氨酸磷酸化位点.在大白菜花蕾败育过程中α-碳酸酐酶3基因不表达,只在保持系B7的大花蕾时期表达.     

日本血吸虫童虫裂解物对东方田鼠肝脏T7噬菌体展示cDNA文库的筛选及阳性克隆分析

东方田鼠对血吸虫具有天然抗性。为筛选和分析东方田鼠抗血吸虫抗性相关基因, 以日本血吸虫童虫可溶性裂解物为探针, 筛选东方田鼠肝脏噬菌体展示cDNA文库。经三轮筛选, 特异性噬菌体得到有效富集(375倍)。随机挑取92个克隆进行序列测定, 获得了19条有效EST序列。其中13个条EST序列与已知基因或表达序列标签同源, 6个EST序列与已知基因或表达序列标签均无同源性, 为新的表达序列标签。将19个EST序列的阳性噬菌体克隆和血吸虫童虫共培养, 其中4号(GenBank Accession No.: EW968294)、13号(GenBank Accession No.: EW968303)、14号(GenBank Accession No.: EW968304)、15号(GenBank Accession No.: EW968305)、18号(GenBank Accession No.: EW968308)克隆均诱导了显著的杀虫效果。综合生物信息学分析结果及体外杀伤试验结果, 编码CASP8和FADD类似性细胞程序性死亡调节蛋白、a-2-HS-糖蛋白、M4蛋白、具有R3H结构域的一种mRNA结合蛋白以及三种未知蛋白的编码基因(14、15、18号克隆)可能是东方田鼠抗血吸虫病抗性相关基因。为进一步研究东方田鼠抗血吸虫机理奠定了基础。     

日本血吸虫童虫裂解物对东方田鼠肝脏T7噬菌体展示cDNA文库的筛选及阳性克隆分析

东方田鼠对血吸虫具有天然抗性。为筛选和分析东方田鼠抗血吸虫抗性相关基因, 以日本血吸虫童虫可溶性裂解物为探针, 筛选东方田鼠肝脏噬菌体展示cDNA文库。经三轮筛选, 特异性噬菌体得到有效富集(375倍)。随机挑取92个克隆进行序列测定, 获得了19条有效EST序列。其中13个条EST序列与已知基因或表达序列标签同源, 6个EST序列与已知基因或表达序列标签均无同源性, 为新的表达序列标签。将19个EST序列的阳性噬菌体克隆和血吸虫童虫共培养, 其中4号(GenBank Accession No.: EW968294)、13号(GenBank Accession No.: EW968303)、14号(GenBank Accession No.: EW968304)、15号(GenBank Accession No.: EW968305)、18号(GenBank Accession No.: EW968308)克隆均诱导了显著的杀虫效果。综合生物信息学分析结果及体外杀伤试验结果, 编码CASP8和FADD类似性细胞程序性死亡调节蛋白、a-2-HS-糖蛋白、M4蛋白、具有R3H结构域的一种mRNA结合蛋白以及三种未知蛋白的编码基因(14、15、18号克隆)可能是东方田鼠抗血吸虫病抗性相关基因。为进一步研究东方田鼠抗血吸虫机理奠定了基础。     

小鼠睾丸生精细胞凋亡相关基因SRG2的分子克隆

从已获得的在隐睾和正常睾丸对照中表达量有明显差异的EST片段 (BE6 4 4 5 4 2 )入手 ,利用网上生物信息学克隆了该基因全长 ,GenBank登录号为AF395 0 83。从小鼠睾丸cDNA文库中分离出该基因完整阅读框cDNA ,SRG2基因的cDNA全长为 10 5 8bp ,编码由 2 95个氨基酸组成、分子量为 335 79、等电点为 9.6 4的蛋白质 ,与人类同源基因TSARG2相似性为 78% ,与已知蛋白质无明显同源性。RT PCR结果表明该基因只在睾丸中有高表达。     

喉癌相关基因LCRG1的克隆和表达分析

喉癌是严重危害人类健康的一类恶性肿瘤。选用在mRNA差异显示研究中获得的在喉癌组织中表达显著下调且代表新基因的EST之一（AF10056）,设计引物进行cDNA文库筛选,得到一全长为3448bp的cDNA序列,其开放读码框编码288个氨基酸,与已知蛋白质无明显同源性,属一新发现的基因,GenBank接受号为AF268387。与GenBank数据库匹配分析,发现该基因包含6个外子,基因组RT－PCR显示有40％（12／30）的喉癌组织表达下调,RT－PCR显示有54．5％（6／11）其他肿瘤细胞系表达缺失。把该基因命名为喉癌相关基因（laryngeal carcinoma related gene1,LCRG1)。这提示LCRG1可能与喉癌的发生发展相关。     

紫花苜蓿果糖-1,6-二磷酸醛缩酶基因全长克隆及分析

根据已知的与盐胁迫相关的EST序列,采用SMART RACE方法克隆了紫花苜蓿果糖-1,6-二磷酸醛缩酶(ALD)全长cDNA,命名为MsALD(GenBank accession No.FJ896113).序列分析结果表明,该cDNA全长1 487 bp,包含一个1 194 bp的最大开放阅读框,编码398个氨基酸.经同源比对和进化树分析,MsALD基因编码的氨基酸与红三叶草、马铃薯、烟草等的果糖-1,6-二磷酸醛缩酶(ALD)氨基酸序列一致性高达90%以上,确定其属于第Ⅰ类果糖-1,6-二磷酸醛缩酶.半定量RT-PCR分析表明,MsALD基因可能与紫花苜蓿抗盐机理相关.     

抑制差减杂交法分离玉米幼苗淹水诱导表达基因

以淹水处理(submergence-treated, ST)的玉米(Zea mays L.)幼苗根部cDNA为目标群体,未处理(untreated, UT)的玉米幼苗根部cDNA为对照群体,进行抑制差减杂交.用经过UT差减的ST cDNA构建了一个含有大约2 000个独立克隆的差减文库.对随机挑取的408个克隆进行差异筛选,获得了184个在ST中特异表达或表达增强的候选克隆.对其中155个cDNA克隆测序并去除重复克隆后,共得到95个差异表达的cDNA片段.GenBank中BLAST查询结果表明:6个克隆为已知的玉米核苷酸序列;68个克隆与已知基因或EST序列部分区域的同源性为60%～90%;21个克隆在GenBank中无法查到对应的同源序列,可能代表了新基因,或者由于序列位于变异丰富的3′端而无法查到与其他物种基因的同源性.     

多发性骨髓瘤细胞中一个表达上调基因的克隆与分析

根据GenBank收录的多发性骨髓瘤细胞株 (ARH 77)表达上调ESTAF4 2 5 30 0设计引物 ,运用RT PCR检测了 5例多发性骨髓瘤患者及 4例正常人骨髓细胞中该EST的表达水平 .Northern印迹杂交分析该EST在多种组织中的表达 .进一步利用该EST作探针 ,筛选ARH 77cDNA文库 ,获得全长cDNA克隆 ,对该序列进行了分析 .结果显示 ,该EST在多发性骨髓瘤患者骨髓细胞中亦有较高的表达 ,而在正常人骨髓细胞中低表达 .经测序证实 ,该cDNA全长为 4 5 2bp(GenBank收录号 :AF4 87338) .预测其编码一个 5 7个氨基酸的小分子量蛋白质 ,属于与DNA复制有关的解旋酶 引物酶基因家族的新成员 .该基因在多发性骨髓瘤细胞中表达上调 ,其表达水平的改变可能与多发性骨髓瘤的发生与发展有关     

人类核糖体S6激酶RPS6KA5的cDNA克隆及组织表达谱分析

Human ribosomal protein S6 kinase includes two protein families: P90RSK and P70S6K, they participate in two different signaling pathways. When the two kinases were inhibited by their antibodies or rapamycin, the proliferation of cells was arrested. However, their analog, the immunosupressant FK-506, can inhibit the proliferation of fibroblast PBL1 without interfering with the activities of P90RSK, P70S6K and MAPK. We take the tactics of "homolog screening" to demonstrate whether there are some novel proteins which can substitute for the known P90RSK and P70S6K or other pathways without interfering with the known P90RSK and P70S6K. With the conserved sequence of mouse p90RSK as a probe, we screened the homologous sequence in NCBI EST database and got three human EST fragments. With the assembled contig as a probe to screen human brain cDNA library, a full-length cDNA of 3833 bp was attained. It contains a completed open reading frame from 165 bp to 2570 bp encoding 802 amino acids. The putative protein has higher homology with other members of p90RSK family. The gene was named RPS6KA5, the accession number in GenBank is AF090421. Northern hybridization showed the gene expressed in 16 human tissues tested, and the gene was localized in 14q31-32.1 by RH mapping. Another novel P70S6K gene has also been cloned. Thus, our initial presumption that there is an analog of known P90RSK and P70S6K in human beings was proved.     

Induction of EGFR-dependent and EGFR-independent signaling pathways by ultraviolet A irradiation

Most of the signal pathways involved in ultraviolet (UV)-induced skin carcinogenesis are thought to originate at plasma membrane receptors. However, UVA-induced signal transduction to downstream ribosomal protein S6 kinases, p70(S6K) and p90(RSK), is not well understood. In this report, we show that UVA stimulation of the epidermal growth factor receptor (EGFR) may lead to activation of p70(S6K)/p90(RSK) through phosphatidyl isositol (PI)-3 kinase and extracellular receptor-activated kinases (ERKs). Evidence is provided that phosphorylation and activation of p70(S6K)/p90(RSK) induced by UVA were prevented in Egfr(-/-) cells and were also markedly inhibited by the EGFR-specific tyrosine kinase inhibitors AG1478 and PD153035. Furthermore, EGFR tyrosine kinase inhibitors and EGFR deficiency significantly suppressed activation of PI-3 kinase and ERKs in regulating activation of p90(RSK)/p70(S6K) but had no effect on activation of c-Jun NH(2)-terminal kinases (JNKs) and p38 kinase in response to UVA. Thus, our results suggest that UVA-induced EGFR signaling may be required for activation of p90(RSK)/p70(S6K), PI-3 kinase, and ERKs but not JNKs or p38 kinase.     

VIIa/tissue factor interaction results in a tissue factor cytoplasmic domain-independent activation of protein synthesis,p70, and p90 S6 kinase phosphorylation

FVIIa binding to tissue factor (TF) and subsequent signal transduction have now been implicated in a variety of pathophysiological processes, including cytokine production during sepsis, tumor angiogenesis and neoangiogenesis, and leukocyte diapedesis. The molecular details, however, by which FVIIa/TF affects gene expression and cellular physiology, remain obscure. Here we show that FVIIa induces a transient phosphorylation of p70/p85(S6K) and p90(RSK) in BHK cells stably transfected with either full-length TF or with a cytoplasmic domain-truncated TF but not in wild type BHK cells. Phosphorylation of these kinases was also observed in HaCaT cells, expressing endogenous TF. Phosphorylation of p70/p85(S6K) coincided with protein kinase B and GSK-3beta phosphorylation. Activation of p70/p85(S6K) was sensitive to inhibitors of phosphatidylinositol 3-kinase and to rapamycin, whereas phosphorylation of p90(RSK) was sensitive to PD98059. FVIIa stimulation of p70/p85(S6K) and p90(RSK) correlated with phosphorylation of the eukaryotic initiation factor eIF-4E, up-regulation of protein levels of eEF1alpha and eEF2, and enhanced [(35)S]methionine incorporation. These effects were not influenced by inhibitors of thrombin or FXa generation and were strictly dependent on the presence of the extracellular domain of TF, but they did not require the intracellular portion of TF. We propose that a TF cytoplasmic domain-independent stimulation of protein synthesis via activation of S6 kinase contributes to FVIIa effects in pathophysiology.     

Extracellular signal-regulated kinase/90-KDA ribosomal S6 kinase/nuclear factor-kappa B pathway mediates phorbol 12-myristate 13-acetate-induced megakaryocytic differentiation of K562 cells

Two signaling pathways, the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)-dependent pathway and the nuclear factor-kappaB (NF-kappaB)-dependent pathway, have been known to mediate megakaryocytic differentiation of K562 cells induced by phorbol 12-myristate 13-acetate (PMA). In this study, we examined whether 90-kDa ribosomal S6 kinase (RSK), known as a substrate of ERK/MAPK and a signal-inducible IkappaBalpha kinase, would link two pathways during the differentiation. RSK1 was activated in a time- and dose-dependent manner during the PMA-induced differentiation. Overexpression of wild-type or dominant inhibitory mutant (D205N) of RSK1 enhanced or suppressed PMA-stimulated NF-kappaB activation and megakaryocytic differentiation as shown by morphology, nonspecific esterase activity, and expression of the CD41 megakaryocytic marker, respectively. In addition, overexpression of the dominant inhibitory mutant (S32A/S36A) of IkappaBalpha inhibited PMA-stimulated and RSK1-enhanced megakaryocytic differentiation, indicating that NF-kappaB mediates a signal for megakaryocytic differentiation downstream of RSK1. PMA-stimulated activation of ERK/MAPK, RSK1, and NF-kappaB and the PMA-induced megakaryocytic differentiation were prevented by pretreatment with PD98059, a specific inhibitor of the mitogen-activated ERK kinase (MEK). Therefore, these results demonstrate that the sequential ERK/RSK1/NF-kappaB pathway mediates PMA-stimulated megakaryocytic differentiation of K562 cells.     

RSK3 encodes a novel pp90rsk isoform with a unique N-terminal sequence: growth factor-stimulated kinase function and nuclear translocation.

A novel pp90rsk Ser/Thr kinase (referred to as RSK3) was cloned from a human cDNA library. The RSK3 cDNA encodes a predicted 733-amino-acid protein with a unique N-terminal region containing a putative nuclear localization signal. RSK3 mRNA was widely expressed (but was predominant in lung and skeletal muscle). By using fluorescence in situ hybridization, the human RSK3 gene was localized to band q27 of chromosome 6. Hemagglutinin epitope-tagged RSK3 was expressed in transiently transfected COS cells. Growth factors, serum, and phorbol ester stimulated autophosphorylation of recombinant RSK3 and its kinase activity toward several protein substrates known to be phosphorylated by RSKs. However, the relative substrate specificity of RSK3 differed from that reported for other isoforms. RSK3 also phosphorylated potential nuclear target proteins including c-Fos and histones. Furthermore, although RSK3 was inactivated by protein phosphatase 2A in vitro, the enzyme was not activated by ERK2/mitogen-activated protein (MAP) kinase. In contrast, the kinase activity of another epitope-tagged RSK isoform (RSK-1) was significantly increased by in vitro incubation with ERK2/MAP kinase. Finally, we used affinity-purified RSK3 antibodies to demonstrate by immunofluorescence that endogenous RSK3 undergoes serum-stimulated nuclear translocation in cultured HeLa cells. These results provide evidence that RSK3 is a third distinct isoform of pp90rsk which translocates to the cell nucleus, phosphorylates potential nuclear targets, and may have a unique upstream activator. RSK3 may therefore subserve a discrete physiologic role(s) that differs from those of the other two known mammalian RSK isoforms.     

In vivo role of the PIF-binding docking site of PDK1 defined by knock-in mutation

PKB/Akt, S6K, SGK and RSK are mediators of responses triggered by insulin and growth factors and are activated following phosphorylation by 3-phosphoinositide-dependent protein kinase-1 (PDK1). To investigate the importance of a substrate-docking site in the kinase domain of PDK1 termed the 'PIF-pocket', we generated embryonic stem (ES) cells in which both copies of the PDK1 gene were altered by knock-in mutation to express a form of PDK1 retaining catalytic activity, in which the PIF-pocket site was disrupted. The knock-in ES cells were viable, mutant PDK1 was expressed at normal levels and insulin-like growth factor 1 induced normal activation of PKB and phosphorylation of the PKB substrates GSK3 and FKHR. In contrast, S6K, RSK and SGK were not activated, nor were physiological substrates of S6K and RSK phosphorylated. These experiments establish the importance of the PIF-pocket in governing the activation of S6K, RSK, SGK, but not PKB, in vivo. They also illustrate the power of knock-in technology to probe the physiological roles of docking interactions in regulating the specificity of signal transduction pathways.     

黄瓜胞质6-磷酸葡萄糖酸脱氢酶基因克隆及序列分析

根据6-磷酸葡萄糖酸脱氢酶(6-phosphogluconate dehydrogenase,6PGDH)基因的保守氨基酸序列设计简并引物,应用RT-PCR技术从黄瓜栽培种品种'北京截头'(Cucumis sativus 'Beijingjietou')叶片中获得了640 bp的特异片段,以该序列在EST数据库进行同源检索筛选,发现甜瓜EST序列AM715537.2与之高度一致,据此设计引物经RT-PCR扩增、分子克隆和序列拼接,获得了黄瓜6-磷酸葡萄糖酸脱氢酶基因全长序列,命名为Cs6PGDH(GenBank登录号FJ610345).序列分析表明,该基因全长1 829 bp,其中开放读码框(ORF)长1 488 bp编码495个氨基酸组成的多肽,编码区内无内含子存在,5'、3'端非翻译区长度各为70 bp和271 bp.Blast同源性分析显示该基因编码的氨基酸序列与拟南芥、大豆、水稻、玉米、菠菜等物种6PGDH 基因有74%以上的一致性.由于与其他物种胞质6PGDH相类似氨基酸N端都缺少长度约为40aa的转运肽,推断Cs6PGDH为黄瓜胞质6-磷酸葡萄糖酸脱氢酶基因.     

Carbachol induces p70S6K1 activation through an ERK-dependent but Akt-independent pathway in human colonic epithelial cells

Stimulation of human colonic epithelial T84 cells with the muscarinic receptor agonist carbachol, a stable analog of acetylcholine, induced Akt, p70S6K1 and ERK activation. Treatment of T84 cells with the selective inhibitor of EGF receptor (EGFR) tyrosine kinase AG1478 abrogated Akt phosphorylation on Ser⁴⁷³ induced by either carbachol or EGF, indicating that carbachol-induced Akt activation is mediated through EGFR transactivation. Surprisingly, AG1478 did not suppress p70S6K1 phosphorylation on Thr³⁸⁹ in response to carbachol, indicating the G protein-coupled receptor (GPCR) stimulation induces p70S6K1 activation, at least in part, via an Akt-independent pathway. In contrast, treatment with the selective MEK inhibitor U0126 (but not with the inactive analog U0124) inhibited carbachol-induced p70S6K1 activation, indicating that the MEK/ERK/RSK pathway plays a critical role in p70S6K1 activation in GPCR-stimulated T84 cells. These findings imply that GPCR activation induces p70S6K1 via ERK rather than through the canonical PI 3-kinase/Akt/TSC/mTORC1 pathway in T84 colon carcinoma cells.     

Cloning and expression of Drosophila melanogaster UDP-GlcNAc:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I

A TBLASTN search of the Drosophila melanogaster expressed sequence tag (EST) database with the amino acid sequence of human UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I, EC 2.4.1.101) as probe yielded a clone (GM01211) with 56% identity over 36 carboxy-terminal amino acids. A 550 base pair (bp) probe derived from the EST clone was used to screen a Drosophila cDNA library in lambda-ZAP II and two cDNAs lacking a start ATG codon were obtained. 5'-Rapid amplification of cDNA ends (5'-RACE) yielded a 2828 bp cDNA containing a full-length 1368 bp open reading frame encoding a 456 amino acid protein with putative N-terminal cytoplasmic (5 residues) and hydrophobic transmembrane (20 residues) domains. The protein showed 52% amino acid sequence identity to human GnT I. This cDNA, truncated to remove the N-terminal hydrophobic domain, was expressed in the baculovirus/Sf9 system as a secreted protein containing an N-terminal (His)6 tag. Protein purified by adsorption to and elution from nickel beads converted Man alpha1-6(Man alpha1-3)Man beta-octyl (M3-octyl) to Man alpha1-6(GlcNAc beta1-2Man alpha1-3)Man beta-octyl. The Km values (0.7 and 0.03 mM for M3-octyl and UDP-GlcNAc respectively), temperature optimum (37 degrees C), pH optimum (pH 5 to 6) and divalent cation requirements (Mn > Fe, Mg, Ni > Ba, Ca, Cd, Cu) were similar to mammalian GnT I. TBLASTN searches of the Berkeley Drosophila Genome Project database with the Drosophila GnT I cDNA sequence as probe allowed localization of the gene to chromosomal region 2R; 57A9. Comparison of the cDNA and genomic DNA sequences allowed the assignment of seven exons and six introns; all introns showed GT-AG splice site consensus sequences. This is the first insect GnT I gene to be cloned and expressed.     

A transcript map of an 800-kb region on human chromosome 11q13, part of the candidate region for SCA5 and BBS1

The exon-amplification method was used to identify putative transcribed sequences from an 800-kb region that includes the genes for phospholipase Cβ3 and PYGM on human chromosome 11q13. The clone contig consisted of ten cosmids, three bacterial artificial chromosomes, and one P1 artificial chromosome. A total of 83 exons were generated of which 23 were derived from known genes and expressed sequence tags (ESTs). Five different EST cDNA clones were identified and mapped on the contig. One is a homolog of the human p70S6 kinase (p70s6 k) gene whose function involves the translational regulation of ribosomal protein synthesis and thereby impacts on ribosomal biogenesis. The gene for p70s6 k is expressed universally, including within adipose cells and retina, and it could play a role in Bardet-Biedl syndrome type 1, which has been mapped to 11q13. Received: 22 July 1998 / Accepted: 24 August 1998