人过敏毒素C5a反义cDNA的克隆、表达和拮抗作用

采用引物二聚体配对搭桥法成功扩增了人过敏毒素C5a全长的反义cNDA ,将扩增的反义cNDA克隆到原核表达载体pPROEXTM HTc上 ,与 6×His头形成融合基因 ,转化E .coliDH5α ,30℃下经IPTG诱导 ,该融合蛋白在大肠杆菌中以可溶性形式表达 ,表达量达 10 % ;利用金属螯合亲和层析一步法纯化 ,得到纯度 80 %的融合蛋白 ;烟草蚀刻病毒蛋白酶酶切超滤浓缩后 ,得到高浓度高纯度人过敏毒素C5a反义肽 ,经N端蛋白测序鉴定 ,其氨基酸序列正确 .髓过氧化物酶 (myeloperoxi dase ,MPO)为单核细胞PMN所特有 ,其活性可以间接反映C5a趋化白细胞的能力 ,C5a刺激血管内皮细胞表达胞间粘附分子 (ICAM 1)、血管间粘附分子 (PCAM 1)等 ,后者能增加对PMN的粘附 ,检测MPO活性可间接反映出C5a的功能 .还观察了纯化的C5a反义肽对C5a刺激内皮细胞胞浆[Ca2 + ]浓度变化的影响 .高浓度高纯度的C5a反义肽对C5a过敏毒素存在拮抗作用 ,说明反义肽在补体系统C5a分子中是存在的 .这为深入研究C5a反义肽的结构与功能关系提供了物质保证     

A novel TNF-inducible message with putative growth suppressor function

We report the nucleotide sequence of a novel cDNA and TNF-induced expression of the corresponding message (mRNA) in human fibroblast cells. This message is also expressed in certain human tumor cell lines and is over-expressed in a colon cancer cell line (HT-29). NIH3T3 cells transfected with the antisense construct of the 5'-region of this novel cDNA formed 20-fold more colonies in culture compared to cells transfected with a sense construct of the same region or the sense and the antisense constructs of the central region of this cDNA. This observation suggests a possible growth suppressor function for the gene represented by this cDNA.     

Antisense RNA of the latent period gene (MER5) inhibits the differentiation of murine erythroleukemia cells

The MER5 cDNA was cloned from RNA preferentially synthesized in murine erythroleukemia (MEL) cells during the early period of MEL cell differentiation. To understand the role of the MER5 gene in the differentiation, we have transferred the MER5 cDNA into MEL cells in both sense and antisense orientations under control of the promoter of the human metallothionein gene. Only in the transformants with the antisense MER5 cDNA, did their elevated expression inhibit differentiation. The result suggests that the MER5 gene product may promote early events in the differentiation of MEL cells.     

Molecular cloning and functional expression of a Talaromyces emersonii derived alpha-amylase encoding genetic determinant in a human cell line

Summary A cDNA fragment encoding a Talaromyces emersonii acid stable alpha-amylase has been cloned into a mammalian cell expression vector system. When human HeLa cells were transformed with this DNA, functional enzyme could be detected in extracellular media and cell lysates derived from stable transformants. Expression of the T. emersonii derived cDNA was verified by northern dot blotting hybridisation analysis of mRNA extracted from transformants, using the labelled amylase encoding cDNA determinant as a probe. Results obtained suggest that expression of the fungus derived alpha-amylase in the transformed HeLa cells is glucocorticoid dependent.     

Transgenic tobacco plants expressing rgp1, a gene encoding a ras-related GTP-binding protein from rice,show distinct morphological characteristics1

The rgp1 gene, originally Isolated from rice seedlings, encodes a small GTP-binding protein which is related to the product of the human proto-oncogene, ras-p21. To determine the physiological role of the rgp1 protein, rgp1-p25, the coding region of rgp1 was introduced into tobacco plants in both sense and antisense orientations. Transformants, which were found to contain the rgp1 gene at up to three loci, showed distinct phenotypic changes. The most notable was a reduction in apical dominance with increased tillering, together with dwarfism or abnormal flower development or both. These effects were similarly observed in both sense and antisense transformants. Northern hybridization analysis showed that rgp1 was expressed only in phenotypically abnormal transformants and not in the apparently normal phenotypes. Furthermore, the R₁ progenies from most transformants co-segregated into a 3:1 ratio for both kanamycin resistance and tillering. The expression of tgp1, a presumed tobacco homologue of rgp1, was markedly reduced in transformants expressing the antisense rgp1, whereas it was apparently unaffected in transformants with sense rgp1. These observations suggest that the phenotypic changes in antisense transformants may be mediated by an effect on native tgp1 mRNA, whereas in sense transformants the changes may be induced by over-production of rgp1-p25. The possibility that the increased tillering may be related to abnormal phytohormone metabolism or response pathways, and that rgp1-p25 may mediate the transmission of signals in these pathways is discussed.     

Alteration of the Amount of the Chloroplast Phosphate Translocator in Transgenic Tobacco Affects the Distribution of Assimilate between Starch and Sugar

Tobacco plants (Nicotiana tabacum L.) transformed with sense and antisense constructs of a cDNA encoding the tobacco phosphate-triose phosphate-3-phosphoglycerate translocator (phosphate translocator) were shown to contain altered amounts of phosphate translocator mRNA and protein. Phosphate translocator activity in intact chloroplasts isolated from transformed plants showed a 15-fold variation, from 20% of the wild-type activity in antisense transformants to 300% of the wild-type activity in sense transformants. However, the maximal rates of photosynthesis and the rates of photosynthetic carbon assimilation in ambient CO2 showed no consistent differences between transformants. Starch content was decreased by 20% and total soluble sugars were increased by 20% in leaves of antisense transformants compared to sense transformants. The 40% decrease in the ratio of starch to total soluble sugars in antisense transformants relative to sense transformants indicates that distribution of assimilate between starch and sugar had been altered. However, the amount of sucrose in the leaves was unchanged. The changes in total soluble sugars were accounted for completely by changes in glucose and fructose, suggesting the existence of a homeostatic mechanism for maintaining sucrose concentrations in the leaves at the expense of glucose and fructose.     

Characterization of cytosolic sialidase from Chinese hamster ovary cells: part I: cloning and expression of soluble sialidase in Escherichia coli

The cDNA of Chinese hamster ovary (CHO) cell cytosolic sialidase was amplified by RT-PCR and cloned into the pGEX-2T plasmid vector encoding for glutathione S-transferase (GST). Screening revealed transformed Escherichia coli clones with the constructed plasmid encoding the CHO cell sialidase sequence. After isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, SDS-PAGE of the total protein extracts revealed a new protein of about 70 kDa, correlating with the molecular weight of a fusion protein composed of the GST (26 kDa) and the cloned cytosolic CHO cell sialidase (43 kDa). A soluble fusion protein was purified from sonified E. coli homogenates by one-step affinity chromatography on Glutathione Sepharose 4B, which showed sialidase activity towards 4-methyl-umbelliferyl-alpha-D-N-acetylneuraminic acid (MUF-Neu5Ac) substrate. Induction of cells with 0.1, 0.5, and 1.0 mM IPTG revealed highest total protein amounts after induction with 1.0 mM IPTG, but highest specific activity for affinity chromatography purified eluates from cultures induced with 0.1 mM IPTG. Therefore, large scale production was performed by inducing cells during exponential growth in a 25 L bioreactor for 3 h with 0.1 mM IPTG after chilling the cell suspension to 25 degrees C. The amount of 26.46 mg of 40-fold purified GST-sialidase with a specific activity of 0.999 U/mg protein was obtained from crude protein extracts by one-step affinity chromatography. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) and Neu5Ac were competitive inhibitors for the sialidase, the former being the more effective one using MUF-Neu5Ac as the substrate. The cytosolic sialidase is capable of desialylating a wide spectrum of different types of gangliosides using a thin-layer chromatography overlay kinetic assay without detergents. This is the subject of the accompanying paper (Müthing, J.; Burg, M. Carbohydr. Res. 2001, 330, 347-356).     

Phenotypic conversion of SV40-immortalized human diploid fibroblasts to senescing cells by introduction of an antisense gene for SV40-T antigen.

Normal human lung fibroblast diploid cells, WI-38, become senescent after a definite number of divisions. VA-13 is a line of immortalized cells established by transformation of WI-38 cells by SV40 virus. To determine whether SV40 large T (SV40-T) antigen is essential for this immortalization of WI-38 cells we introduced an antisense gene for T antigen into VA-13. Two morphologically different types of antisense transformant (VA-AS5-8 and VA-AS37-8) were obtained. In both antisense transformants the expression of T antigen was reduced by more than 70% as compared to that in the parent cells. The morphology of the antisense transformants indicated a partial conversion to the senescent phenotype of WI-38. The relative number of cells in the S phase of the antisense transformants was decreased as compared to that in cultures of VA-13 and about 50% of cells were at G1/0. The doubling time of the transformants was prolonged to close to the doubling time of WI-38. The level of expression of retinoblastoma protein (pRB) complexed with SV40-T antigen of the antisense transformants was significantly decreased although the level of total pRB was much higher than that in VA-13. The pRB was present exclusively in the underphosphorylated form. Thus, the decreased level of formation of the complex between SV40-T and pRB or the underphosphorylation of pRB may explain the suppression of growth of antisense transformants. Together, these results show that an antisense gene for SV40-T antigen can efficiently block the cell proliferation and the cell immortalization of VA-13 cells.     

人白介素24重组蛋白的表达及其抗肿瘤机理的研究

将人白细胞介素24（hIL-24）的cDNA序列克隆至原核高效表达载体pET-21a（+）上,经IPTG诱导后,在大肠杆菌中获高效表达,经纯化、复性后的rhIL-24蛋白具有抑制HeLa细胞生长,诱导HeLa细胞凋亡,刺激外周血单核细胞分泌IL6、TNF-α、IFN-r,抑制血管形成的功能。初探了rhIL-24蛋白能通过下调抗凋亡因子bcl-2表达,激活线粒体凋亡途径引起肿瘤细胞凋亡的机理。     

Antisense Repression of Hexokinase 1 Leads to an Overaccumulation of Starch in Leaves of Transgenic Potato Plants But Not to Significant Changes in Tuber Carbohydrate Metabolism

Potato (Solanum tuberosum L.) plants transformed with sense and antisense constructs of a cDNA encoding the potato hexokinase 1 (StHK1) exhibited altered enzyme activities and expression of StHK1 mRNA. Measurements of the maximum catalytic activity of hexokinase revealed a 22-fold variation in leaves (from 22% of the wild-type activity in antisense transformants to 485% activity in sense transformants) and a 7-fold variation in developing tubers (from 32% of the wild-type activity in antisense transformants to 222% activity in sense transformants). Despite the wide range of hexokinase activities, no change was found in the fresh weight yield, starch, sugar, or metabolite levels of transgenic tubers. However, there was a 3-fold increase in the starch content of leaves from the antisense transformants after the dark period. Starch accumulation at the end of the night period was correlated with a 2-fold increase of glucose and a decrease of sucrose content. These results provide strong support for the hypothesis that glucose is a primary product of transitory starch degradation and is the sugar that is exported to the cytosol at night to support sucrose biosynthesis.     

苎麻CCoAOMT基因cDNA反义转化模式烟草'WS38'

苎麻咖啡酰辅酶A氧甲基转移酶(CCoAOMT)是其木质素合成过程的一种关键酶,运用克隆的该酶基因cDNA及植物表达载体pBI121、pWM101,分别构建了35S启动子控制的苎麻CCoAOMT基因反义cDNA基因质粒(pBI121-antiBnCCoAOMT)和cDNA全长表达质粒(pWM101-BnCCoAOMT),并通过根癌农杆菌介导法将其转化至模式烟草WS38,获得了转基因烟草.对转基因植株进行分子分析和组织学初步研究表明,转反义RNA基因植株叶柄木质素含量较野生烟草或转正义基因烟草叶柄木质素含量降低.说明运用反义RNA技术对CCoAOMT基因的表达进行基因工程调控,一定程度上可以对木质素的合成产生干扰,为获得低木质素或木质素组分改良的苎麻基因工程奠定基础.     

The effect of chimeric transgene architecture on co-ordinated gene silencing

Two gene constructs were made consisting of a 244-bp sense fragment from the 5′ end of a polygalacturonase cDNA, the 3′ end of which was ligated to a 414-bp fragment from the 5′ end of a phytoene synthase cDNA. In the first construct, the phytoene synthase fragment was in a sense orientation (sense/sense chimeric gene) and in the second construct the phytoene synthase fragment was in an antisense orientation (sense/antisense chimeric gene). Both chimeric genes were inserted between a cauliflower mosaic virus promoter and terminator. Tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) plants transformed with each construct gave rise to transformants with three distinct phenotypes: plants with red fruit, plants with pure yellow fruit and plants with red and yellow sectored fruit. For both chimeric constructs, expression of the endogenous polygalacturonase and phytoene synthase genes were found to be co-ordinately suppressed in yellow tissue, but showed normal expression in red tissue. Data from microscopic analyses of fruit chromoplasts, from the three phenotypes, implied that phytoene synthase suppression from each construct predominantly had two states within a cell: on or off. Received: 31 July 1997 / Accepted: 28 August 1997     

Inhibition of human cancer cell growth by inducible expression of human ribonucleotide reductase antisense cDNA

Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis and repair. The enzyme consists of two dissimilar subunits, M1 and M2. It is known that the M2 subunit plays a role in tumorgenicity and metastasis. In this study, we transfected human oropharyngeal KB cancer cells with human RR M1 and M2 antisense cDNA expressed by an inducible vector system. The transfectants were double-selected with hygromycin and G418. The clones, designated KB-M1AS, KB-M2AS and KB-CAT, represented transfectant clones that contained M1 antisense cDNA, M2 antisense cDNA, and a CAT reporter gene, respectively. In a colony-forming assay, colony formation for the KB-M2AS clone decreased approximately 50% when M2 antisense mRNA expression was induced by isopropylthiogalactose (IPTG). However, the KB-M1AS clone revealed no significant inhibition under IPTG induction. RR enzyme activity, as measured by 14CDP reduction assay, revealed a 30% decrease in the IPTG-induced KB-M2AS clone relative to non-IPTG-induced samples at 144 hours. As shown by Northern blot, expression of the M2 antisense mRNA showed peaks at 48 hours and 144 hours after induction by IPTG. M2 antisense mRNA expression induced by IPTG was 33-fold greater than the uninduced control at 144 hours. Western blot analysis showed that the M2 subunit protein level decreased in the KB-M2AS clone beginning at 72 hours after induction and continued to decrease to 50% of the uninduced control at 144 hours, then showed a slight recovery at 168 hours. In conclusion, M2 antisense mRNA expression by an inducible system can effectively decrease RR M2 protein expression, reduce enzyme activity, and inhibit growth. Furthermore, this approach can be employed in future antisense investigations.     

Effect of regulated expression of the fragile histidine traid gene on cell cycle and proliferation

The mechanism of tumor suppressor action of the fragile histidine triad (FHIT) gene is unknown. Disruption of cell cycle regulation leads to the tumor formation and many tumor suppressor genes suppress tumorigenesis through their effect on cell cycle regulation. We examined the expression of FHIT during the cell cycle, and determined whether overexpression of FHIT affects cell cycle kinetics and apoptosis. The FHIT cDNA was cloned into the ecdysone-inducible expression vector in both the sense and antisense orientations. Overexpression of the sense or antisense construct did not affect cell proliferation, cell cycle distribution or apoptosis in human 293T cells. Analysis of the FHIT expression in 293T cells collected at various cell cycle phases showed that the expression of FHIT is not under cell cycle regulation. These results indicate that the tumor suppressor activity of the FHIT gene may be independent of an effect on the cell cycle and apoptosis mechanisms.     

Stable transfection of DAOY cells with a GM3 synthase antisense construct and transient reduction in ganglioside content

Tumor cell gangliosides are bioactive molecules involved in tumor-host interactions. To investigate their role in tumor formation and angiogenesis, we sought to develop an inhibitory model targeting human GM3 synthase, an essential enzyme in the ganglioside synthesis pathway, by antisense transfection. We prepared a number of transfectants from DAOY human medulloblastoma cells and isolated clones that stably expressed a 560-bp fragment of human GM3 synthase cDNA, in either sense or antisense orientation, as well as clones transfected with an empty vector. Both sense and antisense clones permanently incorporated mammalian expression vectors into their genomes. The DAOY cell clones were screened for ganglioside content using total lipid extraction, ganglioside isolation, and HPTLC. One antisense-transfected clone, 7.2A, in which total ganglioside content was reduced by 70%, was selected for further study. All sense-and sham-transfectants had ganglioside levels not different from that of untransfected DAOY cells. After 10 passages however, while antisense mRNA expression was fully maintained, the ganglioside content of 7.2A cells had reverted to normal levels. Antisense RNA transfection can sometimes have a reversible effect on the expression of a target. Possible regulatory mechanisms of this previously unrecognized process of reversion to wild type phenotype are discussed.     

Modulation of the cellulose content of tuber cell walls by antisense expression of different potato (Solanum tuberosum L.) CesA clones

Four potato cellulose synthase (CesA) homologs (StCesA1, 2, 3 and 4) were isolated by screening a cDNA library made from developing tubers. Based on sequence comparisons and the fact that all four potato cDNAs were isolated from this single cDNA-library, all four StCesA clones are likely to play a role in primary cell wall biosynthesis. Several constructs were generated to modulate cellulose levels in potato plants in which the granule-bound starch synthase promoter was used to target the modification to the tubers. The StCesA3 was used for up- and down-regulation of the cellulose levels by sense (SE-StCesA3) and antisense (AS-StCesA3) expression of the complete cDNA. Additionally, the class-specific regions (CSR) of all four potato cellulose synthase genes were used for specific down-regulation (antisense) of the corresponding CesA genes (csr1, 2, 3 and 4). None of the transformants showed an overt developmental phenotype. Sections of tubers were screened for altered cell wall structure by Fourier Transform Infrared microspectroscopy (FTIR) and exploratory Principal Component Analysis (PCA), and those plants discriminating from WT plants were analysed for cellulose content and monosaccharide composition. Several transgenic lines were obtained with mainly decreased levels of cellulose. These results show that the cellulose content in potato tubers can be reduced down to 40% of the WT level without affecting normal plant development, and that constructs based on the CSR alone are specific and sufficient to down-regulate cellulose biosynthesis.     

Specific down-regulation of annexin II expression in human cells interferes with cell proliferation

The protein-tyrosine kinase substrate annexin II is a growth regulated gene whose expression is increased in several human cancers. While the precise function of this protein is not understood, annexin II is proposed to be involved in multiple physiological activities, including DNA synthesis and cell proliferation. Targeted disruption of the annexin II gene affects calcium signaling, tyrosine phosphorylation and apoptosis, indicating the important physiological role of this protein. We used a transient co-transfection assay to regulate annexin II expression in human HeLa, 293 and 293T cells, and measured the effects of annexin II down regulation on DNA synthesis and proliferation. Transfection of cells with an antisense annexin II vector results in inhibition of cell division and proliferation, with concomitant reduction in annexin II message and protein levels. Cellular DNA synthesis is significantly reduced in antisense transfected cells. Replication extracts made from antisense transfected cells have significantly reduced efficiency to support SV40 in vitro DNA replication, while the extracts made from sense transfected cells are fully capable of replication. Our results indicate an important role of annexin II in cellular DNA synthesis and cell proliferation.     

Suppression of villin expression by antisense RNA impairs brush border assembly in polarized epithelial intestinal cells.

We have used an antisense RNA strategy to investigate the role of the actin-associated protein, villin, in the brush-border morphogenesis of human intestinal CaCO2 cells. Stable expression of a cDNA encoding antisense villin RNA resulted in the permanent down-regulation of the endogenous villin message and dramatically affected brush-border assembly. Ultrastructural and immunolocalization studies revealed that epithelial cell polarity was largely maintained. However, in contrast to brush-border markers such as dipeptidyl-peptidase IV, the apical localization of sucrase-isomaltase was specifically impaired. Retransfection of the villin antisense-expressing cell line with a cDNA encoding a partial sense villin RNA restored both brush-border assembly and sucrase-isomaltase apical expression. The suggestion that brush-border morphogenesis may be important for the trafficking of certain proteins is discussed.     

Antisense depletion of death-associated protein kinase promotes apoptosis

Death-associated protein kinases (DAPK) are serine/threonine protein kinases that have an important role in regulating cell death. In this study two antisense approaches were employed to down-regulate expression of the endogenous DAPK-alpha and DAPK-beta proteins. Transient expression of an antisense DAPK cDNA or antisense morpholino oligonucleotides in HeLa, 3T3, or primary human vascular smooth muscle cells demonstrate that decreased DAPK expression promotes a spontaneous, caspase-mediated apoptosis as evidenced by increased activities of caspases-3 and -9. Clonal HeLa cell lines with attenuated levels of DAPK expression, obtained following selection in the presence of antisense DAPK cDNA, are more sensitive to tumor necrosis factor-induced caspase-mediated apoptosis, and their sensitivity is inversely related to DAPK expression. In contrast, HeLa cells with reduced DAPK expression are moderately resistant to cell death induced by interferon-gamma. This finding is consistent with previous studies showing that DAPK has a role in promoting caspase-independent cell death. Together, these studies demonstrate that the cellular activities of DAPK are critical for antagonizing caspase-dependent apoptosis to promote cell survival under normal cell growth conditions.