Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene

A human umbilical vein endothelial cell cDNA library in lambda gt11 was screened for expression of thrombomodulin antigens with affinity-purified rabbit polyclonal anti-thrombomodulin immunoglobulin G (IgG) and mouse monoclonal anti-human thrombomodulin IgG. Among 7 million recombinant clones screened, 12 were recognized by both antibodies. Two of these, lambda HTm10 and lambda HTm12, were shown to encode thrombomodulin by comparison of the amino acid sequence deduced from the nucleotide sequence to the amino acid sequence determined directly from tryptic peptides of thrombomodulin. Thrombomodulin mRNA was estimated to be 3.7 kilobases in length by Northern blot analysis of endothelial cell and placental poly(A)+ RNA. Thrombomodulin mRNA was not detected in human brain, HepG2 hepatoma cells, or the monocytic U937 cell line. Additional cDNA clones were selected by hybridization with the 1.2-kilobase insert of lambda HTm10. One isolate, lambda HTm15, contained a 3693 base pair cDNA insert with an apparent 5'-noncoding region of 146 base pairs, an open reading frame of 1725 base pairs, a stop codon, a 3'-noncoding region of 1779 base pairs, and a poly(A) tail of 40 base pairs. The cDNA sequence encodes a 60.3-kDa protein of 575 amino acids. The predicted protein sequence includes a signal peptide of approximately 21 amino acids, an amino-terminal ligand-binding domain of approximately 223 amino acids, an epidermal growth factor (EGF) homology region of 236 amino acids, a serine/threonine-rich segment of 34 amino acids, a membrane-spanning domain of 23 amino acids, and a cytoplasmic tail of 38 amino acids. The EGF-homology region consists of six tandemly repeated EGF-like domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct detection of beta-1,3-glucanase isozymes on polyacrylamide electrophoresis and isoelectrofocusing gels

A procedure to assay isozymes of beta-1,3-glucanase directly on polyacrylamide gel electrophoresis (PAGE) and isoelectrofocusing (IEF) gels by using 2,3,5-triphenyltetrazolium chloride is described. The reagent reacts with reducing sugars released by beta-1,3-glucanases from the substrate laminarin. Acidic and neutral isozymes of beta-1,3-glucanase were detected and quantified on 17.5% native PAGE gels run with an anodic buffer system. A significant linear relationship (alpha = less than 0.01, R = 0.991) was observed between amounts of beta-1,3-glucanase loaded and intensity of bands stained with the reagent on native PAGE gels. A full isozyme pattern was obtained on 7.5% IEF gels with a pH range of 3.5-9.5. The IEF gels were heated in a microwave oven during the staining process to minimize diffusion.     

Plasmid expression and maintenance during long-term starvation-survival of bacteria in well water.

Strains of enteric bacteria and pseudomonads containing plasmid R388::Tnl721 (Tpr, Tcr) or pRO101 (Hgr, Tcr) were starved for over 250 days in sterile well water to evaluate effects of starvation-survival on plasmid expression and maintenance. Viable populations dropped to between approximately 0.1 and 1% of the initial populations. Escherichia coli(pRO101) and Pseudomonas cepacia(pRO101) lost both viability and plasmid expression at a lower rate than strains containing R388::Tnl721. Three patterns of host-plasmid interaction were detected: (i) no apparent loss of plasmid expression, (ii) loss of plasmid expression on initial recovery with subsequent expression upon resuscitation, and (iii) loss of capability to produce functional plasmid resistance.     

3H-azidopine photoaffinity labeling of high molecular weight proteins in chloroquine resistant falciparum malaria

Using 3H-azidopine, we have succeeded in labeling proteins from chloroquine resistant (CR) human falciparum malaria parasites in the molecular weight range of 155-170 kd. Vinblastine does not compete, but azidopine blocks the labeling using 3H-azidopine. Relatively little or no labeling of the 155-170 kd protein is seen in the chloroquine sensitive strain using 3H-azidopine. Further competition can be seen with nicardipine and reserpine (71%) respectively and verapamil (61%), chloroquine (48%), quinacrine (56%), trifluoperazine (32%) and chlorpromazine (33%). We speculate that this may be the glycoprotein responsible for the resistance to chloroquine in falciparum malaria.     

Carbonyldiphosphonate, a selective inhibitor of mammalian DNA polymerase delta

Twenty-three pyrophosphate analogues were screened as inhibitors of proliferating cell nuclear antigen independent DNA polymerase delta (pol delta) derived from calf thymus. Carbonyldiphosphonate (COMDP), also known as alpha-oxomethylenediphosphonate, inhibited pol delta with a potency (Ki = 1.8 microM) 20 times greater than that displayed for DNA polymerase alpha (pol alpha) derived from the same tissue. Characterization of the mechanism of inhibition of pol delta indicated that COMDP competed with the dNTP specified by the template and was not competitive with the template-primer. In the case of pol alpha, COMDP did not compete with either the dNTP or the polynucleotide substrate. COMDP inhibited the 3'----5' exonuclease activity of pol delta weakly, displaying an IC50 greater than 1 mM.     

Sex determination in the dioecious Melandrium

Summary Melandrium album (2n=24), a dioecious species with heteromorphic sex chromosomes (XY, males and XX, females), has a strong genetic commitment for sex determination. We report here a procedure for obtaining haploid plants from cultured anthers and show that genotype, pollen stage, cold treatment and certain culture media components are essential for a reproducible yield of embryos. Our procedure increased the number of responsive anthers and not the number of responsive microspores per anther. Most likely, our experimental system allows the recovery of competent microspores, and this on a medium containing either an auxin or a cytokinin. All of the 36 anther-derived plants tested expressed a female phenotypic sex instead of the theoretical one male one female ratio. When analysed cytologically, the plants exhibited the corresponding female genetic sex (one or two X chromosomes).     

湖北贝母单鳞片砂培繁殖研究

本文针对湖北贝母生产中存在繁殖系数低的问题,研究了单鳞片砂培繁殖对提高鳞茎繁殖率的效果和原理。试验结果表明:1.单鳞片繁殖率为对照种鳞茎的5—9倍,2.低温(2—10℃)预处理4—8周和暗条件培养,能有效地提高子球形成率,促使子球迅速长大,3.植物激素(6-BA、KT、2,4-D)处理,有利于促进鳞片不定芽原基的分化,繁殖率为种茎繁殖的9—11倍;4.单鳞片繁殖的小鳞茎主要发生在鳞片基部的茎盘上,还可发生在鳞片的远轴面上,但不发生在近轴面。     

一种纯化细菌有机磷水解酶的简便方法

Cibacron blue T_3GA与溴化氰活化的Sepharose 4B偶联后,产生一种能有效地分离有机磷水解酶的吸附剂。用0.15mol/L MgCl_2溶液从黄杆菌P3—2细胞抽提出的粗酶液通过柱层析分离,即可得到纯化8倍、酶活性回收率为269.4％的纯酶制品。该酶制品用凝胶电泳测是均一的。     

Domains required for nucleic acid binding activities in chloroplast ribonucleoproteins.

Five ribonucleoproteins (or RNA-binding proteins) from tobacco chloroplasts have been identified to date; each of these contains an acidic N-terminal domain (24-64 amino acids) and two conserved RNA-binding domains (82-83 amino acids). All five ribonucleoproteins can bind to ssDNA and dsDNA but show high specificity for poly(G) and poly(U). Here we present the nucleic acid binding activity of each domain using a series of deletion mutant proteins made in vitro from the chloroplast 29 kDa ribonucleoproteins. The acidic domain does not have a positive effect on binding activities and proteins lacking this domain show higher affinities for nucleic acids than the wild-type proteins. Mutant proteins containing single RNA-binding domains can bind to poly(G) and poly(U), though with lower affinities than proteins containing two RNA-binding domains. The spacer region (11-37 amino acids) between the two RNA-binding domains does not interact with poly(G) or poly(U) by itself, but is required for the additive activity of the two RNA-binding domains. Proteins consisting of two RNA-binding domains but lacking the spacer have the same activity as those containing only one RNA-binding domain. Possible roles for each domain in chloroplast ribonucleoproteins are discussed.     

马铃薯Y病毒外壳蛋白基因的克隆及序列分析

本文报道应用聚合酶链式反应(PCR)技术,在体外扩增马铃薯 Y 病毒外壳蛋白基因及其克隆和序列分析的结果。病毒 RNA 从马铃薯 Y 病毒感染的烟草叶片中提取,用合成的PCR 3引物及 AMV 逆转录酶合成了单链的 cDNA。利用 PCR 技术,经30个循玎的扩增。得到了一特异的0.8kb 片段。克隆后对此片段进行了限制性内切酶物理图谱分析,并测定了其全序列。实验结果证明,我们克隆到的是完整的马铃薯 Y 病毒的外壳蛋白基因。与国外报道的马铃薯 Y 病毒 N 株相比,其核苷酸序列及推测的氨基酸序列的同源率分别为97.8%和97%。将该基因导入马铃薯以期获得抗 Y 病毒马铃薯的工作正在进行。本文还对 PCR 技术用于扩增植物 RNA 病毒的方法以及用基因工程方法培育抗病毒作物新品种的可行性等进行了讨论。