Integrated regulation of the photosynthetic gene family from Arabidopsis thaliana in transformed tobacco cells

Summary Expression of the three chlorophyll a/b binding protein (cab) genes of Arabidopsis thaliana was studied in transformed tobacco tissues. For each cab gene, approximately 1000 bp of the promoter region plus a portion of the structural gene was inserted into a promoter-expression vector such that a translational fusion between the cab gene and the promoter-less chloramphenicol acetyltransferase (cat) gene was formed. The constructed molecules were introduced into either cultured tobacco cells or tobacco leaves and the promoter activity was monitored as chloramphenicol acetyltransferase activity. The light-grown tissues exhibited 1.5- to 60-fold greater promoter activity than did dark-grown tissues. Expression of the cab promoters was tissue specific: activities were much stronger in green leaves than other tissues. The cab promoters were almost equally active in transformed calli or shoots derived from leaves. However, in cultured tobacco cells, one promoter was two to three times stronger than the other two. The chimeric gene fusion, cab-cat, segregated in the F1 generation as a dominant Mendelian trait.     

Effect of artemisinin (qinghaosu) and chloroquine on drug-sensitive and drug-resistant strains of Plasmodium falciparum malaria: use of [2,8-3H]adenosine as an alternative to [G-3H]hypoxanthine in the assessment of in vitro antimalarial activity

Using [G-3H]hypoxanthine uptake as a radioactive indicator for the growth of malarial parasites, we measured the antimalarial activity of artemisinin (Qinghaosu, QHS) against FCMSU1/Sudan strain (chloroquine-sensitive strain) and FCB K+ strain (chloroquine-resistant strain) of Plasmodium falciparum in continuous culture in vitro. The 50% inhibitory concentrations (IC50) for QHS against FCMSU1/Sudan strain and FCB K+ strain were 2.8 X 10(-8) and 3.0 X 10(-8) M, respectively. On the contrary, the response of the two strains to chloroquine was quite different. The IC50 for chloroquine against FCMSU1/Sudan strain was 5.6 ng/ml, whereas that for the FCB K+ strain was 65.6 ng/ml. Therefore, QHS did not appear to exhibit any cross-resistance with chloroquine. If [2,8-3H]adenosine was used as a radioactive precursor instead of [G-3H]hypoxanthine for the determination of antimalarial activity, virtually identical results were obtained. Therefore, [2,8-3H]adenosine can be used as an alternative to [G-3H]hypoxanthine for the assessment of antimalarial action.     

芦苇胚性愈伤组织的形成及植株的再生

以芦苇种子为外植体,其愈伤组织的诱导率最高。叶鞘和叶片不发生脱分化。培养基中最合适的蔗糖浓度为4%。维生素 B 类、肌醇对愈伤组织的生长起促进作用。而酵母提取物对愈伤组织的诱导和生长具有明显的抑制作用。这种抑制效应,将随酵母提取物浓度的提高而增大。愈伤组织的继代培养,随培养基中2,4-D 浓度的提高,其平均鲜重明显降低。脱分化培养基中2,4-D 浓度对胚性愈伤组织的诱导形成具有一定的相关性。胚性愈伤组织经30代继代培养依然具有90%的分化频率,只是每块愈伤组织的分化苗数减少。反之,非胚性愈伤组织则完全丧失形态发生的能力。对两类愈伤组织进行扫描电镜的观察,发现其表面结构有很大差异。其过氧化物酶、酯酶同工酶谱以及可溶性蛋白的含量均有明显的差别。     

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)     

Three distinct regulatory elements comprise the upstream promoter region of the nopaline synthase gene

Summary Fine deletion mutants were generated in the upstream control region of the nopaline synthase (nos) promoter to define the position and role of upstream regulatory elements. The results indicated that the 8 bp sequence (CAGAAACC) at -106/-113 and its inverted repeat (GGTTTCTG) at -140/-147 are important for promoter function. The downstream element appears more important than the upstream element since deletion of the former reduced promoter activity more significantly than deletion of the latter. Deletion of the element alone, however, did not abolish promoter function, whereas, deletion of the 10 bp potential Z-DNA-forming (Z) element located between the repeat elements nullified promoter activity. Therefore, it appears that the Z element is an essential upstream regulator and the repeated elements are upstream modulators of the nos promoter. These elements are functionally distinct since alteration of stereospecificity or insertion of short oligonucleotides between the elements did not significantly influence promoter activity. These regulatory elements were unable to function from 200 bp upstream of the CCAAT-TATA box region.     

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.     

猕猴桃属美味猕猴桃和毛花猕猴桃种间杂交的胚胎学和胚援救

本文报道美味猕猴桃(Actintdia delictosa cv.Hayward)(6x)和毛花猕猴桃(A.eria-ntha)(2x)杂交当代种子的胚胎学分析和胚援救的结果:1.根据杂交种子外部形态和胚胎发育程度,区分为正常的和败育的两类,其比率接近1:1。正常种子的胚发育分化正常,且含有适量胚乳。败育种子中,除少数不含胚和胚乳外,绝大多数种子的胚发育终止于球形、心形和早子叶阶段,胚体畸形。胚乳处于消失或完全解体。2.在被试8组培养基中,最适合胚萌发和生长的是:MS+IAA0.5ppm+GA_30.5ppm,MS+2ip2ppm+IAA0.5ppm+GA_30.5ppm和 MS+2ip2ppm+GA_30.5ppm。适于实生苗生长的培养基为 MS+GA_30.5ppm 和 MS 培养基。在 MS+BAP2ppm 和 MS+IAA0.5ppm+GA_30.5ppm 培养基上,一些正常种子和少数不正常种子胚的下胚轴直接形成了许多不定芽,从而诱导产生了更多的杂种植株。但是在MS十BAP2ppm+GA_30.5ppm,MS+BAP2ppm+IAA0.5ppm 和 MS+2ip2ppm+GA_30.5ppm培养基上,虽然胚产生了愈伤组织,但都未分化出器官。杂种实生苗根尖细胞近半数的含有近4倍性染色体数(4x=116),约半数为其他倍性。