Chaperone SecB: Conformational changes demonstrated by circular dichroism

The chaperone SecB, which is involved in protein export inEscherichia coli, is shown by circular dichroism measurements to contain a high content of-pleated sheets. Prediction of the secondary structure of SecB is in good agreement with the observed content of-sheet. In accordance with the previous studies in which changes in conformation were assessed indirectly [Randall (1992),Science 257, 241–245], here we show that the conformation of SecB changes with the concentration of salt in the milieu and also when SecB interacts with a peptide ligand.Abbreviations ANS 1-anilino-naphthalene-8-sulfonate - CD circular dichroism - NMR nuclear magnetic resonance - CCA convex constraint analysis     

Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana

Four mutants that show the delayed leaf senescence phenotype were isolated from Arabidopsis thaliana . Genetic analyses revealed that they are all monogenic recessive mutations and fall into three complementation groups, identifying three genetic loci controlling leaf senescence in Arabidopsis . Mutations in these loci cause delay in all senescence parameters examined, including chlorophyll content, photochemical efficiency of photosystem II, relative amount of the large subunit of Rubisco, and RNase and peroxidase activity. Delay of the senescence symptoms was observed during both age-dependent in planta senescence and dark-induced artificial senescence in all of the mutant plants. The results indicate that the three genes defined by the mutations are key genetic elements controlling functional leaf senescence and provide decisive genetic evidence that leaf senescence is a genetically programmed phenomenon controlled by several monogenic loci in Arabidopsis . The results further suggest that the three genes function at a common step of age-dependent and dark-induced senescence processes. It is further shown that one of the mutations is allelic to ein2-1 , an ethylene-insensitive mutation, confirming the role of ethylene signal transduction pathway in leaf senescence of Arabidopsis .     

Green fluorescent protein as a signal for protein-protein interactions.

Green fluorescent protein (GFP) is autofluorescent. This property has made GFP useful in monitoring in vivo activities such as gene expression and protein localization. We find that GFP can be used in vitro to reveal and characterize protein-protein interactions. The interaction between the S-peptide and S-protein fragments of ribonuclease A was chosen as a model system. GFP-tagged S-peptide was produced, and the interaction of this fusion protein with S-protein was analyzed by two distinct methods: fluorescence gel retardation and fluorescence polarization. The fluorescence gel retardation assay is a rapid method to demonstrate the existence of a protein-protein interaction and to estimate the dissociation constant (Kd) of the resulting complex. The fluorescence polarization assay is an accurate method to evaluate Kd in a specified homogeneous solution and can be adapted for the high-throughput screening of protein or peptide libraries. These two methods are powerful new tools to probe protein-protein interactions.     

GI双突变体GIK253RA198C的构建及其性质分析

以双引物法对葡萄糖异构酶（GI）基因进行定点突变,将突变体基因于大肠杆菌中表达,获得了GI双点突变体GIK253RA198C.研究K253R和A198C双点突变对GI的结构和性质的作用,结果表明GIK253RA198C的热稳定性明显下降,最适反应温度降低5℃.文章从结构和机制上解释了为何同是K253R突变,对SM33 GI和密苏里游动放线菌GI的热稳定性产生不同的影响,认为这是由于Lys253在两种GI结构的位置上存在微小差异,从而使引入的Arg对亚基间的相互作用产生了相反效应所引起.     

Cloning and characterization of two catA genes in Acinetobacter lwoffii K24.

Two novel type I catechol 1,2-dioxygenases inducible on aniline media were isolated from Acinetobacter lwoffii K24. Although the two purified enzymes, CD I1 and CD I2, had similar intradiol cleavage activities, they showed different substrate specificities for catechol analogs, physicochemical properties, and amino acid sequences. Two catA genes, catA1 and catA2, encoding by CD I1 and CD I2, respectively, were isolated from the A. lwoffii K24 genomic library by using colony hybridization and PCR. Two DNA fragments containing the catA1 and catA2 genes were located on separate regions of the chromosome. They contained open reading frames encoding 33.4- and 30.4-kDa proteins. The amino acid sequences of the two proteins matched well with previously determined sequences. Interestingly, further analysis of the two DNA fragments revealed the locations of the catB and catC genes as well. Moreover, the DNA fragment containing catA1 had a cluster of genes in the order catB1-catC1-catA1 while the catB2-catA2-catC2 arrangement was found in the catA2 DNA fragment. These results may provide an explanation of the different substrate specificities and physicochemical properties of CD I1 and CD I2.     

Mutational analysis of the linker region of EnvZ, an osmosensor in Escherichia coli.

EnvZ, a transmembrane signal transducer, is composed of a periplasmic sensor domain, transmembrane domains, and a cytoplasmic signaling domain. Between the second transmembrane domain and the cytoplasmic signaling domain there is a linker domain consisting of approximately 50 residues. In this study, we investigated the functional role of the EnvZ linker domain with respect to signal transduction. Amino acid sequence alignment of linker regions among various bacterial signal transducer proteins does not show a high sequence identity but suggests a common helix 1-loop-helix 2 structure. Among several mutations introduced in the EnvZ linker region, it was found that hydrophobic-to-charged amino acid substitutions in helix 1 and helix 2 and deletions in helix 1, loop, and helix 2 (delta14, delta8, and delta7) resulted in constitutive OmpC expression. In the linker mutant EnvZ x delta7, both kinase and phosphatase activities were significantly reduced but the ratio of kinase to phosphatase activity increased, consistent with the constitutive OmpC expression. In contrast, the purified cytoplasmic fragment of EnvZ x delta7 possessed both kinase and phosphatase activities at levels similar to those of the cytoplasmic fragment of wild-type EnvZ. In addition, the linker mutations had no direct effect on EnvZ C-terminal dimerization. These results together with previous data suggest that the linker region is not directly involved in EnvZ enzymatic activities and that it may have a crucial role in propagating a conformational change to ensure correct positioning of two EnvZ molecules within a dimer during the transmembrane signaling.     

Potent inhibition of human immunodeficiency virus type 1 in primary T cells and alveolar macrophages by a combination anti-Rev strategy delivered in an adeno-associated virus vector.

The rate of viral replication appears to play a pivotal role in human immunodeficiency virus type 1 (HIV-1) pathogenesis and disease progression as it outstrips the capacity of the immune system to respond. Important cellular sites for HIV-1 production include T lymphocytes and tissue macrophages. Antiviral strategies, including newer treatment modalities such as gene therapy of HIV-1-susceptible cell populations, must be capable of engendering durable inhibitory effects to HIV-1 replication in both of these primary cell types in order to be effective. Among the potential genetic targets for intervention in the HIV-1 life cycle, the Rev regulatory system, consisting of Rev and its binding site, the Rev-responsive element (RRE), stands out as particularly attractive. Rev is essential for maintaining the stability of the viral genomic RNA as well as viral mRNAs encoding key structural and regulatory proteins. Moreover, it exhibits favorable threshold kinetics, in that Rev concentrations must rise above a critical level to exert their effect. To disable Rev function, primary T cells or macrophages were transduced with anti-Rev single-chain immunoglobulin (SFv) or RRE decoy genes either singly or in combination by employing adeno-associated virus vectors and then challenged with HIV-1. By directing both a protein and a nucleic acid against the normal interaction between Rev and the RRE, this genetic antiviral strategy effectively inhibited infection by either clinical or laboratory virus isolates. These results provide a framework for novel interventions to reduce virus production in the infected host.     

Characterization of molecularly cloned simian-human immunodeficiency viruses causing rapid CD4+ lymphocyte depletion in rhesus monkeys.

In vivo passage of a chimeric simian-human immunodeficiency virus (SHIV-89.6) expressing the human immunodeficiency virus type 1 (HIV-1) tat, rev, vpu, and env genes generated pathogenic viruses (SHIV-89.6P) inducing rapid CD4+ lymphocyte depletion and AIDS-like illness in rhesus monkeys (K. Reimann, J. T. Li, R. Veazey, M. Halloran, I.-W. Park, G. B. Karlsson, J. Sodroski, and N. L. Letvin, J. Virol. 70:6922-6928, 1996). To characterize the molecular changes responsible for this increase in virulence, infectious proviral clones of SHIV-89.6P isolates were derived. Viruses generated from some of these clones caused a rapid and profound decline of CD4+ lymphocytes in a high percentage of inoculated monkeys. Nucleotide changes potentially responsible for the increased virulence of SHIV-89.6P were limited to the env, tat, or long terminal repeat sequences, with most of the observed changes in env. Nucleotide changes in env altered 12 amino acids in the gp120 and gp41 exterior domains, and a 140-bp deletion in env resulted in the substitution of the carboxyl terminus of the SIVmac gp41 glycoprotein for that of the HIV-1 gp41 glycoprotein. The availability of pathogenic proviral clones should facilitate dissection of the molecular determinants of SHIV-89.6P virulence.     

Mutations in the X-linked E1 alpha subunit of pyruvate dehydrogenase: exon skipping, insertion of duplicate sequence, and missense mutations leading to the deficiency of the pyruvate dehydrogenase complex.

Human pyruvate dehydrogenase (PDH)-complex deficiency is an inborn error of metabolism that is extremely heterogeneous in its presentation and clinical course. In a study of 14 patients (7 females and 7 males), we have found a mutation in the coding region of the E1 alpha gene in all 14 patients. Two female patients had the same 7-bp deletion at nt 927; another female patient had a 3-bp deletion at nt 931. Another female patient was found to have a deletion of exon 6 in her cDNA. Two other female patients were found to have insertions, one of 13 bp at nt 981 and one of 46 bp at nucleotide 1078. Two male patients were found to have a 4-bp insertion at nucleotide 1163. The remaining six patients all had missense mutations. A male patient and a female patient both had an A1133G mutation. The other missense mutations were C214T, C615A, and C787G (two patients). Five of these mutations are novel mutations, five have been previously reported in other patients, and two were published observations in other patients in an E1 alpha-mutation summary. In the four cases where parent DNA was available, only one mother was found to be a carrier of the same mutation as her child.