Functional expression of Arabidopsis thaliana anthranilate synthase subunit I in Escherichia coli.

Anthranilate synthase is involved in tryptophan (Trp) biosynthesis. Functional expression of subunit I from Arabidopsis (ASA1) was achieved in bacteria as a protein fused with glutathione S-transferase (GST). The active product was purified in a single step on a glutathione-Sepharose column. The Vmax (45 nmol min-1mg-1), the apparent K(M) for chorismate (180 microM), and the feedback inhibition by Trp (complete inhibition by 10 microM Trp) of the purified fusion product (GST-ASA1) were comparable to anthranilate synthase purified from plants. Polyclonal antibodies raised against the fusion project and purified by affinity chromatography on a GST-ASA1-Sepharose column cross-reacted with a 61.5-kD protein in a partially purified anthranilate synthase preparation from corn seedlings. GST-ASA1 cleavage by thrombin, as well as site-directed mutagenesis modifications of the Trp allosteric site, inactivated the recombinant protein.     

Detection and determination of selenoproteins by nuclear techniques

Microbeam X-ray spectrometry, energy-dispersive X-ray fluorescence analysis, and neutron activation analysis were evaluated for the detection of selenium contained in the selenoprotein glutathione peroxidase. The glutathione peroxidase had been previously separated using polyacrylamide gel electrophoresis. The use of Bragg-reflected polarized X-ray beams was employed in the X-ray fluorescence measurements to minimize the problem of scatter owing to the gel matrix. Current detection limits of selenium in a gel matrix are 2.1 ng in the bench-top microbeam X-ray system and 30–60 ng using XRF with polarized beams. Neutron activation analysis was used for qualitycontrol measurements, with a detection limit here of <0.08 ng. The work has in principle established the feasibility of such an approach.     

牛脑液泡型质子泵70kD和33kD亚基基因的表达

已分离了编码牛脑液泡型质子泵的70kD亚基的cDNA,利用聚合酶链反应(PCR)扩增了70kD亚基的编码片段,同时直接从牛脑cDNA库中得到了33kD亚基的编码片段.分别将相应片段连接到PET载体上完成70kD和33kD亚基基因在大肠杆菌中的表达.SDS聚丙烯酰胺凝胶电泳和蛋白质印迹分析表明70kD和33kD亚基基因均得到明显表达.     

Isolation and Characterization of Two Basic Internal Proteins from the T-Even Bacteriophages

Two species of basic internal proteins were found in osmotic shock supernatant solutions of bacteriophages T4B, T4D, T2H, T2L, and T6. The major species of protein isolated had a molecular weight of approximately 21,000 daltons, whereas the minor protein molecular weight was near 9,500 daltons. The two protein species exhibited unique isoelectric points and amino acid compositions. The 21,000-dalton protein of T2L showed major electrophoretic and compositional differences from the other 21,000-dalton proteins isolated. Similarities between the 21,000-dalton proteins and phage lysozyme are discussed.     

Characterization of T-Even Bacteriophage Substructures: I. Tail Fibers and Tail Tubes

T-even bacteriophages were grown and purified in bulk quantities. The protein coats were disrupted into their component substructures by treatment with 67% dimethyl sulfoxide (DMSO). Tail fibers and tubes were purified on glycerol-CsCl-D(2)O gradients and examined with respect to sedimentation properties, subunit molecular weights, amino acid composition, isoelectric points, and morphology. It was found that intact tail fibers had a sedimentation coefficient of 12 to 13S and that dissociated fibers consisted of three classes of proteins having molecular weights of 150 K +/- 10, 42 K +/- 4, and 28 K +/- 3 daltons. A model was constructed in which the 150-K subunit folded back on itself twice to give a three-stranded rope. Each 150-K subunit then represented a half-fiber and it was proposed that the role of the 42- and 28-K subunits was to hold each half-fiber together as well as serve as a possible link with other substructures. Isoelectric point studies also indicated that there were three different proteins with pI values of 3.5, 5.7, and 8.0. Amino acid analyses indicated that fibers had a composition distinct from other phage substructures. In addition, a striking difference was noted in the content of tryptophan among the phages examined. T4B had three to five times more tryptophan than did T2L, T2H, T4D, and T6. Intact tail tubes had an S(20,w) of 31 to 38S and dissociated tubes consisted of three proteins of molecular weights 57 K +/- 5, 38 K +/- 4, and 25 K +/- 3 daltons. Based on degradation studies with DMSO, it was proposed that these three proteins were arranged in a helical array yielding the tube structure. Isoelectric point studies indicated that there were three major proteins in the tube whose pI values were 5.1, 5.7, and 8.5. No significant differences were observed in the amino acid content of tubes obtained from all the T-even bacteriophages.     

H3-THYMIDINE DERIVATIVE POOLS IN RELATION TO MACRONUCLEAR DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS

The formation of a soluble H³-thymidine derivative pool has been examined in Tetrahymena pyriformis as a function of macronuclear DNA synthesis during the cell life cycle. An autoradiographic technique which allows the detection of water-soluble materials within a cell has shown that these cells do not take up and retain exogenous H³-thymidine during G1 or G2. Uptake of H³-thymidine is restricted to the S period of the cell cycle. Additional autoradiographic experiments show, however, that a soluble pool of H³-thymidine derivatives persists from the end of one DNA synthesis period to the beginning of the next synthesis period in the subsequent cell cycle. Since this persisting pool cannot be labeled with H³-thymidine, the pool does not turn over during non-S periods.