Binding sites of HeLa cell nuclear proteins on the upstream region of adenovirus type 5 E1A gene.

Twenty one binding sites of HeLa cell nuclear proteins were identified on the upstream region of adenovirus type 5 E1A gene using DNase I footprint assay. The proximal promoter region contained five binding sites that overlapped the cap site, TATA box, TATA-like sequence, CCAAT box, and -100 region relative to the E1A cap site(+1). The -190 region was a potential site for octamer-motif binding proteins, such as NFIII and OBP100. An upstream copy of the E1A enhancer element 1 was the site for a factor (E1A-F) with the binding specificity of XGGAYGT (X = A, C; Y = A, T). E1A-F factor also bound to three other sites, one of which coincided with the distal E1A enhancer element. The distal element also contained a potential site for ATF factor. The adenovirus minimal origin of DNA replication competed for DNA-protein complex formation on the CCAAT and TATA box region and the -190 region, suggesting that these regions interacted with a common or related factor.     

Sulfated proteoglycans synthesized by Neuro 2a neuroblastoma cells: Comparison between cells with and without ganglioside-induced neurites

Mouse neuroblastoma Neuro 2a cells are known to extend neurite-like processes in response to gangliosides added to the culture medium. We compared the structural features of proteoglycans (PG) synthesized by conventional Neuro 2a cells with those of neurite-bearing cells. Two different proteoglycans labeled with [³⁵S]sulfate, namely, chondroitin sulfate proteoglycan (CS-PG) and heparan sulfate proteoglycan (HS-PG), were found both in the cell layer and in the culture medium of the conventional cells. CS-PG isolated from the cell layer had a K_av value of 0.38 on Sepharose CL-6B, and had CS side chains with Mr of 27,000. HS-PG in the cell layer was slightly larger (K_av of 0.33) in terms of hydrodynamic size than CS-PG, and the apparent Mr of the heparan sulfate side chains was 10,000. The structural parameters of CS-PG and HS-PG isolated from the medium were almost identical to those of the PGs in the cell layer. In addition to these PGs, single-chain HS, with an average Mr of 2,500, was observed only in the cell layer and this component was the major sulfated component in the cell layers of both control and ganglioside treated cells. The neurite-bearing cells also synthesized both CS-PG and HS-PG which were very similar in hydrodynamic size to those synthesized by the conventional cells, but the size of HS side chains was greater. Radioactivity, as³⁵S, of each sulfated component from the gangliosideteated culture seemed to be slightly less than that of the corresponding component from the control culture. These findings indicate that the marked morphological change in Neuro 2a cells, induced by gangliosides is not accompanied by major changes in the synthesis of PGs.     

Isolation and partial characterization of a novel form of low molecular weight kininogen from guinea pig plasma

Two kinds of low molecular weight kininogen (termed LK1 and LK2) were isolated from pooled plasma of guinea pigs. When polyclonal antisera raised against the individual proteins were used, immunological cross-reactions were observed between LK1 and high molecular weight kininogen (HK), but not either between LK1 and LK2 or between LK2 and HK. After tissue injury, plasma level of LK1 doubled while those of LK2 and HK remained relatively unchanged.     

Possible relationship between poly(ADP-ribose)synthetase and formation of antibody against acetylcholine receptors in patients with myasthenia gravis

We examined the relationship of the serum levels of antibody against acetylcholine receptors to the serum levels of 13 enzymes, including various hydrolytic enzymes, poly(ADP-ribose)synthetase (Poly(ADP-ribose)Syn), and sialyltransferase (NANA-trans), in patients with myasthenia gravis. The patients were divided into two groups, depending on the presence or absence of thymoma. In spite of the absence of significant difference in the absolute levels of individual enzymatic activities between the two groups, the network relationships of such enzymes were quite different between the two groups. Of the 13 enzymes examined, only Poly(ADP-ribose)Syn showed a weak but significant correlation with the level of the antibody in the patients without thymoma. A multivariate study more clearly suggested the relationship between the antibody formation and Poly(ADP-ribose)Syn in the patients without thymoma. Such observations were not found in the patients with thymoma.     

In isolated DNA, formamidopyrimidine-DNA glycosylase-sensitive sites determined by electrophoresis correspond to the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine by HPLC-ECD.

Several methods have been developed for determining the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in DNA. In the present study, we compared an electrophoretic method that uses formamidopyrimidine-DNA glycosylase (FPG protein) with a HPLC-ECD method. Firstly, we produced 8-oxodG in lambda DNA with methylene blue and visible light and cleaved it in one-half of the modified DNA enzymatically with FPG protein. Then, we determined the number of FPG protein-sensitive sites by electrophoresis (Y) and the number of 8-oxodGs by HPLC-ECD (X) per 10(5)dG of isolated DNA. Simple regression analysis of the data showed Y=1.07X+1.52 to be the most likely relationship. The correlation coefficient was 0.97. The values obtained by the two methods were very similar. This result is noteworthy because the number of FPG protein-sensitive sites determined by other methods have not yet come close to the number obtained by HPLC-ECD. Thus, this method might be more quantitative than other methods that measure FPG protein-sensitive sites. Another reason this electrophoresis method might be more useful than HPLC-ECD is that we can determine some other types of oxidative DNA damage well, by changing the DNA glycosylase.     

Genome analysis of Agrobacterium tumefaciens: construction of physical maps for linear and circular chromosomal DNAs, determination of copy number ratio and mapping of chromosomal virulence genes.

The phytopathogenic bacterium Agrobacterium tumefaciens is unique in that it possesses both linear and circular DNA chromosomes in addition to a plant-tumor-inducing (Ti) plasmid. We analyzed the two chromosomal DNA molecules in strain MAFF301001, whose Ti plasmid has already been sequenced completely. Physical maps of the chromosomal DNAs were constructed by Southern hybridization experiments using Pme I and Swa I fragments and short fragments bridging the Swa I fragments with special care to avoid any missing fragment. Hybridization with 16S rDNA probe showed one rDNA locus on the linear chromosome and two loci on the circular chromosome. For this bacterium to be pathogenic, not only Ti plasmid but also chromosomal genes are required. The chromosomal virulence (chv) genes (chvA, chvB, chvD, chvE, chvG, chvH, and chvI) and the chromosomal genes affecting the virulence [acvB, pgm(exoC), glgP, miaA, and ros] were successfully mapped onto 5 different regions in the chromosomal physical maps. These chv genes and the chromosomal genes affecting the virulence other than pgm and glgP were found on the circular chromosome, whereas the pgm and glgP genes were located on the linear chromosome. In contrast to the large terminal inverted repeats of Streptomyces linear chromosomal DNA, no hybridization signal was detected between left and right terminal fragments of the linear A. tumefaciens chromosome. Quantitative analysis of DNA fragments indicated that the copy numbers of the two chromosomal DNAs and the Ti plasmid are identical.     

Efficient folding of the insect neuropeptide eclosion hormone by protein disulfide isomerase.

Eclosion hormone is an insect neuropeptide that consists of 62 amino acid residues including three disulfide bonds. We have previously reported its hypothetical 3D structure consisting mainly of three alpha-helices. In this paper, we report the effects of chaperone proteins on the refolding of denatured eclosion hormone in a redox buffer containing reduced and oxidized glutathione. Urea-denatured eclosion hormone was spontaneously reactivated within 1 min with a yield of more than 90%, while beta-mercaptoethanol-denatured eclosion hormone was reactivated in a few minutes with a yield of 75%. Under the same experimental conditions, eclosion hormone treated with beta-mercaptoethanol and urea was reactivated slowly with a yield of 47% over a period of 2 h. Protein disulfide isomerase, a eucaryotic chaperone protein, markedly increased the reactivation yield and rate of the totally denatured hormone. GroE oligomers slightly improved the reactivation yield but peptidyl prolyl isomerase had no influence on yield or rate. We propose that the folding pathway of eclosion hormone involves at least two rate-limiting steps, and that protein disulfide isomerase is likely to be involved in the folding in insect neuronal cells.     

Three-dimensional structural model analysis of the binding site of lithocholic acid, an inhibitor of DNA polymerase beta and DNA topoisomerase II.

The molecular action of lithocholic acid (LCA), a selective inhibitor of mammalian DNA polymerase beta (pol beta), was investigated. We found that LCA could also strongly inhibit the activity of human DNA topoisomerase II (topo II). No other DNA metabolic enzymes tested were affected by LCA. Therefore, LCA should be classified as an inhibitor of both pol beta and topo II. Here, we report the molecular interaction of LCA with pol beta and topo II. By three-dimensional structural model analysis and by comparison with the spatial positioning of specific amino acids binding to LCA on pol beta (Lys60, Leu77, and Thr79), we obtained supplementary information that allowed us to build a structural model of topo II. Modeling analysis revealed that the LCA-interaction interface in both enzymes has a pocket comprised of three amino acids in common, which binds to the LCA molecule. In topo II, the three amino acid residues were Lys720, Leu760, and Thr791. These results suggested that the LCA binding domains of pol beta and topo II are three-dimensionally very similar.