Masao Kawakita

Nucleotide sequence of the hemagglutinin-neuraminidase gene of Newcastle disease virus avirulent strain D26: evidence for a longer coding region with a carboxyl terminal extension as compared to virulent strains

The nucleotide sequence of DNA clones complementary to the genomic RNA of an extremely avirulent strain D26 of Newcastle disease virus was analyzed, and the sequence of 2102 nucleotides directly following F gene reported previously (Sato et al., 1987, Virus Res. 7, 241-255), and corresponding to HN0 gene was determined. A long open reading frame coding for the HN0 peptide of 616 amino acid residues was found in this sequence. It was flanked by the consensus sequences N1 and N2 (Ishida et al., 1986, Nucleic Acids Res. 14, 6551-6564), and the former was shown by the primer extension method to serve as the transcriptional initiation site. The deduced amino acid sequence of the HN0 peptide was highly homologous to that of the HN peptides of strains Beaudette C and B1, but had a carboxyl terminal extension of 39 amino acid residues with a potential glycosylation site in it. The terminal extension is likely to be excised during the processing, and this is consistent with the observation that unglycosylated HN0 is larger in size than unglycosylated HN. A microheterogeneity among the cDNA clones in the nucleotide sequence was also noted which may be relevant to the synthesis of a small amount of an HN-sized peptide in strain D26-infected cells.

Neutralizing monoclonal antibody against ras oncogene product p21 which impairs guanine nucleotide exchange.

The neutralizing monoclonal antibody Y13-259 severely hampers the nucleotide exchange reaction between p21-bound and exogenous guanine nucleotides but does not interfere with the association of GDP to p21. These results suggest that the nucleotide exchange reaction is critical for p21 function. Interestingly, the v-ras p21 has a much faster dissociation rate than the p21 of the c-ras proto-oncogene.

Characterization of a factor that stimulates hydrolysis of GTP bound to ras gene product p21 (GTPase-activating protein) and correlation of its activity to cell density

The postmicrosomal fraction of the extract from NIH 3T3 and BALB/c 3T3 cells stimulated the hydrolysis of GTP bound to H-ras gene product p21 by severalfold. The stimulation was observed with normal p21 but not with p21 with valine as the 12th residue. This specificity is similar to that of GTPase-activating protein (GAP) for N-ras p21 described by M. Trahey and F. McCormick (Science 238:542-545, 1987). Consistent with this specificity, analysis of p21-bound nucleotides in living cells revealed that almost all normal p21 bound GDP, whereas oncogenic mutant p21s bound both GTP and GDP. Similar activity was also found in various mouse tissues, with brain tissue showing the highest specific activity. When cell extracts were prepared from cultured cells, there was a linear relationship between GAP activity and cell density. These results suggest the factor is involved in the regulation of cell proliferation.

Interaction of guanosine 5′-diphosphate, 2′-(or 3′-) diphosphate(ppGpp) with elongation factors from E. coli

Abstract The interactions of guanosine 5′-diphosphate, 2′-(or 3′-) diphosphate(ppGpp) with the polypeptide elongation factors Tu(EF Tu) and G(EF G) have been studied. The data indicate that ppGpp binds with EF Tu to form an EF Tu-ppGpp complex, and inhibits, in a competitive manner, the exchange reaction of Tu-GDP and 3 H-GDP. The ribosome-dependent GTPase reaction catalyzed by EF G is also depressed by ppGpp.

The UDP-galactose translocator gene is mapped to band Xp11. 23-p11. 22 containing the Wiskott-Aldrich Syndrome Locus

We have cloned a segment of the human gene encoding UDP-galactose translocator by genetic complementation of its defective mutant in mouse FM3A cells. Chromosome mapping using fluorescentin situ hybridization revealed that the cloned gene hybridized to the Xp11.23-11.23 region of the X chromosome. This region is shared by the locus of Wiskott-Aldrich syndrome, an X-linked recessive immunodeficiency disorder, characterized by defective sugar chains on cell surface components. Genetic and phenotypic similarities suggest a possible link between UDP-galactose translocator and the Wiskott-Aldrich syndrome (WAS).

Inhibitor of Interferon-Induced Double-Stranded RNA-Dependent Protein Kinase and Its Relevance to Alteration of Cellular Protein Kinase Activity Level in Response to External Stimuli

In FL cells, interferon (IFN)‐induced dsRNA‐dependent protein kinase (PK‐I) was found to be present in a form complexed with a potent inhibitor of its dsRNA‐dependent activation. The inhibitor was readily dissociated from PK‐I by DEAE‐cellulose chromatography to yield a dsRNA‐responsive PK‐I. The inhibitor was also dissociated easily from PK‐I by gel filtration through Sephacryl S‐200. The apparent molecular mass of the inhibitor as estimated by gel filtration was more than 160 kilodaltons. Activity of the inhibitor was decreased on IFN treatment for 8.5 hr or on Sindbis virus infection with concomitant increase in the amount of dsRNA‐activatable form of PK‐I. This result implies that the inhibitor may be one of the regulatory factors of cellular PK‐I activity. Longer IFN treatment (24 hr) led to recovery of the inhibitor activity, but it was overridden by an extensive net synthesis of the PK‐I protein.

ANTIBODY AGAINST NEUROFIBROMATOSIS TYPE 1 GENE PRODUCT REACTS WITH A TRITON-INSOLUBLE GTPASE ACTIVATING PROTEIN TOWARD RAS P21

Cellular fractionation of GTPase activating protein (GAP) activity using bovine cerebral cortex revealed that about half of GAP activity was found in membrane fraction. GAP activity of membrane was not solubilized with 0.5% (v/v) triton X-100 and was immunoprecipitated with antibody against carboxy-terminus of neurofibromatosis type 1 (NF1) gene product. In contrast, soluble GAP activity was precipitated with antibody against GAP but not with anti-NF1. These results suggest that NF1 gene product is a GTPase activating protein toward ras p21 with completely different intracellular distribution from that of GAP.

Identification of the two 3H-GTP binding proteins in E. coli supernatant as Tu-Ts and Tu FACTORS

Abstract Of the two 3 H-GTP-binding components separated on DEAE-Sephadex column chromatography of an E. coli supernatant fraction, the one was identified as Tu-Ts complex and the other as Tu. This assignment was based on the observation that the former rapidly binds with 3 H-GDP whereas the latter binds with it only slowly. This was further confirmed by elution pattern of the two components from Sephadex G-IOO column. Molecular weight of Tu, Ts and Tu-Ts was estimated to be approximately 5 × 10 4 , 5 × 10 4 , and 1 × 10 5 respectively.

Crystallization of phenylalanyl-tRNAPhe-EF-Tu-GTP complex

Abstract A method for the preparation of crystalline([14C]phenylalanyl-tRNAPhe)-Tu-[32P]GTP complex has been described. The ternary complex containing unfractionated mixture of tRNA charged with twenty amino acids has also been crystallized.

CONFORMATIONAL CHANGES OF Ca 2+ ,Mg 2+ -ADENOSINETRIPHOSPHATASE AS DETECTED BY SITE-SPECIFICALLY INTRODUCED FLUORESCENT AND PARAMAGNETIC PROBES AND BY SUSCEPTIBILITY TO TRYPTIC DIGESTION

Publisher Summary By using fluorescent and paramagnetic probes that are introduced to specific locations on the ATPase molecule, systematic information concerning the conformational similarity and dissimilarity among various intermediates of ATPase was gathered. There was some indication that the same probe when attached to distinct sites on ATPase behaves differently depending on the local environment of its attachment site. By virtue of its high sensitivity and resolving power, this approach might enable to analyze ATPase conformation in terms of the interaction between sub-molecular domains. Accumulating evidence suggests that the ATPase molecule is composed of a few relatively hydrophilic stretches connected by intra-membranous hydrophobic core regions and that each of these might be regarded as a distinct domain. During the catalytic cycle, each domain might behave as an individual unit working in concert with the others to carry out the overall transport reaction.