Michio Himeno

Characterization of internal DNA-binding and C-terminal dimerization domains of human centromere/kinetochore autoantigen CENP-C in vitro : role of DNA-binding and self-associating activities in kinetochore organization

Human centromere protein C (CENP-C), a chromosomal component of the inner plate of kinetochores, was originally identified as one of the centromere auto-antigens. In a previous study, we showed that it possesses DNA-binding activity in vitro. Recently, centromere-binding activity was suggested at the C-terminal region in vivo. However, little is known about the role of CENP-C in kinetochore organization. Here, to characterize its biochemical properties, three separate antigenic regions of human CENP-C were expressed in Escherichia coli, affinity purified and used in South-western blotting and chemical cross-linking analyses. We found that the internal DNA-binding domain was composed of two kinds of elements: the ‘core’ and two flanking ‘stabilizing’ elements that support the activity. When cross-linked with disuccinimidyl suberate (DSS), the N-terminal region produced the ladder bands of dimerand tetramer: the C-terminal region exclusively produced the dimer band, whereas the internal region was not affected at all. Dimer formation at the C-terminus in the native state was also indicated by gel filtration and the presence of conformation-specific autoantibodies in the patients sera. These results suggest that human CENP-C consists of three functional units required for ‘kinetochore assembly’: a putative N-terminal oligomerization domain, an internal DNA-binding domain and a C-terminal dimerization domain.

Mode of action of Bacillus thuringiensis δ-endotoxin: Effect on TN-368 cells

Abstract TN-368 cells swelled and burst upon treatment with the dissolved δ-endotoxin of Bacillus thuringiensis . However, the cytotoxic response was greatly affected by the ionic conditions of the solutions employed for the toxin tests. Ions, in addition to K + , seemed to participate in the cytotoxic expression of δ-endotoxin, if their concentration was sufficient (>100 m m ). Although similar swollen cells were observed with valinomycin treatment, some differences appeared on the ultrastructural level, especially in the mitochondria. Those of the toxin-treated cells were transformed into the “condensed” form, while those of valinomycin-treated cells were transformed into the “swollen” form. Therefore, the cytotoxic effect of δ-endotoxin, unlike that of valinomycin, seemed to be a general breakdown of ion regulation on the cell level.

Specific Toxicity of δ-Endotoxins from Bacillus thuringiensis to Bombyx mori

Two δ-endotoxins, CryIA(a) and CryIA(b), from Bacillus thuringiensis subsp.. aizawai were used to investigate the specificity in insecticidal activity. CryIA(a) was 17-fold more toxic to Bomhyx mori than CryIA(b). After in vitro solubilization and digestion of these δ-endotoxins, the specificity of toxicity was retained. Trypsin-activated CryIA(a) and CryIA(b) showed specific, high affinity and saturable binding to brush border membrane vesicles (BBMV) from B. mori midguts. These two toxins competed for the same binding site. Dissociation constant for CryIA(a) and CryIA(b) binding to B. mori BBMV was O.89nM and 1.46 nM, respectively. In both toxins, dissociation reaction followed a biphasic process with a fast and a very slow component, suggesting that binding of the toxins proceeds through a reversible component and an apparently irreversible component. In the CryIA(a) dissociation reaction, the irreversible component comprised a large portion of total binding. On the other hand in that of CryIA(b), the reversible component was major. These results suggest that the specific toxicity of the toxins to B. mori may depend mainly on irreversibility.

Formation of nuclear polyhedral bodies and nuclear polyhedrosis virus of silkworm in mammalian cells infected with viral DNA

Abstract The infectious DNA of nuclear polyhedrosis virus of silkworm ( Bombyx mori Linne) caused production of characteristic nuclear polyhedral bodies in cultured FL cells of human amniotic origin, which did not support virus growth. The activity of the viral DNA in producing polyhedral bodies in FL cells was lost by pretreatment of the inoculum with DNase and by heating, but RNase was inactive. The polyhedral bodies formed in the cells were identical with those formed in cells of the silkworm with respect to their morphological and serological character. The polyhedral bodies isolated from FL cells and dissolved in alkali caused nuclear polyhedrosis when injected into silkworm pupae.

The nucleic acid of nuclear-polyhedrosis virus of the silkworm

Viral DNA was isolated from virus particles of nuclear-polyhedrosis of the silkworm, Bornbyx mori L., with 0·1 M -Na 2 CO 3 -NaHCO 3 buffer solution (pH 10·0), and then fractionated and purified on methylated albumin columns. Viral DNA showed a single peak on methylated albumin column fractionation as well as in analytical centrifugation, and the S 20,w of viral DNA was 13·1 s. The purified viral DNA was infectious. While DNase-treated DNA lost its infectivity, RNase-treated DNA was unaffected. The evidence that the ratio in viral DNA of adenine to thymine as well as of guanine to cytosine was equal to unity, that the DNA gave a sharp melting curve and that heat-denatured DNA was observed to react with formaldehyde showed that the DNA isolated was double-stranded DNA.

A virus-inactivating protein isolated from the digestive juice of the silkworm, Bombyx mori

Abstract A protein that can precipitate nuclear polyhedrosis virus (NPV) in vitro was isolated from the digestive juice of silkworm larvae ( Bombyx mori ) by the procedures of gel filtration and ion-exchange and hydroxylapatite column chromatography. The SDS-polyacrylamide gel electrophoretic and the ultracentrifugal analyses showed that the purified substance was a homogenous simple protein. The molecular weight of the purified protein was 27,000–28,000 and the sedimentation coefficient was 2.61 S. This protein had an additional activity to inactivate NPV of B. mori in vitro, somewhat analogous to serological neutralization by serum proteins. Electron microscope observations showed that amorphous materials could be found on the surface of envelopes and that the nucleocapsids disappeared.

Properties of flacherie virus of the silkworm, Bombyx mori

Abstract The flacherie virus of the silkworm, Bombyx mori , was isolated from infected larvae reared under aseptic conditions. Two types of infectious particles, tentatively designated FVS I and FVS II, were separated by density gradient centrifugation. Some properties of the separated particles were investigated. Electron micrographs showed that FVS I and FVS II were spherical particles with diameters of 27 ± 2 nm and 22 ± 2 nm, respectively. The sedimentation coefficients of FVS I and FVS II were 180 S and 134 S, respectively. It was concluded from experiments of incorporation of 3 H-uracil inoculated into diseased larvae at late stage of flacherie disease that the nucleic acid of FVS II was RNA. The two types of particles were present in Sakaki and Wadayama strains of flacherie virus.

Functional cloning of centromere protein B (CENP-B) box-enriched alphoid DNA repeats utilizing the sequence-specific DNA binding activity of human CENP-Bin vitro

The centromere is a distinctive portion of the chromosome consisting of ‘centromere DNA’ and ‘centromere proteins’. Recently, a direct molecular interaction was discovered between human centromere protein B (CENP-B) and human centromeric alphoid repeats. This enabled us to isolate the CENP-B-targeted centromeric DNA sequences by positively utilizing the biologic activity of CENP-Bin vitro. In the previous model experiment, we found that oligonucleotides covering the CENP-B binding sequences were enriched by the DNA immunoprecipitation procedure. Here we apply the same technique to the direct isolation of a functional part of human centromeric DNA from a genomic DNA library. Restriction digestion of two isolated clones showed the typical repeating pattern of an alphoid family that is known to localize at the centromeric region of all human chromosomes. Sequence analysis showed that these two clones frequently contain the authentic CENP-B binding motif, CTTCGTTGGAAACGGGA, or a new one with one base replaced, CTTCGTTGGAAACGGGT. The frequent distribution of these motifs suggests that the isolated sequences are directly involved in the organization of centromeric heterochromatin at the primary constriction in conjunction with CENP-B.

Infectivity of the components of a nuclear polyhedrosis virus of the silkworm, Bombyx mori

Abstract The polyhedra of the nuclear polyhedrosis virus (NPV) of Bombyx mori were dissolved under controlled dissolution with weak alkali. Five components were separated by centrifugation of the dissolved solution in a sucrose gradient. The assay of the separated components in B. mori pupae showed that two types of rods were almost equally, highly infectious, whereas the small spherical particles were much less infectious than the rods by an order of 4 log units. The infectious unit of this virus appeared to be the virus rod without the outer membrane, and the small spherical particle did not represent any infectious functional unit but was a degradation product of the viral internal substance and the outer membrane may not be essential for the virus infection in the pupal hemocoel.

Studies on lipids in the hemolymph of the silkworm, Bombyx mori, during the course of nucleopolyhedrosis.

Abstract Total lipids were determined in the hemolymph of the silkworm, Bombyx mori, infected with nucleopolyhedrosis virus. In the phospholipid fraction lecithin, phosphatidylserine, and phosphatidylethanolamine were detected. Hydrolysis of phospholipid gave two substances which were not identified. One of them gave a positive reaction with the iodine vapor test and the other gave positive reactions both with the iodine vapor and the ninhydrin tests. Cholesterol was identified as the only component of the cholesterol fraction in the body fluid of diseased as well as of normal silkworms. Fatty acids increased when polyhedra were released into the body fluid. Palmitic and stearic acids were detected, together with an unidentified fatty acid, as the components of the fatty acid fraction.