|Budget Amount *help
¥6,600,000 (Direct Cost : ¥6,600,000)
Fiscal Year 1993 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1992 : ¥5,400,000 (Direct Cost : ¥5,400,000)
A double-stranded ribonuclease (dsRNase), which exists in the midgut epithelium and the digestive juice of the silkworm, Bombyx mori, was partially purified by using DEAE, CM and G-75 column chromatography. On the elution profile of nuclease activity of each chromatogram, the elution region of dsRNase activity was coincided with those of DNase and ssRNase activities. In order to isolate the dsRNase from DNase and single-stranded RNase (ssRNase), dsRNase activity fraction on CM colum chromatography was applied to native DNA-cellulose column chromatography, and then eluted by the stepwaise method of the increasing of NaCl concentration. The dsRNase was still eluted with DNase and ssRNase from 0.25 through 0.5 M NaCl. Further investigation clarify that both DNase and dsRNase was eluted from 0.25 through 0.3 M NaCl, and that ssRNase with the DNase and dsRNase from 0.4 through 0.5 M NaCl. The activities of DNase and dsRNase, which were obtained from nuclease activity fraction on DNA cellulo
se colum chromaography, were inhibited with the increasing of single-stranded RNA (ssRNA) content, and degraded a circular DNA (pUC 119). These results suggest that an endonuclease, which will be capable of attacking DNA, dsRNA and ssRNA, exists in the digestive juice of B.mori.
The endonuclease indicates a high activity in the midgut and the digestive juice during larval-pupal metamorphosis. At this period, chromatin droplets were observed in the content of the inside of the midgut, and also an analysis of DNA extracted from the content exhibited a ladder-like electrophoretic pattern on an agarose gel. On the basis of these data, an experiment was undertaken to investigate whether the endonuclease in the digestive juice will be concerned with the fragmentation of DNA.The in vitro incubation of the partially purified digestive endonuclease with the chromatin from 6 day-old larvae of the 5th instar resulted in the fragmentation of DNA.The digestive endonuclease was activated by Mg^<2+>/Ca^<2+>, and inhibited by Zn^<2+>, indicating that it will contribute to the process of apoptosis in the midgut during larval-pupal metamorphosis. Less