|Budget Amount *help
¥2,900,000 (Direct Cost : ¥2,900,000)
Fiscal Year 1999 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1998 : ¥2,200,000 (Direct Cost : ¥2,200,000)
Nuclear envelope precursor vesicle fractions, PV1 and PV2, were affinity-purified from a Xenopus egg extract by a chromatin binding method. An in vitro nuclear assembly system constituted with thus affinity-purified vesicle fractions was established. PV1 and PV2 contained larger and smaller vesicles, respectively. Chromatin was incubated in a Xenopus egg cytosol fraction supplemented with PV1, PV2, or PV1+ PV2 vesicles, then reconstituted nuclei were observed by fluorescence and electron microscopy. From these and other results, followings were suggested. 1. Two vesicle populations, PV1 and PV2, are necessary for the assembly of normal sized nuclei, 2. PV1 contains a chromatin targeting molecule(s) and membrane fusion machinery, 3. PV2 contains a chromatin targeting molecule(s) and a molecule(s) necessary for nuclear pore complex assembly, and 4. PV1 has the ability to assemble a nuclear membrane, and PV2 is necessary for the assembly of nuclear pore complexes and for nuclei to grow to
the normal size.
Sera of autoimmune diseases such as primary biliary cirrhosis, rheumatoid arthritis and Sjogren's syndrome were used to study the nuclear envelope assembly mechanism. It was shown that most sera of these diseases inhibit the nuclear envelope assembly to various degrees, by means of an in vitro nuclear envelope assembly system involving a Xenopus egg extract. Sera which strongly inhibit the nuclear envelope assembly were frequently obtained from Sjogren's syndrome patients. Methods for the monitoring and purification of proteins which are reactive with these sera and responsible for the nuclear envelope assembly were developed. These methods were applied to Sjogren's syndrome sera, K32 and K199, and antigens participating in the nuclear envelope assembly were characterized and purified. It was shown that antigens are a 66k integral membrane protein and a 48k soluble protein, respectively. Both proteins participated in the step(s) of fusion of nuclear envelope precursor vesicles. Less