| Budget Amount *help |
¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
Apoptosis is a cellular suicidal program by which damaged or no longer needed cells are individually eliminated to maintain healthy homeostasis in multicellular organisms. It has long been suggested that apbptotic DNA laddering is produced by a nuclear Ca^<2+>/Mg2^<+->-dependent endonuclease, and we identified a novel 33 kDa Ca^<2+>/Mg^<2+>-dependent endonuclease, named DNase γ, in the nuclei of apoptotic rat thymocytes. To date, we have determined the nucleotide sequences of the CDNA encoding human, rat, mouse, andXenopus laevis DNase γ. DNase γ is a member of the mammalian DNase I family ofDNases. It shows high activity under neutral pH conditions and is strongly inhibited by certain metal ions, such :as Zn^<2+> and Ni^<2+>. A previous study revealed that DNase γ is stored within the inner space of the nuclear envelope. When cells are induced to undergo apoptosis, DNase γ translocates into the nucleus and hydrolyzes genomic DNA into nucleosomal fragments. Thus, DNase γ is shown to have the ability to produce apoptotic DNA fragmentation in mammalian cells ; however, the apoptotic situations under which DNase γ is indispensable for DNA laddering have yet to be identified in vivo.
In other to investigate the physiological function of DNase γ in vivo, we tried to generate DNase γ null mice. We screened a mouse genomic library and isolated a partial clone encoding the 5th exon containing the active His residue of DNase γ. Using the genomic clone, we prepared a targeting construct and succeeded in generating DNase γ null mice. DNase γ knock-out (KO) mice were born at expected Mendelian frequencies and showed normal viability. The loss of DNase y activity has been confirmed by activity gel assay. The mutant mice will be a powerful tool to clarify the physiological role of DNase γ in apoptosis in vivo. Furthermore, the mice may be used as novel animal models for some apoptosis deficiency-related diseases, such as autoimmune disease and neuronal degeneration disease.
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