Molecular cloning of a cDNA encoding a silkworm protein that contains the conserved BH regions of Bcl-2 family proteins.

Abstract

To search for a Bcl-2 family homologue in the posterior silk gland of Bombyx mori, Western blot analysis was performed with the anti-rat Bcl-XL antiserum preabsorbed with a XL1-Blue MRF' lysate. The antiserum was shown to cross-react specifically with a silkworm protein of 80000 mol. wt (BmP80). The level of BmP80 increased dramatically during the spinning stage and decreased rapidly after the formation of a cocoon, implying that the silkworm protein was involved in histolysis of posterior silk gland as a stimulator. Screening a B. mori cDNA library with the same preabsorbed antiserum, a cDNA clone contaiing a cDNA fragment that is presumably large enough to encode the entire BmP80 protein was identified. The cDNA fragment contained 127 nucleotides (nt) of untranslated sequence at the 5' end, 2895nt of presumptive coding sequence and 625nt of untranslated sequence including a poly(A) tail at the 3' end. The calculated mol. wt of the presumptive protein (BmP109) was 108800. BmP109 shared sequence homology with the antiapoptotic proteins within the four conserved regions, BH1, BH2, BH3 and BH4, which were located at the C-terminal half of the protein and resided in the same characteristic order as Bcl-2 family proteins. Comparison of amino acid content revealed that BmP109 contained much more cysteine and lysine but less glycine and arginine than the antiapoptotic proteins. Northern blot analysis indicated that the mRNA for BmP109 is about 4.0kb. Reverse transcription-polymerase chain reaction experiments showed that the mRNA level for BmP109 increased dramatically during the spinning stage and decreased rapidly after formation of a cocoon, suggesting the involvement of transcriptional regulation.