DNA replication is one of key event in cell-cycle progression, yet due to their importance and lethality, the chronological phenotypes of DNA synthesis machineries after the depletion of corresponding genes have proved difficult to study. In the present study, mRNAs for three DNA polymerases, a clamp, and three clamp loaders were gradually depleted from cultured silkworm cells by soaking RNAi. Interestingly, the depletion of these DNA synthesis factors had different effects on the cell growth rate and arrest of cell-cycle progression during time-lapse observation. The depletion of DNA polymerases immediately arrested the cell-cycle progression at the S phase, while that of PCNA, a DNA clamp, required more time to slow cell growth and finally induced apoptosis. Surprisingly, silkworm cells continued to undergo several rounds of cell division when the components of clamp loaders were knocked down.
Here we report the inhibitory effects on silk fibroin production by the expression of mutants of B. mori phenylalanyl-tRNA synthetase α-subunit (BmPheRS-α) with relaxed amino acid recognition capacities in posterior silk glands (PSG) of transgenic B. mori larvae. We succeeded previously in incorporating unnatural amino acids (UAAs), 4-substituted analogues of phenylalanine (Phe), into silk fibroin by PSG-specific expression of BmPheRS-α mutants bearing a T407A or A450G mutation on its amino acid binding pocket. We had expected that the repertoire of UAAs incorporated into silk fibroin could be further expanded by expressing BmPheRS-α mutants with more highly relaxed amino acid recognition capacities. Hence, in this study, we generated two novel transgenic B. mori lines expressing BmPheRS-α mutants bearing more drastic side-chain mutations, T407G and T407A/A450G. The newly generated transgenic larvae spun thinner cocoons with substantially lower production of silk fibroin. We found that the PSG dissected from 5th instar transgenic larvae were degenerated into smaller sizes compared to the wild-type larvae. Mass analysis of the silk fibroin indicated misincorporation of tryptophan (Trp) in place of some Phe residues. Recognition of Trp by the T407G or T407A/A450G mutant of BmPheRS-α was verified by in vitro aminoacylation assay. From these observations, we speculated that misincorporation of Trp into protein biosynthesis due to excess relaxation of the amino acid recognition capacity of BmPheRS-α caused the degeneration of PSG in the transgenic larvae.
The Okushiri virus (OKV), isolated from Aedes larvae collected on Okushiri Island, Hokkaido, Japan, is a member of the Negevirus family, a newly proposed insect-specific virus group (genus). To enable genetic manipulation of the genome, we constructed an infectious cDNA clone of OKV. RNA synthesized in vitro from pFBOKV, a full-length OKV cDNA, in the presence or absence of cap analogue produced infectious progeny viruses effectively in mosquito C6/36 cells. Subsequently, ORF3 in pFBOKV was replaced with a GFP coding sequence to generate a construct designated as pO2GFP. C6/36 cells transfected with pO2GFP-derived RNA successfully expressed GFP, but failed to produce progeny viruses. Co-transfection of C6/36 cells with pO2GFP- and pFBOKV-derived RNA revealed that it is possible to produce infectious pO2GFP-derived progeny viruses by supplying OKV genetic elements and/or gene product deleted in pO2GFP even though the infectivity appeared to be low. Although improvements are required to construct viral vectors that propagate more efficiently, this is, to our knowledge, the first report of construction of a negevirus-based foreign gene expression system. The cDNA clones constructed and biological insights obtained in this study will be powerful tools allowing for reverse genetics of OKV and providing a basis to develop efficient negevirus-based expression vector systems.
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