phiC31-integrase-mediated, site-specific integration of transgenes in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Transgenic silkworms can be useful for investigating the functions of genes in the post-genomic era. However, the common method of using a transposon as an insertion tool may result in the random integration of a foreign gene into the genome and suffer from a strong position effect. To overcome these problems, it is necessary to develop a site-specific integration system. It is known that phiC31 integrase has the capacity to mediate recombination between the target sequences attP and attB. To test the availability of site-specific integration in the silkworm, we first examined the efficiency of recombination between the target sites of the two plasmids in silkworm embryos and found that the frequency of recombination was very high. Then we constructed a host strain that possessed the target sequence attP using the common method. We injected the donor plasmid together with the phiC31 integrase mRNA into the embryos of the host strain and obtained positive lines. Structural analysis of the lines showed that site-specific integration occurred by recombination between the genomic attP site and the attB site of the donor plasmid. We can conclude from the results that phiC31 integrase has the ability to mediate the site-specific integration of transgenes into the silkworm chromosome.     

PhiC31 integrase-mediated cassette exchange in silkworm embryos

To construct an effective site-specific integration system in the silkworm, we examined if phiC31 integrase works in silkworm embryos. As an assay system, we constructed an extrachromosomal cassette exchange reaction system between two attP sites of an acceptor plasmid and two attB sites of a donor plasmid. To evaluate the activity, integrase mRNAs synthesized from three different plasmids were used. We injected a mixture of the acceptor and donor plasmids with the mRNA synthesized in vitro from one of the three plasmids into silkworm embryos at 4-6?h after oviposition and recovered plasmid DNAs from the embryos 3?days after injection. The resultant plasmids were transformed into Escherichia coli and spread on selection medium plates containing the appropriate antibiotics. A colony-forming assay and restriction enzyme digestion of the plasmids purified from the colonies showed that the phiC31 integrase worked very efficiently in the silkworm embryos. Notably, a phiC31 integrase mRNA synthesized from two of the plasmids produced cassette exchange plasmids at a high frequency, suggesting that the mRNA can be used to construct a targeted integration system in silkworms.     

UV-B radiation induces epithelial tumors in mice lacking DNA polymerase eta and mesenchymal tumors in mice deficient for DNA polymerase iota

DNA polymerase eta (Pol eta) is the product of the Polh gene, which is responsible for the group variant of xeroderma pigmentosum, a rare inherited recessive disease which is characterized by susceptibility to sunlight-induced skin cancer. We recently reported in a study of Polh mutant mice that Pol eta is involved in the somatic hypermutation of immunoglobulin genes, but the cancer predisposition of Polh-/- mice has not been examined until very recently. Another translesion synthesis polymerase, Pol iota, a Pol eta paralog encoded by the Poli gene, is naturally deficient in the 129 mouse strain, and the function of Pol iota is enigmatic. Here, we generated Polh Poli double-deficient mice and compared the tumor susceptibility of them with Polh- or Poli-deficient animals under the same genetic background. While Pol iota deficiency does not influence the UV sensitivity of mouse fibroblasts irrespective of Polh genotype, Polh Poli double-deficient mice show slightly earlier onset of skin tumor formation. Intriguingly, histological diagnosis after chronic treatment with UV light reveals that Pol iota deficiency leads to the formation of mesenchymal tumors, such as sarcomas, that are not observed in Polh(-/-) mice. These results suggest the involvement of the Pol eta and Pol iota proteins in UV-induced skin carcinogenesis.     

Recent development in GAMMA 10 experiment

After the attainment of the density doubling due to the potential confinement, GAMMA 10 experiments have been directed to obtain a high-density plasma with potential confinement and also to study the dependence of the confining potential and confinement time on the plasma density. These problems are important to understand the physics of potential formation in tandem mirrors and also for the development of a tandem mirror reactor. GAMMA 10 experiments have advanced greatly after the Sorrento IAEA Conference, where high-density plasma production by using an ICRF heating at a higher harmonic frequency was reported. Recently, a high-density plasma was attained and the reproducibility of high-density plasma production was much improved by adjusting the spacing of the conducting plates installed in the anchor transition regions. In this paper, we report the production of a high-density plasma and the dependence of the confining potential and confinement time on the density up to a density of 4×10¹² cm^?3.     

Phosphorylation of Rho-associated kinase (Rho-kinase/ROCK/ROK) substrates by protein kinases A and C

Rho-associated kinase (Rho-kinase/ROCK/ROK) is a serine/threonine kinase and plays an important role in various cellular functions. The cAMP-dependent protein kinase (protein kinase A/PKA) and protein kinase C (PKC) are also serine/threonine kinases, and directly and/or indirectly take part in the signal transduction pathways of Rho-kinase. They have similar phosphorylation site motifs, RXXS/T and RXS/T. The purpose of this study was to identify whether sites phosphorylated by Rho-kinase could be targets for PKA and PKC and to find peptide substrates that are specific to Rho-kinase, i.e., with no phosphorylation by PKA and PKC. A total of 18 substrates for Rho-kinase were tested for phosphorylation by PKA and PKC. Twelve of these sites were easily phosphorylated. These results mean that Rho-kinase substrates can be good substrates for PKA and/or PKC. On the other hand, six Rho-kinase substrates showing no or very low phosphorylation efficiency (<20%) for PKA and PKC were identified. Kinetic parameters (K(m) and k(cat)) showed that two of these peptides could be useful as substrates specific to Rho-kinase phosphorylation.     

Construction of a piggyBac-based enhancer trap system for the analysis of gene function in silkworm Bombyx mori

Enhancer trapping and insertional mutagenesis are powerful tools for analyzing genetic function. To construct an enhancer trap system in the silkworm Bombyx mori, we developed efficient jumpstarter strains by inserting the piggyBac transposase gene under the control of Bombyx cytoplasmic actin gene (BmA3) promoter into the genome. To stabilize the inserted transgene, the jumpstarter strains were constructed using the Minos transposon as a vector. The ability of each of the 13 jumpstarter strains to remobilize their respective transposons was tested by crossing the jumpstarters with a mutator strain carrying a GAL4 construct containing the BmA3 promoter. Four strains with high remobilization activity were then selected and used to produce enhancer trap lines by crossing with the mutator strains and hybridizing the F1 progeny with a UAS-EGFP strain. Several enhancer trap lines showing characteristic expression patterns at the embryonic, larval, pupal, and adult stages were detected in the subsequent generation. Approximately 10-40% of the silkworms from each cross in the hybridized brood had a remobilized mutator. An analysis of the insertion positions in 105 lines by inverse PCR using a silkworm genome database revealed that remobilization occurred randomly in each chromosome. The frequency of insertion of the remobilized mutator into putative exons, introns, intergenic regions, and repetitive sequences was 12, 9, 36, and 40%, respectively. We concluded that the piggyBac-based GAL4 enhancer trap system developed in this study is applicable for large-scale enhancer trapping in the silkworm.