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The integrase from the Streptomyces bacteriophage ?C31 carries out efficient recombination between an attP site in the phage genome and an attB site in the host chromosome. In the present study, we have used the ?C31 integrase system to mediate site-specific recombination in the cultured silkworm cell line BmN4. A plasmid containing a cDNA encoding DsRed flanked by two ?C31 attP sites was co-transfected together with a helper plasmid encoding the ?C31 integrase into a cell line in which ?C31 attB sites inserted between a baculovirus IE2 promoter, and a polyadenylation signal are present in one chromosome. Seven days after transfection, expression of DsRed was observed in transformed cells. Nucleotide sequence analysis demonstrated that the expected recombination between the attB and attP sites had been precisely carried out by the ?C31 integrase. These results indicate that the ?C31 site-specific recombination system should be widely applicable for efficient site-specific gene integration into silkworm chromosomes. [ABSTRACT FROM AUTHOR]

Abstract Nonhomologous end-joining (NHEJ) is one of the repair pathways for double-strand breaks (DSBs) in eukaryotic cells. By using linearized plasmid substrates, we have detected intramolecular NHEJ activity in a cell-free extract from the cultured silkworm cell line BmN4. The efficiency of NHEJ differed according to the structure of DNA ends; approximately 1% of input DNA was repaired when the substrate had cohesive ends. The reaction required the hydrolysis of nucleotide triphosphate; interestingly, all of four rNTPs or four dNTPs could support the reaction. A substrate with non-complementary DNA ends was mainly repaired by the DNA polymerase-mediated pathway. These results indicate that the present cell-free system will be useful to analyze the molecular mechanisms of DSB repair and NHEJ in insect cells. [ABSTRACT FROM AUTHOR]

The sericulture industry plays a very important role in our national economy. Silkworm ( Bombyx mori) is always regarded as a model animal and biological reactor. There have been detailed studies on the structure, expression and control and molecular evolution of silk genes. However, few, if any, reports are available on the localization of structural genes in silkworm by molecular cytogenetics. The present experiment has tentatively localized the Fib-H gene at the distal end of the 25th linkage group, namely at the 25-0.0 position, and verified that Fib-H has only one locus, thus providing a temporary solution to the problem about its localization. [ABSTRACT FROM AUTHOR]