Molecular characterization of a stably transformed Bombyx mori cell line: identification of alternative transcriptional initiation sites of the A3 cytoplasmic actin gene.

Abstract

We have isolated a stably transformed Bormbyx mori cell line containing a novel selectable marker gene, puromycin N-acetyl transferase, under control of transcriptional regulatory signals from the A3 cytoplasmic actin gene. By using this cell line we have identified alternative transcriptional initiation sites for the A3 actin gene. One of these start sites is located approximately 35 bp upstream from the previously determined transcription initiation site. The two mRNA start sites are utilized with a similar efficiencies in the BmN cell line. In addition, we detected transcripts that initiated in the first intron of the A3 actin gene. These transcripts may be synthesised under control of an alternative promoter. The stably transformed B. mori cell line used in this study was also extensively characterized. Integration of the plasmid molecules into the host genome was demonstrated by Southern and in situ hybridization analyses. Establishment and characterization of stably transformed insect cell lines, like the one described here, represents an important step in the development of nonlytic insect expression systems.