A new continuous cell line from larval ovaries of silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

A new continuous cell line from ovarian tissue of commercial variety “Kolar Gold” of silkworm, Bombyx mori, was established and designated as DZNU-Bm-12. The tissue was grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat-inactivated B. mori hemolymph at 25 ± 1°C. The migration of partially attached small round refractive cells from the fragments of ovarioles began from the beginning of explantation. The cells multiplied partially attached in the primary culture initially, and some of them become freely suspended after 20 passages. The cells were adapted to MGM-448 and TNM-FH media each with 10% FBS and the population doubling time of cell line was about 36 and 24 hr, respectively. The chromosome number was near diploid at initial passages and slightly increased at 176th passage, but a few tetraploids and hexaploids were also observed. DNA profiles using simple sequence repeat loci established the differences between DZNU-Bm-12 and DZNU-Bm-1 and most widely used Bm-5 and BmN cell lines. The cell line was found susceptible to B. mori nucleopolyhedrovirus (BmNPV) with 85–90% of the cells harboring BmNPV and having an average of 3–17 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV-based baculoviral expression system and also for studying in vitro virus replication.     

Serum-free culture of an embryonic cell line from <Emphasis Type="Italic">Bombyx mori</Emphasis> and reinforcement of susceptibility of a recombinant BmNPV by cooling

We established the first continuous cell line that uses a serum-free culture from the embryo of Bombyx mori (Lepidoptera: Bombycidae), designated as NIAS-Bm-Ke17. This cell line was serially subcultured in the SH-Ke-117 medium. The cells adhere weakly to the culture flask, and most cells have an oval shape. The cell line was subcultured 154 times, and the population doubling time is 83.67 ± 5.22 h. Random amplification of polymorphic DNA-polymerase chain reaction with a tenmar single primer for discrimination of insect cell lines recognized the NIAS-Bm-Ke1 cell line as B. mori. This cell line does not support the growth of the B. mori nuclear polyhedrosis virus (BmNPV) in the absence of the heat-inactivated hemolymph of B. mori. However, the heat-inactivated hemolymph in 1% volume of the medium supported a high level of susceptibility to BmNPV. In addition, the cooling treatment of the cells at 2.5°C also enhanced the susceptibility. We report a new serum-free culture system of the B. mori cell line for the baculovirus expression vector system.     

Evidence for Gene Conversion Between Tandemly Duplicated Cytoplasmic Actin Genes of Helicoverpa armigera (Lepidoptera:Noctuidae)

Tandemly duplicated actin genes have been isolated from a Helicoverpa armigera genomic library. Sequence comparisons with actin genes from other species suggest they encode cytoplasmic actins, being most closely related to the Bombyx mori A3 actin gene. The duplicated H. armigera actin genes, termed A3a and A3b, share 98.3% nucleotide sequence identity over their entire putative coding region. Analysis of the distribution of nucleotide differences shows the first 763 bp are identical between the two coding regions, with the 18 nucleotide changes occurring in the remaining 366 bp. This observation suggests a gene conversion event has taken place between the duplicated H. armigera A3a and A3b actin genes. Translation of the open-reading frames indicates the products of these genes are identical, apart from a single amino acid difference at codon 273. Polymerase chain reaction and northern blot analysis have shown both H. armigera A3a and A3b genes are expressed during pupal development and in the brain of newly eclosed adults. A region 5′ of the H. armigera A3a actin gene start codon has been identified which contains regulatory sequences commonly found in the promoter region of actin genes, including TATA, CAAT, and CArG motifs. Received: 10 January 1996 / Accepted: 12 March 1996