Backgroundlin-4 and let-7, the two founding members of heterochronic microRNA genes, are firstly confirmed in Caenorhabditis elegans to control the proper timing of developmental programs in a heterochronic pathway. let-7 has been thought to trigger the onset of adulthood across animal phyla. Ecdysone and Broad-Complex are required for the temporal expression of let-7 in Drosophila melanogaster. For a better understanding of the conservation and functions of let-7, we seek to explore how it is expressed in the silkworm (Bombyx mori).ResultsOne member of let-7 family has been identified in silkworm computationally and experimentally. All known members of this family share the same nucleotides at ten positions within the mature sequences. Sequence logo and phylogenetic tree show that they are not only conserved but diversify to some extent among some species. The bmo-let-7 was very lowly expressed in ova harvested from newborn unmated female adult and in individuals from the first molt to the early third instar, highly expressed after the third molt, and the most abundant expression was observed after mounting, particularly after pupation. The expression levels were higher at the end of each instar and at the beginning of each molt than at other periods, coinciding with the pulse of ecdysone and BR-C as a whole. Using cultured ovary cell line, BmN-SWU1, we examined the effect of altered ecdysone levels on bmo-let-7 expression. The expression was also detected in various tissues of day 3 of the fifth instar and of from day 7 of the fifth to pupa, suggesting a wide distributing pattern with various signal intensities.Conclusionbmo-let-7 is stage- and tissue-specifically expressed in the silkworm. Although no signals were detected during embryonic development and first larval instar stages, the expression of bmo-let-7 was observed from the first molt, suggesting that it might also function at early larval stage of the silkworm. The detailed expression profiles in the whole life cycle and cultured cell line of silkworm showed a clear association with ecdysone pulse and a variety of biological processes.

Cyclin B is an important regulator of the cell cycle G2 to M phase transition. The silkworm genomic database shows that there are two Cyclin B genes in the silkworm (Bombyx mori), BmCyclin B and BmCyclin B3. Using silkworm EST data, the cyclin B3 (EU074796) gene was cloned. Its complete cDNA was 1665 bp with an ORF of 1536 bp derived from seven exons and six introns. The BmCyclin B3 gene encodes 511 amino acids, and the predicted molecular weight is 57.8 kD with an isoelectric point of 9.18. The protein contains one protein damage box and two cyclin boxes. RNA interference-mediated reduction of BmCyclin B and BmCyclin B3 expression induced cell cycle arrest in G2 or M phase in BmN-SWU1 cells, thus inhibiting cell proliferation. These results suggest that BmCyclin B and BmCyclin B3 are necessary for completing the cell cycle in silkworm cells. Cyclin B is a mitotic cyclin protein. It is expressed and gradually accumulates from the G1 late phase, reaches a maximum level at the G2 late phase, continues to be maintained to the middle of metaphase, and is then immediately degraded. The accumulation and degradation of cyclin B plays a critical role in mitosis and karyokinesis [1][2][3]. A cyclin box and conserved cdk-binding domains are present in all cyclins. A-and B-type cyclins possess a destruction box, a motif containing ~nine amino acids required to target cyclins to ubiquitin-dependent degradation during mitosis [46]. Cyclin B genes can be placed in two evolutionary branches, the B-type and B3-type cyclin families [7]. B-type cyclins are present in both yeast and higher eukaryotes. Although a single Cyclin B gene has been found in invertebrate species, a pair of very closely related B-type cyclins is present in vertebrates. These vertebrate cyclin B1 and B2 proteins differ in their subcellular localization [8,9]. The Cyclin B3-type family is conserved in metazoans and has a similarity of 39%-46% in the cyclin protein box [1012].Both cyclin B and cyclin B3 have a conserved amino acid sequence, which is known as the cell cyclin box. This domain can combine with CDKI to form complexes, which, when activated by CDK-activating kinase, drives cells from the G2 phase limit point to the M phase. The Cyclin B gene has previously been cloned from silkworm eggs [13]. The expression of Cyclin B was reduced in silkworm ovarian BmN cells, 24 h after infection by nucleopolyhedrovirus (BmNPV), and the cells then arrested in G2/M phase [14].The silkworm undergoes complete metamorphosis, and is a good model organism of Lepidoptera. A full understanding of its cell-cycle regulation mechanism would improve