Identification and expression analysis of the replication factor C protein in the silkworm, Bombyx mori

Abstract

The silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), is an agriculturally crucial animal for silk production. Approximately 820 silkworm mutants have been deposited and categorized according to the color of the pupa, skin color, patterns of the larva, and color of the eggs (http://shigen.nig.ac.jp/silkwormbase/top.jsp). The phenotypes available for B. mori have been used in genetic studies. The recent focus in silkworm research has been on using the silkworm as an animal model in drug discovery and development instead of mice or other animals to confirm drug efficacy, studying the function of silkworm proteins, and utilizing it as an expression host for recombinant proteins. However, the cell cycle, including initiation of DNA replication, has not yet been investigated in B. mori. As an initial step toward understanding this process for this species, we attempted to identify factors involved in DNA replication. Replication factor C (RFC) fulfills essential functions in DNA replication and repair of eukaryotic cells (Mossi and Hubscher, 1998). Eukaryotes have five RFC subunits, designated RFC1 to RFC5, and all of them contribute to RFC activity and chromosome replication (Ohta et al., 2002). These subunits belong to the AAA+ protein family (Ohta et al., 2002) and feature seven well-conserved motifs, the RFC boxes II through VIII, which are crucial for the RFC complex formation (Mossi and Hubscher). Previously, we identified a minichromosome maintenance protein in silkworms, which is one of the proteins involved in DNA replication (Yamamoto et al., 2010). In the process of identifying components important for DNA replication in this species, a cDNA encoding RFC, another DNA replication factor, was obtained. In the present study, sequence alignment and the corresponding phylogenetic tree identified the RFC cDNA as a homologue of the B. mori RFC subunit 2 (bmRFC2). Furthermore, the expression pattern of the RNA encoding bmRFC2 was determined by real-time PCR.