Proliferating Cell Nuclear Antigen from the mulberry silkworm Bombyxmori : Cloning and characterisation

Abstract

In a proliferating eukaryotic cell, duplication of the genetic complement occurs once per cell cycle during the S phase. DNA replication in a single cell must be coordinated with other cell cycle processes such as mitosis and cytokinesis in surrounding cells within a tissue. Much of this regulation occurs at the level of initiation of DNA replication via interaction with pathways that control cell cycle progression. Furthermore, DNA replication in eukaryotic cells must be coupled to DNA repair and chromatin assembly. Replication fork proteins also play a role in coordinating the cell cycle.

Proliferating Cell Nuclear Antigen (PCNA) is an important protein involved in S-phase progression. It interacts cooperatively at the replication fork with the leading strand synthesis enzyme, DNA polymerase . This interaction enhances the processivity of polymerase by about 3-5 fold. In recent years, identification of proteins that interact with PCNA has established PCNA as a key protein required for coordinated regulation of replication and other events at the replication fork, such as DNA methylation and DNA repair (Tsurimoto, 1998).

In the present study, the mulberry silkworm Bombyx mori has been used as an experimental model to characterize and analyze the role of PCNA, for the following reasons: the silk gland cells in B. mori undergo endoreplication, where multiple rounds of DNA replication occur without cell or nuclear division. In this terminally differentiated tissue, cells grow enormously during larval development without division. Each silk gland, made up of about 1000 cells and measuring 1-2 mm at the first instar, grows up to several centimeters in length by the fifth instar without change in cell number. Although the cells or nuclei do not divide, DNA continues to replicate for 18-19 rounds throughout larval development. Consequently, the highly ramified nuclei accumulate DNA amounting to ~300,000× the haploid genomic content in late larval stages (Niranjanakumari and Gopinathan, 1992). Concomitant with DNA doubling, the activity of DNA polymerase also doubles. Therefore, it was of interest to examine the status of an associated protein like PCNA in a non-proliferating but replicating cell.

The thesis addresses the following broad aspects:

Cloning, characterization, and analysis of pcna expression in silk glands of B. mori.

Role of host PCNA in viral development using cultured cell lines of B. mori and Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) as a model system.

Identification of tissue- and stage-specific expressed sequence tags (ESTs) from silk gland compartments using Differential Display PCR (DD-PCR) and RNA Arbitrary Primed PCR (RAP-PCR).