Functional analysis of Drosophila DNA polymerase ε p58 subunit.

DNA polymerase ε (polε) plays a central role in DNA replication in eukaryotic cells, and has been suggested to the main synthetic polymerase on the leading strand. It is a hetero-tetrameric enzyme, comprising a large catalytic subunit (the A subunit ~250 kDa), a B subunit of ~60 kDa in most species (~80 kDa in budding yeast) and two smaller subunits (each ~20 kDa). In Drosophila, two subunits of polε (dpolε) have been identified. One is the 255 kDa catalytic subunit (dpolεp255), and the other is the 58 kDa subunit (dpolεp58). The functions of the B subunit have been mainly studied in budding yeast and mammalian cell culture, few studies have been performed in the context of an intact multicellular organism and therefore its functions in this context remain poorly understood. To address this we examined the in vivo role of dpolεp58 in Drosophila. A homozygous dpolεp58 mutant is pupal lethal, and the imaginal discs are less developed in the third instar larvae. In the eye discs of this mutant S phases, as measured by BrdU incorporation assays, were significantly reduced. In addition staining with an anti-phospho histone H3 (PH3) antibody, (a marker of M phase), was increased in the posterior region of eye discs, where usually cells stop replicating and start differentiation. These results indicate that dpolεp58 is essential for Drosophila development and plays an important role in progression of S phase in mitotic cell cycles. We also observed that the size of nuclei in salivary gland cells were decreased in dpolεp58 mutant, indicating that dpolεp58 also plays a role in endoreplication. Furthermore we detect a putative functional interaction between dpolε and ORC2 in discs suggesting that polε plays a role in the initiation of DNA replication in Drosophila.

Differential requirement for the N-terminal catalytic domain of the DNA polymerase ε p255 subunit in the mitotic cell cycle and the endocycle.

In Drosophila, the 255kDa catalytic subunit (dpolεp255) and the 58kDa subunit of DNA polymerase ε (dpolεp58) have been identified. The N-terminus of dpolεp255 carries well-conserved six DNA polymerase subdomains and five 3'→5' exonuclease motifs as observed with Polε in other species. We here examined roles of dpolεp255 during Drosophila development using transgenic fly lines expressing double stranded RNA (dsRNA). Expression of dpolεp255 dsRNA in eye discs induced a small eye phenotype and inhibited DNA synthesis, indicating a role in the G1-S transition and/or S-phase progression of the mitotic cycle. Similarly, expression of dpolεp255 dsRNA in the salivary glands resulted in small size and endoreplication defects, demonstrating a critical role in endocycle progression. In the eye disc, defects induced by knockdown of dpolεp255 were rescued by overexpression of the C-terminal region of dpolεp255, indicating that the function of this non-catalytic domain is conserved between yeast and Drosophila. However, this was not the case for the salivary gland, suggesting that the catalytic N-terminal region is crucial for endoreplication and its defect cannot be complemented by other DNA polymerases. In addition, several genetic interactants with dpolεp255 including genes related to DNA replication such as RFC, DNA primase, DNA polη, Mcm10 and Psf2 and chromatin remodeling such as Iswi were also identified.

[Stereotactic vacuum-assisted breast biopsy (Mammotome biopsy) for non-palpable microcalcification on mammography].

PURPOSE: The purpose of this study was to assess the benefits of stereotactic vacuum-assisted breast biopsy in patients with non-palpable microcalcification detected on mammography. METHODS: Between October 2001 and November 2003, stereotactic Mammotome biopsies were performed for 150 microcalcified lesions on mammography using the prone-type stereotactic vacuum-assisted breast biopsy system (Mammotest and Mammovision, Fischer, Denver, USA) . The mammography findings were classified according to the guidelines of The Japan Radiological Society/The Japan Association of Radiological Technologists. Ninety-eight cases were category 3, 38 were category 4, and 14 were category 5. RESULTS: All cases were determined to be cases of microcalcification by specimen radiography or histology. Complications were negligible. One hundred twenty of the cases were mastopathy, and 30 of them were breast cancer (14 were ductal carcinoma in situ, 7 were ductal carcinoma in situ with microinvasion, and 9 were invasive ductal carcinoma). Twenty-seven breast cancers were diagnosed as category 4 or 5 (51.9%) on mammography. The operative stages of 27 cases were as follows: 7 were stage 0, 17 were stage 1, and 3 were stage 2A. Twenty-four of 27 (88.9%) were early breast cancers. CONCLUSION: Mammotome biopsy is a safe and useful modality for the histological diagnosis of non-palpable microcalcifications.