Development of a non-immunological system for the study of the cellular localization of BRCA1 gene product in living cells

Abstract

BRCAl, is a familial breast and ovarian cancer susceptibility gene that has been cloned and shown to be either lost or mutated in families with breast and ovarian cancer. BRCAl, has been postulated to encode a tumor suppressor, a protein that acts as a negative regulator of tumor growth. To explore the biolo^cal function of BRCAl, several studies have been performed for the identification of cellular localization of BRCAl gene product. Results obtained from these immunofluorescent/ immunohistochemical studies generated two opposing views, cytoplasmic localization versus nuclear localization. Here, we describe a non-immunological system employing the Eukaiyotic Green fluorescent Protein (EGFP) tag for the study of the cellular localization of BRCAl gene product in living cells. Proteins carrying the green fluorescent protein (GFP) of Aequorea victoria provide a powerful system to analyze protein expression and targeting in living cells. Fusion proteins containing the GFP tag are therefore valuable tools to analyze nuclear trafficking in living cells. Here, we reporte the use of a mutant GFP, namely Eukaryotic Green Fluorescent Protein (EGFP), as a marker for the protein import into mammalian nuclei. We have analyzed the behavior of a protein domain of the BRCAl, that contains five putative nuclear localization signals (NLSs), in vivo using a chimera constructed from this polypeptide and the EGFP. This in vivo studies showed that EGFP was distributed uniformly throughout the cytoplasm and the nucleus. When EGFP was fused to NLSs containing domain of the BRCAl protein, fluorescent was predominantly detected in the nucleus, showing that these potential NLSs consensus sequences may destínate the full-lengh BRCAl producy into the nucleus of mammalian cell. This study has also shown that EGFP can be used as a potential fluorescent tag for visualization of gene expression and cellular protein localization in living cells.