Identification of two evolutionarily conserved and functional regulatory elements in intron 2 of the human BRCA1 gene
Section snippets
Intronic sequence conservation between human and mouse BRCA1 identifies nine potential regulatory regions
As an initial approach to identifying novel sequence elements involved in regulation of BRCA1 expression, we performed a comparative analysis of the human and mouse BRCA1 genomic sequences. Based on the observation that these elements are usually composed of long sequences (≥100 bp in length) and are highly conserved (≥70% identity) [14], nine CNS matching these criteria were identified in six different introns of the BRCA1 gene (unpublished data). A BLAST search of the conserved sequences
Discussion
Comparative genomics offers a potential strategy for identifying important regulatory elements on the basis that evolutionary conservation implies functional constraints. In this study, we utilized human–mouse genomic comparisons to identify potential noncoding regulatory elements in the BRCA1 gene. The evolutionary distance been humans and mice is reported to be sufficient for identification of functionally important elements [48]. This premise is supported by studies identifying functional
Cell culture
Cervical adenocarcinoma HeLa cells (ATCC CCL-2) and SV40 T-antigen-immortalized human mammary epithelial SVCT cells (ECACC 94122105) were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum (Invitrogen). SVCT growth medium was also supplemented with insulin (Sigma–Aldrich Pty. Ltd., Sydney, Australia; 10 μg/ml) and hydrocortisone (Sigma–Aldrich; 0.5 μg/ml). Cells were maintained at 37°C in a 5% CO2 humidified incubator.
Comparative sequence analysis
BRCA1 genomic DNA from the following species
Acknowledgments
The authors are grateful to Panayiotis Ioannou (Murdoch Institute, Melbourne) for the pGETrec plasmid, the ICRF (Cancer Research UK) for the use of the SVCT cell line, Matthew Wakefield (ANU, Canberra) for the marsupial genomic DNA, Sylvie Mayozer (IARC, Lyon) for primers and help with mapping BRCA1 deletions, Georgia Chenevix-Trench and Aaron Lewis (QIMR, Brisbane) for help with mutation analysis, and Ibtissam Abdul Jabbar (UQ, Brisbane) for MoFlo FACS. The authors thank the kConFab research
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