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Our group uses genetic selection in yeast (yeast one hybrid and yeast two hybrid cloning: see Figure) as a tool to aid our analysis of protein-DNA and protein-protein interactions, respectively. Malathi Raman, a full-time PhD student in the group since 2003 funded by NHLI and the British Heart Foundation, is conducting a one hybrid screen for two cardiac-specific transcription factors that bind a CACC-box in the promoter of the human cardiac troponin I (TnIc) gene, which is only ever expressed in the heart ( in collaboration with Dr. Paul Barton, Molecular Biology). These factors, which we call HCB1 & 2 for Heart CACC-box Binding Factors 1 & 2, were originally identified by us as proteins present in heart muscle cell extracts that specifically bound to the TnIc CACC box. Preliminary tissue distribution suggested they might be cardiac–specific, as they are not detected in extracts of liver, kidney or skeletal muscle-derived cell lines (Dellow et al , 2001). As their cDNAs have not been cloned, we have no idea at present to which family of transcription factors HCB1 & 2 belong. Five copies of the TnIc CACC-box have been cloned upstream of His3 and LacZ reporter genes and introduced into the genome of a yeast strain. This strain will be transformed with a cardiac myocyte cDNA library in order to identify proteins capable of binding the CACC-box and activating reporter expression. The cloned cDNA will be isolated from positive colonies and it's identity determined by sequencing and bioinformatics. In this way, we can determine if HCB1 & 2 represent known transcription factors that have not been previously linked to regulation of the TnIc gene, or if they are, in fact, novel proteins that have not been described before. Once cloned, the HCB1 & 2 factors will be further characterised with regard to expression and function.
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Further reading
Dellow KA, Bhavsar PK, Brand NJ, & Barton PJ (2001). Identification of novel, cardiac-restricted transcription factors binding to a CACC-box within the human cardiac troponin I promoter. Cardiovasc Res 50 , 24-33. |
