A LIGAND ARRAY APPROACH TO IDENTIFY CIS-REGULATORY DNA AND BINDING PROTEINS

California State University Los Angeles, Los Angeles CA

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Abstract

Our long-range goal is to develop a surface array-based detection system to rapidly characterize regulatory[unreadable] molecular interactions. The objectives of this proposal are to develop a high-throughput, double-stranded[unreadable] oligonucleotide array-based DNA binding protein analysis system to identify cis-regulatory DNA binding[unreadable] motifs operative in bacterial and plant cells, and to use matrix-assisted laser-desorption ionization time-offlight[unreadable] mass spectrometry (MALDI-TOF-MS) to identify the interacting proteins. The central hypothesis of this[unreadable] proposal is that specific protein-DNA interactions can be detected by surface plasmon resonance using[unreadable] arrays of 48 different double-stranded DNAs and cell or nuclear extracts. We expect that the hundreds to[unreadable] thousands of different transcription factors present in the extracts will find and bind tightly to their target[unreadable] sequences in a single experiment. We formulated this hypothesis on the basis of our previous work and[unreadable] strong preliminary data. We have demonstrated in our own labs that SPR can sensitively detect specific[unreadable] protein-DNA interactions on surfaces, obviating the need for a labeling step, and we can differentiate[unreadable] between protein and DNA samples spotted in an arrayed pattern. The rationale for the proposed research is[unreadable] that by developing a method to identify cis-regulatory sequences in a high-throughput manner in any cell[unreadable] type, we can make significant gains in our understanding of sequence-driven gene regulation. Such[unreadable] knowledge will be beneficial to all of biology, from informing basic mechanisms in organismic development[unreadable] and responses to the environment, to a clearer view of the diseased state. In addition, all biological[unreadable] processes that contribute to minority health disparities have some uncharacterized component of[unreadable] transcriptional control. The approach will thus be useful in identifying those fundamental regulatory[unreadable] responses that contribute to health disparities in minority communities. We are well prepared to undertake[unreadable] the proposed research, as we have the breadth of expertise in the PI and collaborators to meet our[unreadable] objectives in a conducive, particularly well-equipped research environment.