Search

The current focus of many functional genomic studies is on the elucidation of gene regulatory networks. The functional analyses of transcription factors and their DNA-binding sites, in conjunction with genome-wide expression profiling, are crucial in understanding of gene regulatory networks. This paper describes an efficient and easy method for characterizing the DNA-binding sequence specificity of novel plant transcription factors. This new method is based on the fusion of a DNA-binding protein (DBP) to 6×His-tagged cellulase D (CELD), which serves both as a means for affinity purification of DBP-DNA complex in the selection of binding sites from a pool of biotinylated random-sequence oligonucleotides and as a reporter for measurement of DNA-binding activity. Thus, it eliminates the use of radioactivity and gel electrophoresis techniques currently used for purification of DBP-DNA complexes and assays of DNA-binding activity. The effectiveness of this method was demonstrated by the success of simultaneous selection of the binding sites of nine plant DBPs from four superfamilies (AP2, bHLH, NAC and MYB). The high-throughput capacity of CELD-based DNA-binding assays allows the quantitative analysis of the binding sequence specificity from a large number of DBP-selected oligonucleotides. The binding sequence specificity of three novel transcription factors (rice OsbHLH66, wheat TaNAC69 and TaMYB80), determined with this method, is presented. This new method provides the capacity of high-throughput analysis on the DNA-binding sequence specificity of a large number of putative transcription factors, predicted on the basis of conserved DNA-binding domains. [ABSTRACT FROM AUTHOR]

In June 2001, when the Human Genome Project and Celera completed the first maps of the human genome, Francis Collins, head of the government sponsored HGP, warned that only then would the real race begin. Proteomics is more complex by several orders of magnitude than genomics, with no one company, laboratory or consortium remotely able to run the race alone, acknowledged Brian D. Chait, head of the mass spectrometry laboratory at Rockefeller University in New York City. With the publication of the draft of the human genome in the February 16, 2001 issues of Science and Nature came what many had already suspected: instead of the earlier estimate of about 100 000 human genes, the actual count reduced this figure by 75 percent.