Since sequence-specific DNA-binding proteins control gene expression in bacteria, and may serve the same crucial function in higher organisms, regulation should be studied at the molecular level (among others) to understand the signals responsible for differentiation and development. One way to approach these problems is to identify eukaryotic sequence-specific DNA-binding proteins. Hybridization mapping of Drosophila melanogaster nucleosomes has revealed a protein (D1) specific for AT-rich heterochromatic sequences. This approach will be continued with hybridization probes for lower abundance sequences, to investigate the generality of D1 binding to AT-rich, nonsatellite sequences, and to search for more highly sequence-specific DNA-binding proteins of lower copy number genes. Reconstitution hybridization mapping, a new approach designed to separate overlapping nucleoprotein and DNA species, should unambiguously identify minor particles which are """"""""cryptic"""""""" in a two-dimensional pattern obtained from unfractionated nucleosomes. DNA-cellulose chromatography will be used to purify the proteins, which will be mixed with salt-stripped nucleosomes, spread on two-dimensional gels, and analyzed by hybridization and by correlating DNA and protein patterns. Protein-protein cross-linking should reveal the interactions between D1 molecules, the histones, and other elements of nuclear structure. Cross-linking will also stabilize proteins which might otherwise redistribute between nucleosomes. The kinetics and structural effects of D1 binding to DNA in vitro will be investigated using nucleoprotein electrophoretic methods and protection (footprinting) experiments. These studies will reveal how D1, a nucleosomal, sequence-specific DNA-binding protein of the most general variety, recognizes DNA, and how widespread is its distribution throughout the D. melanogaster genome.
| Iyer, R K; Levinger, L F (1988) Visualization of RNA binding proteins by sequential gel shift and ultraviolet cross-linking. Gene Anal Tech 5:125-9 |
