Eukaryotic cellular factors that control gene activity are being identified in an increasing number of regulated systems. In the Drosophila Heat Shock system such a factor has been purified (the HAP, Heat Shock Activating Protein) and assays show that it binds specifically to DNA in the promoter region of heat shock genes. In order to fully understand the complete regulatory circuit, it will be necessary to identify, isolate and characterize the genes which code for these regulatory proteins. This can be accomplished by probing genomic libraries with gene specific cDNA probes. A first step will be the preparation of such a probe and will involve the construction of a cDNA expression library. Purified mRNA from cultured Drosophila embryonic SL2 cells will be used as templates for full length cDNA synthesis. These will be ligated to a high efficiency expression vector, the recently developed lambda gtll cloning vector, and plated on host cells to produce the library. Transcription of the insert DNA's will be induced and the proteins coded for will be expressed as part of a B-galactosidase fusion protein. Two methods will be used to screen this library for the presence of HAP coding sequences. The first will make use of labeled anti-HAP antibodies using standard methods developed for use with lambda gtll. A second, novel, method will also be attempted. A specific oligonucleotide, whose sequence corresponds to the known DNA binding sequence of the HAP protein, will serve as the probe. It will be labeled and incubated with plaque containing filters under known binding conditions. This should serve to identify HAP, and thus HAP cDNA, containing plaques. The HAP specific cDNA, once it has been identified and successfully cloned will be sequenced and used in several ways. Successful clones can be used as a readily available source for the HAP facilitating the study of DNA-protein interactions. This cDNA will also be used to probe a genomic library for the HAP gene itself and will thus aid in the cloning of such a gene. Such regulatory genes may themselves be regulated and knowledge of their structure, especially those sequences upstream from the coding region, should yield clues to their function.
