The unkempt gene in Drosophila melanogaster encodes a product that contains five so-called Cys(3)His finger motifs thought to bind or cleave RNA's of several types. Dr. Mohler and his students will try to confirm the interaction with RNA and the presence of chelating zinc atoms in these motifs and, secondly, assess the role of the wild type unkempt gene product in ovarian and embryonic development, in the developing eye disc and in blood cell proliferation. In the first investigations, full length, and N and C terminal halves of the peptides will be expressed in and purified from E. coli. Their ability to bind DNA and RNA will be assessed by column retention and gel shift assays. RNase activity will also be examined using substrates with single 5' or 3' terminal hairpin loops to assess 5' and 3' exonucleolytic activity, or, in the case of an RNA substrate with both 5' and 3' hairpins, endonucleolytic activity. Zinc binding will be determined renatured peptides bound to nitrocellulose membranes in the presence of radiolabelled zinc atoms. Unkempt's role in eye development will be determined in mitotic clones with homozygous null alleles. Specific cell losses during development will be assessed both cytologically and by staining with cell type specific antibodies. To see if unkempt mutants affect blood cell proliferation, mutant lymph glands will be transplanted to wild type hosts. The cells from these mutant glands will be identified by LacZ staining, and their proliferative and differentiative responses assessed by host lethality and cytology respectively. By using the FLP-FRT system and the ovoD mutant, germ line clones of homozygous unkempt null mutants will be produced. If eggs are not laid, the status of the arrested germ line cells will be examined, after being identified immunologically with an antibody to a myc-epitope expressed by a transgene on the same chromosome as the unkempt null mutant allele. If eggs are laid but do not develop, the stage of developmental arrest will be determined and certain early embryonic structures will be examined cytologically.
