THE ORGANIZATION AND SEQUENCE OF THE 5S RIBOSOMAL GENES OF THE DIPTERAN FLY CALLIPHORA ERYTHROCEPHALA AND THE ISOLATION OF GENES SPECIFICALLY TRANSCRIBED DURING OOGENESIS IN THE DIPTERAN FLY CALLIPHORA ERYTHROCEPHALA
Doctor of Philosophy
The genomic organization and sequence of the 5S RNA genes of the Dipteran fly Calliphora erythrocephala have been determined. The 5S RNA genes consist of a tandem array of 480 base pair (bp) repeating units which are clustered at a single locus 4b on chromosome 2. The repeating units show only minimal sequences and length heterogeneity, and a consensus sequence for a cloned series of 5S repeat units was determined. The coding sequence for the mature 5S RNA contains a single residue change from the known gene sequences from three Drosophila species (19,30). A precursor RNA containing additional 3' sequences with some homology to the equivalent sequences of the Drosophila species (19,30) is indicated. Partial pseudogenes homologous to the extreme 3' end of the transcribed region and the adjacent termination sequence are found at two positions in the spacer. Comparison of the 5S RNA genes of C. erythrocephala to those of three Drosophila species (19,39) identified a striking series of perfectly conserved homologies identically positioned ((+OR-)1 nucleotide) within the 5' flanking DNA of all four Dipteran 5S RNA coding regions. One of the Dipteran homology blocks is highly conserved in sequence and position in all but one of the eukaryotic 5S RNA gene sequences examined (17/18 genes). A cloned library of genomic DNA sequences of C. erythrocephala was prepared and screened by differential hybridization to ('32)P-cDNA made against poly(A)('+) RNA from embryos and two stages of oogenesis to detect sequences specifically transcribed during oogenesis. Four phage clones showed transcriptional activity in specific stages of oogenesis but not during embryogenesis. Each clone hybridized to a single poly(A)('+) RNA transcript which differed in size and stage-specificity for the four clones. All four clones contain DNA sequences which are highly repeated within the C. erythrocephala genome, but the transcriptional activity, if any, of the repetitive elements in each clone is undetermined. Transcribed fragments from each clone failed to hybridize to D. melanogaster genomic DNA at hybridization conditions allowing 10% sequence divergence. Whole follicles were used in the identification of these oogenesis-specific phage clones, and the cell specificity (follicle cell or nurse cell) is, as yet, undetermined.