Vertebrate species, including man, are believed to be phylogenetically polyploid. The resulting duplicated genes often return to a diploid functional level by """"""""diploidization."""""""" Nothing is known as to the molecular mechanism(s) operating to regulate the expression of these duplicated genes to the diploid level, although such knowledge would surely advance our understanding of eukaryotic genetic control. Molecular study of the structure of a diploidizing gene may provide clues as to where we should be looking in order to elucidate such mechanisms.
The aim of the feasibility studies in this proposal is to characterize a sub-set of rapidly diverging ribosomal RNA (rRNA) genes we have discovered within the genome of the diploidizing tetraploid treefrog Hyla versicolor, a gene sub-set which we believe my itself be returning to the diploid functional level, and to compare parameters with those in a closely related diploid progenitor species, H. chrysoscelis. Restriction enzyme mapping of genomic rRNA genes in several different sympatric populations of the diploid/tetraploid species pair H. chrysoscelis/H. versicolor will be done, to evaluate the possible contribution of gene flow to the origin of the marked sequence heterogeneity we have thus far observed in one natural population of the tetraploid. If a gene is """"""""diploidizing,"""""""" then by definition it is losing function. We will prepare recombinant libraries, from which rRNA gene will be isolated from the diploids and also the several differing classes of tetraploid rRNA gene sub-sets we have identified, and utilize these to determine whether, and to what extent, the rapidly diverging sub-set in the tetraploid is functionally significant. These experiments will also be extended to determination of the degree to which the various sub-sets are transcribed, in an effort to localize some site which may be important for genetic control of diploidization: for example, at the level of gene structure or a processing step deficiency. Finally, we will initiate experiments aimed at characterizing potneital control regions within the various gene sub-sets. In the proposed preliminary experiments, this will limited to determination of the precise nucleotide sequence of the transcription initiation site and surrounding putative promoter region, but will be extended to the identification of important upstream transcription factor binding sites, if time permits.
