Under this grant we developed the technique of DNA reannealing and rebanding in NaI gradients to identify molecules of DNA in different organizations, regarding the relationship between unique and repeated sequences. We were able to show that genomic reorganizations have occurred pariodically during primate evolution. We propose to test the hypothesis that some ancestral structural genes reside in a newly-formed, long-range DNA organization and that the DNA organizational state affects gene expression. We will identify ancestral structural genes flanked by new-amplified repeated DNA. We will then select structural genes found in the """"""""flexible"""""""" DNA organization revealed by the reannealing-rebanding technique. We will then clone the homologous gene(s) from baboons and examine the organization of its flanking sequences, especially regarding newly-evolved repeated sequences. We will examine the position of this gene in reannealing-rebanding gradients of baboon DNA. Finally, we will sequence all relevant DNA regions. All these experiments will serve to determine the structure of regions of DNA which have been newly reorganized. We will examine the expression of the human and baboon structural genes in tissue cluture and in vivo. We will use in situ molecular hybridization to evaluate the expression of these structural genes in vivo. We will add, remove or change repeated sequences neqr the structural genes and evaluate changes in expression after insertion into cultured animal cells. This work will add significant knowledge about the structure and evolution of the human genome. It will also result in the characterization of many structural gene clones obtained by other investigators. And it will provide continually refined methods for phylogenetic classification.
| Thompson, J; Solomon, R; Pellegrino, M et al. (1989) A noise-free molecular hybridization procedure for measuring RNA in cell lysates. Anal Biochem 181:371-8 |
