1. This research will contribute to the construction of a detailed cytological map of the human genome in which the locations of genes and linkage groups are established with respect to visible features on the chromosomes. It will focus on assignment of genes involved in growth control, in development and in genetic disorders. This mapping information will lead to testable hypotheses regarding the involvement of specific genes in certain types of malignancies, in the pathogenesis of malformation syndromes and in specific Mendelian phenotypes. These efforts will be extended to the mouse genome in order to elucidate mammalian karyotype evolution and to develop mouse models for human genetic disorders based on the mapping of putative homologous mutations to conserved chromosomal regions. The approach will be to hybridize cloned gene probes to DNA from somatic cell hybrid panels that contain reduced sets of human or mouse chromosomes; and directly (in situ) to normal or rearranged human and mouse metaphase chromosomes. 2. The hypothesis that complex developmental disorders can be caused by DNA sequence rearrangements that give rise to deletion or abnormal expression of several genes will be tested. Pulsed-field-gradient gel electrophoresis and Southern blotting of large (100-1000kb) restriction fragments will be applied to DNA samples from patients with visible or suspected (submicroscopic) chromosome rearrangements, and specific disorders such as the Beckwith-Wiedemann syndrome, the Rett syndrome, cystic fibrosis with developmental abnormalities and others. Probes to be used will include """"""""candidate genes"""""""" and other DNA fragments that map nearby or are obtained by screening """"""""jumping libraries"""""""". Thus, a high-resolution physical map of the region involved in the clinical syndrome will be constructed that will then allow the identification of structural genomic abnormalities. The expression of genes mapped to this region can then be examined in the patients. 3. In two series of rat hepatoma x human fibroblast hybrids human liver-specific functions have been activated, and the responsible genes have been mapped. The molecular mechanisms of these gene activation phenomena will now be examined using cloned gene sequences. Parameters associated with differential gene activity will be tested, such as CpG methylation, chromatin structure (DNase I sensitivity) and the presence of trans-acting DNA binding proteins that interact with specific sequences of the cloned genes.
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