IDENTIFICATION AND CHARACTERIZATION OF THE H19 LOCUS WHICH IS UNDER THE CONTROL OF THE MURINE RAF AND RIF GENES

The mouse $\alpha$-fetoprotein (AFP) gene is under the control of two loci, raf and Rif, in the mouse liver. The raf locus is thought to be responsible for the repression of the AFP locus after birth, and the Rif gene is part of the mechanism that induces the expression of the AFP gene during liver regeneration. The experiments described in this thesis establish that raf and Rif are not specific for the AFP locus but they control at least another murine structural locus, H19. The H19 locus was identified by constructing a fetal liver cDNA library and examining fetal liver specific clones for the diagnostic levels in C3H/He, BALB/cJ and C57BL/6 inbred mouse strain. The H19 gene is expressed in visceral endoderm, fetal liver and fetal gut, tissues that express also high levels of AFP. In addition, the H19 gene is expressed in fetal and adult myocardium and skeletal muscle. These tissues do not express detectable levels AFP. The expression of the H19 gene in myocardium and skeletal muscle is not under the control of the raf locus. Northern blot analysis was used to examine the expression of the H19 gene in cells in culture. The structure and the sequences of the H19 gene, which is highly conserved among mammalian species, were determined and compared to those of the AFP gene. The results suggest that although the AFP and H19 genes do not share a common, evolutionary ancestor, they contain in their $5\sp\prime$ flanking regions homologous sequences which could potentially mediate the coordinated regulation of the two genes. In order to study in detail the regulatory sequences of the H19 gene, constructs that contain the entire gene along with various amounts of $5\sp\prime$ and $3\sp\prime$ flanking sequences were introduced into cells into culture and the transcriptional efficiency of the introduced sequences were analyzed by Northern blot or S1 protection analysis. These experiments strongly suggest that both $5\sp\prime$ and $3\sp\prime$ flanking sequences play an important role in the regulation of the expression of the H19 gene