Most genes in mammals are expressed equally from the maternally and paternally inherited alleles. However, some genes are parentally imprinted, and are expressed exclusively from a single parent's allele. One consequence of imprinted genes is that a single mutation or deletion, in the expressed allele, will result in the absence of a gene product. These types of mutations are most likely involved in genetic diseases such as Beckwith-Wiedemann Syndrome, Prader-Willi Syndrome and Angelman Syndrome. In contrast, relaxation of the imprinting of two genes, IGF2 and H19, has been associated with gene activation in Wilms' tumor and may be part of a new genetic mechanism involved in the development of cancer. The objective of the studies described in this proposal is to learn how parental identity of imprinted genes is assigned. The studies will employ the H19 gene, which is expressed from the maternally derived allele in mice and humans. The recent demonstration that the inactive allele of H19 is highly methylated suggests that methylation may be involved in the mediation of imprinting. Furthermore, the analysis of H19 transgenes, which recapitulate the imprinting of the endogenous locus, indicates that cis-acting sequences can be tested for the conferral of imprinting using transgenic mice. The experiments designed in this proposal will: (i) determine if allele-specific methylation designates and perpetuates parental identity by first assessing allele-specific methylation in germ cells and the early embryo, and then mutagenizing CpG dinculeotides that remain differentially methylated and testing the altered DNA in transgenic mice; (2) use transgenic mice to identify the cis-acting sequences that cause the H19 gene to be imprinted and subsequently determine if the minimal identified sequences will imprint another mammalian gene; and (3) assess the role of strain-specific trans-acting modifiers in the imprinting of the newly derived transgenic mouse lines. These experiments will eventually allow the identification of trans-acting factors that determine imprinting. An understanding of how the imprinting process normally operates will allow greater understanding of how imprinting is altered in cancers such as Wilms' tumor.
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