Experiments are proposed to investigate at the molecular level the mechanism of an important biological phenomenon; parental imprinting in mammals. Genetic data and the results of micromanipulation experiments have indicated that portions of the paternal and maternal genomes in mice are functionally non-equivalent, and play complementary roles during development. Thus, the must be differentially modified (""""""""imprinted"""""""") before or during gametogenesis. Available evidence suggests that imprinting could be related to some human genetic diseases and could play a role in the appearance of human embryonic tumors. Our recent results of targeted mutagenesis experiments demonstrated that the gene encoding insulin-like growth factor II (IGF-II) is parentally imprinted. Heterozygous animals carrying a paternally-derived inactivated allele and a maternally-derived intact allele (P-heterozygotes) exhibit a growth-deficiency phenotype. In contrast, when the inactivated gene is maternally derived, heterogous progeny have normal size. There is no apparent phenotypic difference between P-heterozygotes and homozygous mutant animals. Molecular analysis (based on the presence of unique molecular tags that can be used to discriminate between the mutant and wild-type alleles) has indicated that the paternal IGF-II gene is expressed, whereas the maternal allele is practically silent. The gamete-of-origin-dependent """"""""imprint"""""""" switches when the allele passes through the germ line of opposite sex. Because the IGF-II locus is currently the only known model gene system that is subject to imprinting, we propose to use as a resource the available mutant animals and ask a series of questions that should provide insights into the unknown biochemical nature of an """"""""imprint"""""""". Specifically, transgenic animal experiments have been designed to investigate whether an imprinting signal exists on DNA, and, if so, in what location. Moreover, gene transfer experiments using appropriate recipient cultured cells are proposed, which should demonstrate whether the imprint is a covalent DNA modification. If so, additional experiments with cultured cells and whole animals will demonstrate whether an imprint corresponds to methylation of cytosine residues. Finally, other aspects of simple and testable models of imprinting, which are discussed in detail will also be examined.
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