Naoki Miyoshi

Peg1/Mest imprinted gene on chromosome 6 identified by cDNA subtraction hybridization

Parthenogenesis in the mouse is embryonic lethal partly because of imprinted genes that are expressed only from the paternal genome. In a systematic screen using subtraction hybridization between cDNAs from normal and parthenogenetic embryos, we initially identified two apparently novel imprinted genes, Peg1 and Peg3. Peg1 (paternally expressed gene 1) or Mest, the first imprinted gene found on the mouse chromosome 6, may contribute to the lethality of parthenogenones and of embryos with a maternal duplication for the proximal chromosome 6. Peg1/Mest is widely expressed in mesodermal tissues and belongs to the alpha/beta hydrolase fold family. A similar approach with androgenones can be used to identify imprinted genes that are expressed from the maternal genome only.

Identification of an imprinted gene, Meg3/Gtl2 and its human homologue MEG3, first mapped on mouse distal chromosome 12 and human chromosome 14q.

The paternal duplication of mouse distal chromosome 12 leads to late embryonal/neonatal lethality and growth promotion, whereas maternal duplication leads to late embryonal lethality and growth retardation. Human paternal or maternal uniparental disomies of chromosome 14q that are syntenic to mouse distal chromosome 12 have also been reported to show some imprinting effects on growth, mental activity and musculoskeletal morphology. For the isolation of imprinted genes in this region, a systematic screen of maternally expressed genes (Megs) was carried out by our subtraction‐hybridization method using androgenetic and normally fertilized embryos.

Mouse Peg9/Dlk1 and human PEG9/DLK1 are paternally expressed imprinted genes closely located to the maternally expressed imprinted genes: mouse Meg3/Gtl2 and human MEG3

Genomic imprinting significantly influences development, growth and behaviour in mammals. Systematic screening of imprinted genes has been extensively carried out to identify the genes responsible for imprinted phenotypes and to elucidate the biological significance of this phenomenon. In this study, we applied DNA chip technology for isolating paternally expressed imprinted genes (Pegs). We compared the resulting expression profiles of parthenogenetic and fertilized control embryos to identify novel imprinted genes.

Subtraction-hybridization method for the identification of imprinted genes.

Imprinted genes show monoallelic expression from either the paternal or maternal genome (1,2), and their regulated expression is usually associated with the existence of parentally differentially methylated regions on genomic DNAs (3,4). Because of this, essentially two different approaches, using either cDNA or genomic DNA as starting material (5) have been developed for systematic isolation of imprinted genes. In this chapter, we describe a subtraction-hybridization method (6-8) as an example of the former approach. Both parthenogenetic embryos and androgenetic embryos (9,10) are the most suitable biological materials for the subtraction or detection of imprinted genes. However, it is difficult to obtain a large amount of such special materials because only a small number of these embryos develop to the d 10 stage (9,10). Thus, polymerase chain reaction (PCR)-based techniques, such as the differential display (11-13) and subtraction-hybridization methods, are necessary to accomplish this experiment. The subtraction-hybridization method has been successfully applied for isolation of both paternally expressed genes (Pegs) (6,14,15) and maternally expressed genes (Megs) (7), and it allows cDNA libraries to be made from a very small amount of biological material. We are convinced that this method can be applied in many fields of biological science.