International Mammalian Genome Society


The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

A15 Nubp2 Gene of Mouse t-Complex Shows Random Monoallelic Expression in Single Cells

Y. Sano1, T. Shimada2, H. Nakashima2, J. Eliason3, T. Kocarek4, and M. S. H. Ko1. 1Laboratory of Genetics, National Institute on Aging, NIH, Baltimore, MD21224, USA; 2Center for Molecular Medicine and Genetics; and 3Karmanos Cancer Institute; 4Institute of Chemical Toxicology, Wayne State University, Detroit, MI 48201, USA

Nucleotide binding protein 2 (Nubp2), a gene isolated among cDNA clones from the extraembryonic tissues of E7.5 mouse embryos, is mapped to the t-complex on chromosome 17 along with other nine genes from the same cDNA collection. The gene encodes a protein with ATP/GTP-binding motif and belongs to NUBP/MRP gene family.

Since an imprinted region of the t-complex is nearby, we screened for possible imprinting using the 50% methylation assay. Southern blotting was performed on genomic DNA isolated from the spleens of 129SV/J mice. The Nubp2 showed at least one hemizygously methylated CpG site, suggesting genomic imprinting. To examine whether the gene shows true parental origin-specific monoallelic expression, F1 hybrid mice between C57BL/6J and M. Spretus were studied. In all organs examined, the Nubp2 gene showed a biallelic expression pattern. This is not consistent with genomic imprinting.

One possible alternative is that although the gene is located on an autosome, it may show random monoallelic gene expression at the single cell level, like that seen in X-chromosome inactivation. To test this, gene-specific RT-PCR and subsequent Single Nucleotide Primer Extension (SNuPE) assays were performed on single hepatocytes and fibroblast cells of F1 hybrid animals. The Nubp2 gene showed a random monoallelic expression pattern that is very similar to control results for the Hprt gene, which is X-inactivated.

In general, epigenetic phenomena that achieve dosage control fall into three categories: 1) random X chromosome inactivation in female, 2) parental specific imprinting of selected autosomal genes, and 3) random autosomal inactivation, e.g. immunoglobulin and olfactory receptor genes. Based on methylation assay and allele-specific gene expression results with single cells, we conclude that Nubp2 belongs to the third category.

 


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