Reconstruction of the 204 Mb human DMD-gene bhy homologous YAC recombination
| Autor: | P.M. Grootscholten, H. Y. Steensma, J.T. den Dunnen, Alison J. Coffey, Anthony P. Monaco, G.J.B. van Ommen, R. Butler, R. Anand, David R. Bentley, J.G. Dauwerse, A.P. Walker |
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| Rok vydání: | 1992 |
| Předmět: |
Genetics
Yeast artificial chromosome congenital hereditary and neonatal diseases and abnormalities Mutation biology Becker's muscular dystrophy Duchenne muscular dystrophy fungi Saccharomyces cerevisiae General Medicine Molecular cloning medicine.disease biology.organism_classification medicine.disease_cause medicine Homologous recombination Molecular Biology Gene Genetics (clinical) |
| Zdroj: | Human Molecular Genetics. 1:19-28 |
| ISSN: | 1460-2083 0964-6906 |
| DOI: | 10.1093/hmg/1.1.19 |
| Popis: | The human dystrophin gene, mutations of which cause Duchenne and Becker muscular dystrophy, measures 2.4 Mb. This size seriously limits its cloning as a single DNA fragment and subsequent in-vitro expression studies. We have used stepwise in-vivo recombination between overlapping yeast artificial chromosomes (YACs) to reconstruct the dystrophin gene. The recombinant YACs are mitotically stable upon propagation in haploid yeast cells. In contrast, specific combinations of YACs display a remarkable mitotic and meiotic instability in diploid cells. Non-disjunction is rare for overlapping YACs, but increases upon sporulation of diploid cells containing non-overlapping molecules. We have exploited this feature in a three-point recombination to bridge a 280 kb gap between two non-overlapping YACs for which no YAC of proper polarity existed. Our largest recombinant YAC measures 2.3 Mb and contains the entire muscle specific DMD-gene with the exception of a 100 kb region containing the in-frame exon 60. The latter segment has a high tendency to undergo deletions in multi-molecular interactions, probably due to the presence of as yet unidentified instability-enhancing sequences. Fluorescent in situ hybridizations confirmed that the 2.3 Mb DMD YAC contained Xp21-sequences only and indicated a compact tertiary structure of the DMD-gene in interphase lymphocyte nuclei. We conclude that the yeast system is a flexible, efficient and generally applicable tool to reconstruct or build genomic regions from overlapping YAC constituents. Its application to the human dystrophin gene has provided many possibilities for future studies. |
| Databáze: | OpenAIRE |
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