Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination.
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 ch...
| Hauptverfasser: | , , , , , , , , , |
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| Format: | Journal article |
| Sprache: | English |
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1992
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| _version_ | 1826257661913464832 |
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| author | Den Dunnen, J Grootscholten, P Dauwerse, J Walker, A Monaco, A Butler, R Anand, R Coffey, A Bentley, DR Steensma, H |
| author_facet | Den Dunnen, J Grootscholten, P Dauwerse, J Walker, A Monaco, A Butler, R Anand, R Coffey, A Bentley, DR Steensma, H |
| author_sort | Den Dunnen, J |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-06T18:21:41Z |
| format | Journal article |
| id | oxford-uuid:068205d7-c87e-428f-9a14-ce37ddb352ad |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T18:21:41Z |
| publishDate | 1992 |
| record_format | dspace |
| spelling | oxford-uuid:068205d7-c87e-428f-9a14-ce37ddb352ad2022-03-26T09:03:01ZReconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:068205d7-c87e-428f-9a14-ce37ddb352adEnglishSymplectic Elements at Oxford1992Den Dunnen, JGrootscholten, PDauwerse, JWalker, AMonaco, AButler, RAnand, RCoffey, ABentley, DRSteensma, HThe 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. |
| spellingShingle | Den Dunnen, J Grootscholten, P Dauwerse, J Walker, A Monaco, A Butler, R Anand, R Coffey, A Bentley, DR Steensma, H Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination. |
| title | Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination. |
| title_full | Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination. |
| title_fullStr | Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination. |
| title_full_unstemmed | Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination. |
| title_short | Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination. |
| title_sort | reconstruction of the 2 4 mb human dmd gene by homologous yac recombination |
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