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

D28 Comparative Sequencing of Mouse X Chromosome: Generating a Sequence-Ready Map F8 to Dmd

Straw, R. N. A.1, Botcherby, M.1, Clarke, D.1, Weston, P.1, Greystrong, J.1, Bate, R.2, Denny, P.2, Rhodes, M.1, Campbell, R.1, Brown, S. D. M.2. 1MRC Human Genome Mapping Project Resource Centre, Hinxton, Cambridge, CB10 1SB, UK; 2 MRC UK Mouse Genome Centre and Mammalian Genetics Unit, Harwell, Oxon, UK

The 2-3 Mb region that spans from F8 (Factor VIII) to Dmd (Duchenne muscular dystrophy) on mouse X chromosome crosses two evolutionary breakpoints with F8 and Dmd residing on human Xq28 and Xp21 respectively. Recent mapping (1) has also placed Tbl1 (Transducin beta-like 1) in-between F8 and Dmd, which maps to human Xp22.3. Current opinion is that this region generally becomes gene-poor as you move distally towards Dmd.

A genetic map has been created across the complete region using the EUCIB Backcross (http://www.hgmp.mrc.ac.uk/MBx/MBxHomepage.html). Work is currently focused on generating a sequence-ready map between F8 and Dmd, with particular interest in identifying and ultimately sequencing across the evolutionary breakpoints. A physical contig has been generated around Tbl1 and significant portions of this region are being sequenced.

Due to the paucity of available genes within the F8 to Dmd region, microsatellites and STSs are the marker types used to anchor clones to the map. A PCR based approach has been employed to screen pools generated from the RPCI Mouse PAC library, firstly with MIT microsatellites, then with primers designed from clone end-sequence data. In addition filters have been screened by hybridisation using labelled probes generated from short over-lapping oligos (Overgos). Initial analysis of the end-sequence data suggests that the region is high in mouse L1 repeats. Clones are fingerprinted as an aid to generating a minimal tiling path using a HindIII digestion followed by agarose separation and visualisation using SYBR Green. The fingerprints are analysed using IMAGE (2) and contigs constructed using FPC (3).

This work is part of a large collaboration aiming to sequence large contiguous regions within the 5 Mb Ids (Iduronate 2-sulphatase) to Dmd region of mouse X chromosome (see abstract by Bate et al), with the intention of investigating the benefits of comparative sequencing as a strategy for the identification of coding regions and regulatory elements.

(1) C. M. Disteche, M. B. Dinulos, M. T. Bassi, R. W. Elliott and E. I. Rugarli. Mamm. Genome 9, 1062-1064 (1998).

(2) J. Sulston, F. Mallet, R. Durbin and T. Horsnell. Image analysis of restriction enzyme fingerprint autoradiograms. Comput. Appl. Biosci. 5, 101-106 (1989).

(3) C. Soderlund, I. Longden and R. Mott. FPC: a system for building contigs from restriction fingerprinted clones. Comput. Appl. Biosci. 13, 523-535 (1997).

 


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