HOW THE DYT1 GENE WAS FOUND

     Finding the DYT1 gene involved a chromosome walking strategy requiring several stages and many different procedures. The first step that was used to find this gene was the construction of an ordered collection of cloned chromosome 9 fragments spanning the 150-kb target region between markers D9S2161 and D9S63. In the May of 1997 study, a YAC (yeast artificial chromosome) was constructed for a region, which included this 150kb region, and found four YACs spanning this target region (8H12, 183D9, 251H9, and 22A4) (Ozelius, 1997). An array of cosmids were isolated from two human, chromosome-9 specific libraries, the Lawrence Library and the Los Alamos Library, and were initially screened with these YACs. Cosmids are bacteriophage lamda vectors that can contain cloned DNA up to 45,000 base pairs in length. The cosmids, from each end, that were used to initiate the cosmid walk were LL09NC0150H11 on the centromeric side, which hybridized to YAC 8H12, and 37F5LA and 18D5LA on the telomeric end, which hybridized to the other three YACs. Starting with the end sequences on both ends, a cosmid walk was initiated until the cosmids spanned the whole region (Fig 2). This collection of adjacent, overlapping cosmids is called a contig.
 

This entire process is chromosome walking. An overlapping subset of eleven
cosmids was found after analysis with the restriction enzymes EcoR1, Xhol, and Notl. Using gene markers, the cosmid end clones, and the cloned exons and oligonucleotides from unique regions, the cosmids were aligned by size and hybridization patterns spanning the 150-kb region.


 

      The second step was performing exon trapping so genes could be identified in this target region. Exon trapping is a method that rapidly and efficiently isolates exon sequences from cloned genomic DNA (cosmids) by way of selection for functional splice sites. Exon trapping involves cloning genomic DNA into an exon trap vector containing splice sites, a promoter, and polyadenylation signals. The promoter facilitates high levels of transcription. The purified recombinant DNA is then transfected into COS-7 cells, transcribed, and then spliced. After extraction of total RNA and the generation of cDNA, two rounds of PCR (polymerase chain reaction), using vector specific primers permit amplification of the trapped exons (fig. 3).

The PCR products are then cloned to generate an exon trap library, or they are used as complex probes of cDNA libraries. By this approach, 28 unique putative exons were trapped from cosmids in the target region. These cloned exon fragments were used to screen human fetal and adult cDNA libraries by colony hybridization. Five cDNAs were found : DQ1, DQ2, DQ3, DQ4, and DQ5. These cDNAs were extended and sequenced in multiple clones and aligned across the cosmid contig.


 

       The third step was sequence analysis of cDNA and genomic DNA and mutational screening. The four cDNAs: DQ1, DQ2, DQ3, and DQ4, were sequenced and then screened for heterozygous mutations in affected individuals with torsion dystonia. DQ5 was excluded because only the 3' untranslated region overlapped the critical interval. These cDNAs were screened for mutations by PCR (polymerase chain reaction) of 100-300-bp fragments, followed by SSCP (single-stranded conformation polymorphism). A number of sequence variations were found but all these nucleotide changes except one, the GAG deletion in DQ2, were confirmed as polymorphisms by their presence in control samples..


 

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