|Genome Sequencing Section
DOE Human Genome Program Contractor-Grantee Workshop
2. Genomic Sequencing of 3 Mb of Human Chromosome 16p13.3 Containing 4 Disease Genes
M.O. Mundt, D.O. Ricke, D.C. Bruce, A.C.
Munk, D.L. Robinson, M.D. Jones, J.M. Buckingham, L.A. Chasteen, E.H. Saunders,
L.S. Thompson, L.A. Goodwin, A.L. Williams, J.L. Longmire, P.S. White,
L.L. Deaven, and N.A. Doggett
We have nearly completed genomic sequencing of a 3.0 Mb cosmid/P1 contig of the human chromosome region in 16p13.3 extending from the tuberous sclerosis disease (TSC2) locus to the CREB binding protein (CREBBP) locus [responsible for Rubinstein-Taybi Syndrome and implicated in acute myeloid leukemias associated with translocations t(8;16)(p11;p13.3) and t(11;16)(q23;p13.3)]. This contig also encompasses the polycystic kidney disease 1 (PKD1), the familial Mediterranean fever gene (MEFV) and the syntenic breakpoint between mouse chromosomes 16 and 17. The average overlap between clones in the contig is about 25%. Our earlier sample sequencing (SASE) of this region had revealed that it is gene rich and G+C rich (>50% G+C), with the gene density approaching one gene/10 kb in some stretches. These observations are consistent with the cytogenetic designation of 16p13.3 as a G+C rich "T" band (Dutrillaux, 1973; Holmquist, 1992). Our strategy for sequencing involved nebulization to randomly break DNA, size selection of 3 kb fragments, double adapter cloning into bluescript KS+ plasmid, and sequencing of both ends to 6X random sequencing coverage. Sequencing reactions were predominately Big dye terminators (ABI). Assembly of sequence contigs was assisted by the inherent relationship of the end sequences being approximately 3 kb apart. Closure and finishing was achieved by a combination of primer walking, longer reads, and alternate chemistry reactions. Sequence analysis and annotation is semi-automated with use of the SCAN program (developed by Ricke). We have achieved 100% closure of all 58 clones which we have attempted to sequence from this region. Three gaps remain but clones have now been found which span these. One of these "gaps" in the cosmid contig map is in the same region of a breakpoint cluster in the CREB binding protein gene, which occurs in leukemias. This region was stably maintained in BACs however. The maximum G+C content found in a finished clone is 57%. Alu content has also been high, with up to 30 Alu's in a finished cosmid. Supported by the US DOE, OBER under contract W-7405-ENG-36.
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