YAC fragmentation with repetitive and single-copy sequences: detailed physical mapping of the presenilin 1 gene on chromosome 14.
We constructed new LYS2 fragmentation vectors that allow direct acentric and centric fragmentation of yeast artificial chromosomes (YACs) and selection of fragmented YACs in yeast strain AB1380. The fragmentation vectors were used efficiently with repetitive (e.g., Alu), low-copy (e.g., CA-repeats) and single-copy (e.g., exons) sequences. High recombination efficiencies were obtained in fragmenting two different CEPH YACs with the Alu consensus sequence as target sequences for homologous recombination. Analysis of the acentric Alu fragmentation panel of 788H12, containing the presenilin 1 (PSEN1) gene for familial Alzheimer's disease (AD), indicated that high-resolution YAC fragmentation panels covering the entire parent YAC are obtained. Also, marker content analysis of the fragmentation panel indicated that fragmented YACs were propagated stably without rearrangements. The same fragmentation vectors were used efficiently for fragmentation of 788H12 with unique sequences, i.e., exons 3 and 12 of PSEN1 and D14S77, a polymorphic CA repeat, as target sequences. Together, our YAC fragmentation data of 788H12 provided a size estimate for the coding region of PSEN1 of 60kb and a more precise localization of D14S77 at 25kb upstream of PSEN1.
Del-Favero
J
J
Department of Molecular Genetics, Laboratory of Psychogenetics, Flanders Interuniversity Institute for Biotechnology (VIB), University of Antwerp (UIA), Department of Biochemistry, Antwerp, Belgium.
Goossens
D
D
Van den Bossche
D
D
Van Broeckhoven
C
C
D020087:Alu Elements / Q000235:genetics; D000544:Alzheimer Disease / Q000235:genetics*; D002874:Chromosome Mapping; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D002883:Chromosomes, Human, Pair 14 / Q000235:genetics*; D016521:Electrophoresis, Gel, Pulsed-Field; D005819:Genetic Markers / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D018895:Microsatellite Repeats / Q000235:genetics; D020161:Physical Chromosome Mapping; D053764:Presenilin-1
D005819:Genetic Markers; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D053764:Presenilin-1