Meiotic recombination between yeast artificial chromosomes yields a single clone containing the entire BCL2 protooncogene.
The common translocation found in human follicular lymphoma, t(14;18)(q32;q21), results in deregulation of the BCL2 protoonocogene. The isolation of the intact gene would provide an essential substrate to analyze the molecular basis of this malignancy. Pulsed-field gel electrophoresis suggested that this three-exon gene was several hundred kilobases (kb) long. Therefore, a library of yeast artificial chromosome (YAC) clones was screened to isolate the intact BCL2 gene. Two clones, yA85B6 (200 kb) and yB206A6 (700 kb), were isolated by using polymerase chain reaction (PCR) assays specific for exon I/II and exon III, respectively. However, neither YAC contained the entire BCL2 locus. Since the two YACs were found to overlap by 60 kb, we sought to take advantage of the high recombination frequency in yeast and induce physical recombination between the two clones. Cells containing each YAC were mated and induced to undergo meiotic division and sporulation. Analysis of the resulting tetrads revealed a spore containing a single recombinant YAC of 800 kb. PCR assays and Southern blotting demonstrated that this recombined YAC contained the entire approximately 230-kb BCL2 gene. Furthermore, probe order was conserved and there was no evidence of overt rearrangements or deletions. These results indicate the feasibility of reconstructing large genomic segments with overlapping YAC clones to study genes spanning hundreds of kilobases.
Silverman
G A
GA
Department of Pediatrics, University School of Medicine, St. Louis, MO 63110.
Green
E D
ED
Young
R L
RL
Jockel
J I
JI
Domer
P H
PH
Korsmeyer
S J
SJ
D015825:Chromosomes, Fungal; D002883:Chromosomes, Human, Pair 14; D002887:Chromosomes, Human, Pair 18; D003001:Cloning, Molecular; D003434:Crossing Over, Genetic; D006801:Humans; D008224:Lymphoma, Follicular / Q000235:genetics; D008540:Meiosis; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2; D011519:Proto-Oncogenes; D011995:Recombination, Genetic; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D014178:Translocation, Genetic
D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011505:Protein-Tyrosine Kinases