Meiotic Recombination Between Yeast Artificial Chromosomes Yields a Single Clone Containing the Entire BCL2 Protooncogene

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 87; no. 24; pp. 9913 - 9917
• Main Authors: Silverman, Gary A., Green, Eric D., Young, Robert L., Jockel, Jennifer I., Domer, Peter H., Korsmeyer, Stanley J.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.12.1990; National Acad Sciences
• Subjects: Biological and medical sciences; Chromosomes, Fungal; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 18; Cloning, Molecular; Crossing Over, Genetic; DNA; DNA probes; Exons; Fundamental and applied biological sciences. Psychology; Genes; Genes. Genome; Genomics; Humans; Introns; Libraries; Lymphoma, Follicular - genetics; Meiosis; Molecular and cellular biology; Molecular genetics; Polymerase Chain Reaction; Protein-Tyrosine Kinases - genetics; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogenes; Recombination, Genetic; Restriction Mapping; Saccharomyces cerevisiae - cytology; Saccharomyces cerevisiae - genetics; Spores; Translocation, Genetic; Yeasts; Human; Recombination; C-Onc gene; Chromosome DNA; Meiosis; Method; Yeast artificial chromosome; Molecular cloning
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.87.24.9913

The common translocation found in human follicular lymphoma, t(14;18)(q32;q21), results in deregulation of the BCL2 protooncogene. 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 ≈ 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.