Genetic transfer and expression of reconstructed yeast artificial chromosomes containing normal and translocated BCL2 proto-oncogenes.

Abstract

The goal of this study was to determine whether it will be feasible to study the expression of a large, human gene, such as the BCL2 proto-oncogene, by DNA transfection. The BCL2 proto-oncogene is 230 kb in size and is deregulated in tumor cells by translocation into the immunoglobulin heavy-chain locus. Yeast artificial chromosomes (YACs) containing the human BCL2 gene were altered by homologous recombination in Saccharomyces cerevisiae to yield replicas of the normal and translocated alleles. Constructions containing either allele and ranging in size from 360 to 800 kb were integrated stably into a mouse tumor line. Fifty-eight percent of the clones contained a copy of the entire YAC insert. Over 50% of these clones expressed appropriate levels of human BCL2 RNA and protein. These studies suggested that the expression of large human genes and their pathologic rearrangements can be studied by transfection techniques employing YACs propagated in S. cerevisiae.

Cloning of the entire FL11 gene, disrupted by the Ewing’s Sarcoma translocation breakpoint on 11q24, in a yeast artificial chromosome

“…The human c-abl oncogene (ABL1), located at 9q34, where the consistent t(9;22)(q34;ql 1) translo cation in chronic myelocytic leukemia occurs, has its most 5' exon located 200 kb upstream of the remaining c-abl exons and shows a remarkable genomic structure, and transcription and splicing organization (Bernards et al, 1987). The Bcl2 proto oncogene (BCL2), identified at the breakpoint in lymphoma cells bearing the t( 14; 18) translocation, spans 230 kb of DNA (Silverman et al, 1993). The molecular analysis of YAC B45C11 has shown that FLI1 spans about 120 kb of DNA and will greatly expedite the structural study of this gene, disrupted by the ES and PNE chromosomal translocation.…”

“…Interestingly, FLI 1 is a very large gene: other genes, previously localized at the site of consistent chromosomal translocations, have been shown to span large segments of DNA and to have complex organizations (Bernards ct al., 1987;Silverman et al, 1993). The human c-abl oncogene (ABL1), located at 9q34, where the consistent t(9;22)(q34;ql 1) translo cation in chronic myelocytic leukemia occurs, has its most 5' exon located 200 kb upstream of the remaining c-abl exons and shows a remarkable genomic structure, and transcription and splicing organization (Bernards et al, 1987).…”

SERPINB12 Is a Novel Member of the Human ov-serpin Family That Is Widely Expressed and Inhibits Trypsin-like Serine Proteinases

“…Protein bands were visualized after staining in a solution containing 0.25% Coomassie Brilliant Blue R-250, 45% methanol, and 10% acetic acid. For immunodetection, proteins separated by SDS-PAGE were electroblotted at 100 V for 1 h at 4°C onto reinforced nitrocellulose (NitroPure, Osmonics Inc., Westborough, MA) as described (23). The transfer buffer was 25 mM Tris-base, pH 8.0, and 190 mM glycine, pH 8.3.…”

Squamous Cell Carcinoma Antigen 2 Is a Novel Serpin That Inhibits the Chymotrypsin-like Proteinases Cathepsin G and Mast Cell Chymase

“…Proteins separated by SDS-PAGE were electroblotted at 100 V for 1 h at 4°C, onto reinforced nitrocellulose (NitroPlus, Micron Separations, Inc., Westborough, MA) as described (32). The transfer buffer was 25 mM Tris base, pH 8.0, 190 mM glycine (pH 8.3).…”