Isolation, characterization and targeted disruption of mouse ppia: cyclophilin A is not essential for mammalian cell viability.
Cyclophilins (CyPs) are a family of proteins found in organisms ranging from prokaryotes to humans. These molecules exhibit peptidyl-prolyl isomerase activity in vitro, suggesting that they influence the conformation of proteins in cells. CyPs also bind with varying affinities to the immunosuppressive drug cyclosporin A (CsA), a compound used clinically to prevent allograft rejection. The founding member of the family, cyclophilin A (CyPA), is an abundant, ubiquitously expressed protein of unknown function that binds with nanomolar affinity to CsA. Here, we describe the isolation and characterization of mouse Ppia (mPpia), the gene encoding CyPA. Ppia was isolated using a PCR screen that distinguishes the expressed gene from multiple pseudogenes present in the mouse genome. mPpia consists of 5 exons and 4 introns spanning roughly 4.5 kb and maps to chromosome 11 near the centromere. Sequence analysis of a 369-bp fragment from the proximal promoter region of mPpia revealed the presence of a TATA box and sites recognized by several transcriptional regulators, including Sp1, AP-2, GATA factors, c-Myb, and NF-IL-6. This region is sufficient to drive high-level reporter gene expression in transfected cells. Both copies of Ppia were disrupted in murine embryonic stem (ES) cells via gene targeting. Ppia(-/-) ES cells grow normally and differentiate into hematopoeitic precursor cells in vitro, indicating that CyPA is not essential for mammalian cell viability.
Colgan
J
J
Department of Microbiology, Department of Medicine, Columbia University College of Physicians and Surgeons, 701 West 168th Street, New York, New York, 10032, USA.
Asmal
M
M
Luban
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics; D002874:Chromosome Mapping; D002875:Chromosomes / Q000235:genetics; D021984:Cyclophilin A / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015342:DNA Probes; D005091:Exons; D005796:Genes / Q000235:genetics; D020043:Genes, Essential; D007438:Introns; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009115:Muridae; D016296:Mutagenesis; D011401:Promoter Regions, Genetic / Q000235:genetics; D011544:Pseudogenes; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D015342:DNA Probes; D011993:Recombinant Fusion Proteins; D004247:DNA; D008156:Luciferases; D021984:Cyclophilin A