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Functional Cloning Using a Xenopus Oocyte Expression System
Published on: January 30, 2016
Cloning of a cDNA encoding a human protein which binds a sequence in the c-myc gene similar to the
Y Z Stasiv1, T D Mashkova, B K Chernov
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.
Abstract:
A human cDNA clone encoding a c-myc promoter-binding protein (IRLB) was selected by screening a human fibroblast lambda gt11 phage library with the hexamer oligodeoxyribonucleotide (oligo) 5'-GGCGGGAAAAAGAACGGA, corresponding to the protein-binding element of human c-myc similar to the interferon-stimulated response element (ISRE). The lambda gt11 phage clone, encoding a fusion protein which bound the probe oligo, was used to create an strain of Escherichia coli. The deduced amino-acid sequence of the cloned protein contains a putative alpha-helix which is expected to act as the DNA-binding domain. DNase footprinting analysis and oligo-binding specificity assays showed that the cloned factor recognizes the ISRE-like element of the P2 promoter region of human c-myc.
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