Cyclic amplification and selection of targets (CASTing) for the myogenin consensus binding site - PubMed (original) (raw)

Cyclic amplification and selection of targets (CASTing) for the myogenin consensus binding site

W E Wright et al. Mol Cell Biol. 1991 Aug.

Abstract

The consensus binding site for the muscle regulatory factor myogenin was determined from an unbiased set of degenerate oligonucleotides using CASTing (cyclic amplification and selection of targets). Stretches of totally random sequence flanked by polymerase chain reaction priming sequences were mixed with purified myogenin or myotube nuclear extracts, DNA-protein complexes were immunoprecipitated with an antimyogenin antibody, and the DNA was amplified by polymerase chain reaction. Specific binding was obtained after four to six cycles of CASTing. The population of selected binding sites was then cloned, and a consensus was determined from sequencing individual isolates. Starting from a pool with 14 random bases, purified myogenin yielded a consensus binding site of AACAG[T/C]TGTT, while nuclear extracts retrieved the sequence TTGCACCTGTTNNTT from a pool containing 35 random bases. The latter sequence is consistent with that predicted from combining an E12/E47 half-site (N[not T]CAC) with the purified myogenin half-site ([T/C] TGTT). The presence of paired E boxes in many of the sequences isolated following CASTing with nuclear extracts proves that myogenin can bind cooperatively with other E-box-binding factors.

PubMed Disclaimer

Similar articles

Cited by

References

    1. Science. 1990 Nov 23;250(4984):1104-10 - PubMed
    1. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1089-93 - PubMed
    1. J Biol Chem. 1988 Oct 15;263(29):14724-31 - PubMed
    1. Gene. 1988 Jul 15;67(1):31-40 - PubMed
    1. Genes Dev. 1989 May;3(5):628-40 - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources