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Nucleic Acids Research, 1991, Vol. 19, No. 5 1029-1034
© 1991

MOLECULAR BIOLOGY

An avian 40 KDa nucleoprotein binds preferentially to a promoter sequence containing one single pair of methylated CpG

Andre Pwlak, Margaret Bryans and Jean-Pierre Jost*

Friedrich Miescher Institut P0 Box 2543, CH-4002 Basel, Swilzerland

* To whom correspondence should be addressed

Received December 13, 1990. Revised January 29, 1991. Accepted January 29, 1991.

In vitro transcription competition with ollgonucleotides has shown that a down regulating factor can be displaced by a methylated oligonucleotide covering a specific region of the avian vitellogenln ll gene promoter (Proc. Natl. Acad. Sci USA, (1990) 87, 3047 – 3051). Gel mobility shift and competition assays show that a protein binding preferentially to methylated DNA (MDBP-2) is present in fractionated hen and rooster nuclear extracts. The protein(s) bind to the methylated sequence 5' TTCACCTTmCGCTATG AGGGGGATCATACTGG' 3' (nucleotide positions + 2 to + 32) of the vitellogenin ll promoter and not to other methylated DNA sequences. Contact points of the MDBP-2 with DNA were studied by DNA binding interference experiments with partially depurinated and depurimidinated oligonucleotides. The protein has an approximate molecular weight of 40 KDa and is mainly found in the liver and oviduct. Proteolytic clipping bandshift assays of the MDBP-2 from rooster and hen liver nuclear extracts indicate that the protein from the two sources are different. In vitro transcription experiments show that the addition of a purified nuclear fraction containing the MDBP-2 inhibits In a dose dependent manner the transcription of viteliogenin ii gene.


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