Figure 7

At least one ACS like element is required for p35 binding. (A) ARS consensus sequence. (B) 240 bp yeast S. cerevisiae ARS1 fragment containing several functional elements required for ARS activity (Boxes A, B1, B2 and B3 respectively). Several oligos spanning the entire ARS1 as indicated in the figure were designed and subsequently cloned into p21N fragment. Double stranded oligo 'a' (ARSa) does not contain any ARS like element. Oligo 'b' (ARSb) contains one 11/11 match and one 9/11 match to ACS element. Oligo 'c' (ARSc) and oligo 'd' (ARSd) contain one 9/11 match to ACS element each. In the fragment dmut, 'A's and 'T's within the core ARS were mutated to 'G's and 'C's. (C) Nucleotide sequences of ARSd and ARSdmut. (D) Gel shift assay was performed either using ARSwt (lanes 1,8), or p21N (lanes 2,3 and 9) or the constructs derived from p21N by cloning fragments a-d and dmut (lanes 4–7, 10,11) as described in (B). p35 does not bind to p21N or p21N + ARSa or p21N + ARSdmut. All the other DNA fragments showed DNA binding activity.