TRANSCRIPTION THROUGH THE YEAST ORIGIN OF REPLICATION ARS1 ENDS AT THE ABFI BINDING-SITE AND AFFECTS EXTRACHROMOSOMAL MAINTENANCE OF MINICHROMOSOMES

When the function of origins of replication in yeast was compromised b y placing ARS sequences downstream of strong promoters, ARS activity m ight have been affected either by transcription or by an altered chrom atin configuration induced by the construct. To distinguish between th ese possibilities, derivatives of the yeast TRP1ARS1 minichromosome we re constructed that contained either the DED1 or the PET56 promoter fi ring against ARS1 (DEDARS and PETARS constructs). PETARS constructs tr ansformed yeast at high frequencies and were maintained as minichromos omes consistent with efficient ARS1 function, but DEDARS constructs tr ansformed at low frequencies and had to be rescued as minichromosomes by insertion of a second ARS (H4-ARS). Chromatin analysis revealed tha t the ARS1 regions in PETARS and H4-DEDARS constructs were indistingui shable from the ARS1 region of the host TRP1ARS1 circle showing a nucl ease sensitive region flanked by a nucleosome. However, RNA-analysis i n the ARS region showed high and low levels of transcripts in H4-DEDAR S and PETARS, respectively. Transcription elongated through the A, B1, and B2 elements and ended in B3, the binding site for ABFI. We conclu de that transcription through ARS1 and not an altered chromatin struct ure affected ARS activity in these constructs.