Specific signals at the 3' end of the DHFR gene define one boundary of the downstream origin of replication.
The Chinese hamster dihydrofolate reductase (DHFR) origin of replication consists of a 55-kb zone of potential initiation sites lying between the convergently transcribed DHFR and 2BE2121 genes. Two subregions within this zone (ori-beta/ori-beta' and ori-gamma) are preferred. In the DHFR-deficient variant, DR8, which has deleted a 14-kb sequence straddling the 3' end of the DHFR gene, early-firing origin activity in the downstream ori-beta/ori-beta' and ori-gamma regions is completely suppressed. We show that the critical deleted sequences reside within a 168-bp segment encompassing the intron 5/exon 6 boundary, exon 6, 54 bp of the 3' untranslated region (UTR), but not the three natural polyA sites. In wild-type cells, this sequence efficiently arrests transcription in a region a few kilobases downstream, which coincides with the 5' boundary of the replication initiation zone. In DR8, DHFR-specific transcripts efficiently use an alternative sixth exon (6c) and polyA signals near the middle of the former intergenic region to process primary transcripts. However, transcription proceeds to a position almost 35 kb downstream from these signals, and replication initiation can only be detected beyond this point. When the wild-type 168-bp 3' element is inserted into DR8 at the same position as alternative exon 6c, transcription is arrested efficiently and initiations occur almost immediately downstream. Thus, the normal 3' end of the DHFR gene constitutes a boundary element not only for the gene but also for the local origin of replication.
Mesner
Larry D
LD
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908-0073, USA.
Hamlin
Joyce L
JL
D024509:3' Flanking Region / Q000235:genetics*; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus / Q000235:genetics*; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional; D040461:Gene Components; D009693:Nucleic Acid Hybridization; D011995:Recombination, Genetic / Q000235:genetics; D018741:Replication Origin / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics* / Q000502:physiology
D017931:DNA Primers; D013762:Tetrahydrofolate Dehydrogenase