Analysis of the mouse Dhfr/Rep-3 major promoter region by using linker-scanning and internal deletion mutations and DNase I footprinting.
The mouse dihydrofolate reductase (Dhfr) promoter region is buried within a CpG island (a region rich in unmethylated CpG dinucleotides), has a high G+C content, and lacks CAAT and TATA elements. The region contains four 48-bp repeats, each of which contains an Sp1-binding site. Another gene, Rep-3 (formerly designated Rep-1), shares the same general promoter region with Dhfr, being transcribed in the direction opposite that of Dhfr. Both genes appear to be housekeeping genes and are expressed at relatively low levels in all tissues. The 5' termini of the major Dhfr transcripts are separated from the 5' termini of the Rep-3 transcripts by approximately 140 bp. This curious structural arrangement suggested that the two genes might share common regulatory elements. To investigate the promoter sequences driving bidirectional transcription, a series of promoter mutations was constructed. These mutations were assayed by a replicating minigene system and by promoter fusions to the chloramphenicol acetyltransferase gene. Linker-scanning mutations that spanned the four repeats produced a variety of mRNA transcript phenotypes. The effects were primarily quantitative, generally reducing the abundance of transcripts for one or both genes. Some mutations affected Dhfr in a qualitative manner, such as by changing the startpoint of one of the major Dhfr transcripts or changing the relative abundance of the two major Dhfr transcripts. Additionally, protein transcription factors that bind to sequences in the mouse Dhfr/Rep-3 major promoter region, potentially affecting expression of either or both genes, were investigated by DNase I footprinting. The results indicate that multiple protein-DNA interactions occur in this region, reflecting potentially complex transcriptional control mechanisms that might modulate expression of either or both genes under different physiological conditions.
Smith
M L
ML
Department of Biology, Emory University, Atlanta, Georgia 30322.
Mitchell
P J
PJ
Crouse
G F
GF
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002872:Chromosome Deletion; D003850:Deoxyribonuclease I; D005796:Genes; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D013762:Tetrahydrofolate Dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I