An intragenic region downstream from the dihydrofolate reductase promoter is required for replication-dependent expression.
The gene encoding dihydrofolate reductase (DHFR) is down-regulated as myoblasts withdraw from the cell cycle and commit to terminal differentiation. To localize cis-acting elements involved in regulating DHFR gene expression, the DHFR promoter and upstream region, together with differing amounts of contiguous intragenic sequence, were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The resulting fusion genes were stably transformed into muscle cells, and CAT mRNA levels were measured in proliferative myoblasts and committed myocytes. A gene consisting of the -850/+465 region of DHFR (numbers refer to distance in base pairs from transcription initiation site) fused to CAT was efficiently expressed in proliferating myoblasts and was appropriately down-regulated during commitment. A gene consisting of the -850/+60 region of DHFR fused to CAT was poorly expressed in proliferating myoblasts and was not down-regulated during commitment. When inserted between the Rous sarcoma virus promoter and CAT sequence of RSVpCAT, the +61/+465 region of the DHFR gene augmented CAT mRNA expression in muscle cell transformants but did not confer a regulated pattern of expression. Our data indicate that DHFR sequences between +60 and +465 are required but are not sufficient for replication-dependent expression. The DHFR sequences may be operating at either a transcriptional or posttranscriptional level.
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331.
E E
EE
Schmidt
R A
RA
Owen
G F
GF
Merrill
D000818:Animals; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D015971:Gene Expression Regulation, Enzymologic; D007438:Introns; D051379:Mice; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014170:Transformation, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D014764:Viral Proteins; D013762:Tetrahydrofolate Dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus