Differential effects of simple repeating DNA sequences on gene expression from the SV40 early promoter.
The influence of simple repeat sequences, cloned into different positions relative to the SV40 early promoter/enhancer, on the transient expression of the chloramphenicol acetyltransferase (CAT) gene was investigated. Insertion of (G)29.(C)29 in either orientation into the 5'-untranslated region of the CAT gene reduced expression in CV-1 cells 50-100 fold when compared with controls with random sequence inserts. Analysis of CAT-specific mRNA levels demonstrated that the effect was due to a reduction of CAT mRNA production rather than to posttranscriptional events. In contrast, insertion of the same insert in either orientation upstream of the promoter-enhancer or downstream of the gene stimulated gene expression 2-3-fold. These effects could be reversed by cotransfection of a competitor plasmid carrying (G)25.(C)25 sequences. The results suggest that a G.C-binding transcription factor modulates gene expression in this system and that promoter strength can be regulated by providing protein-binding sites in trans. Although constructs containing longer tracts of alternating (C-G), (T-G), or (A-T) sequences inhibited CAT expression when inserted in the 5'-untranslated region of the CAT gene, the amount of CAT mRNA was unaffected. Hence, these inhibitions must be due to posttranscriptional events, presumably at the level of translation. These effects of microsatellite sequences on gene expression are discussed with respect to recent data on related simple repeat sequences which cause several human genetic diseases.
Department of Biochemistry, School of Medicine, University of Alabama, Birmingham 35294.
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D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis*; D002522:Chlorocebus aethiops; D004276:DNA, Satellite / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
D004276:DNA, Satellite; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase