High level accumulation of an aberrantly spliced human DHFR RNA species.
Cells transduced with either of two human DHFR minigenes express an RNA product which is considerably shorter than what would be predicted from the size of an unspliced transcript expressed from its DNA template. RNA blotting analysis has shown that this short transcript accumulated to exceedingly high levels which were comparable to the levels reached by the highly abundant endogenous actin mRNA, or MoMLV RNA expressed in chronically infected cells. RNA blotting, RNase mapping, primer extension, RT-PCR, and sequencing have shown that this highly expressed transcript, termed TBN, is a spliced RNA product which utilizes cryptic splice signals present in the normally spliced DHFR mRNA. Subcloning experiments have demonstrated that all the information required for the generation and high level accumulation of the TBN transcripts is encoded in the 1.6 kb DHFR DNA minigene. TBN transcripts were generated with comparable efficiency from DNA templates containing either the human ADA or the early SV40 promoters. Since neither the ADA nor the SV40 promoter are considered to be particularly "strong" promoters, this observation argues that initiation of transcription is not the rate limiting step in determining the amount of the TBN transcripts which accumulate in the cell. Insertion of a foreign sequence into the DHFR DNA minigene led to the expression and high level accumulation of a chimeric transcript, suggesting that the unusual properties of this expression system may be used for high level expression of foreign sequences. These observations offer new insights into the mechanisms which control the accumulation of translatable mRNA in the cell, and have potentially important implications for experiments involving optimization of gene expression for gene therapy applications.
Lee
S W
SW
Department of Molecular Biology, Dankook University, Seoul, Korea.
Gilboa
E
E
D016475:3T3 Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D017422:Sequence Analysis, DNA; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014157:Transcription Factors / Q000032:analysis; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000166:cytology / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014157:Transcription Factors; D012313:RNA; D013762:Tetrahydrofolate Dehydrogenase