PMID: 7544133Aug 1, 1995Paper

Use of reverse ligation-PCR to identify transcriptional start sites in GC-rich TATA-less genes: application to the rat IGFBP-2 gene

DNA and Cell Biology
Y R Boisclair, A L Brown

Abstract

TATA-less genes are often GC-rich in the region of transcriptional initiation and the corresponding mRNAs are prone to the formation of secondary structure. These properties have made it difficult to determine unambigously the start sites of transcription for some of these genes by conventional assays such as primer extension and nuclease protection. Using the TATA-less rat IGFBP-2 gene, we demonstrate that tobacco acid pyrophosphatase-reverse ligation polymerase chain reaction (TAP-RLPCR), a novel and sensitive assay, can be used to map the start sites of these genes. First, the validity of TAP-RLPCR was demonstrated by mapping the transcription start site of the rat insulin-like growth factor 1 (IGFBP-1) gene to the correct position (nucleotides -173 relative to ATG, +1). Using total RNA obtained from the rat liver cell line BRL-3A, the transcription start sites of the rat IGFBP-2 gene were mapped to a narrow cluster extending from nucleotides -86 to -90 (ATG, +1), 39 bp downstream of three adjacent GC boxes that are essential to the transcriptional activity of the gene. The assay was also used to map the start sites of a luciferase reporter gene driven by the fragment -1,295 to -32 of the rat IGFBP-2 promoter after transfect...Continue Reading

References

Jun 30, 1992·Biochemical and Biophysical Research Communications·T G UntermanR G Goswami
Feb 22, 1991·Cell·T M DeChiaraA Efstratiadis
Apr 1, 1990·Molecular Endocrinology·C McKeonS I Taylor
Jan 1, 1993·Vitamins and Hormones·M M Rechler
Jan 1, 1994·Molecular and Cellular Biology·R JavaheryS T Smale
Dec 15, 1993·Proceedings of the National Academy of Sciences of the United States of America·S L AckermanC Y Yeung
Apr 11, 1993·Nucleic Acids Research·M Fromont-RacineT Grange
Jun 1, 1993·Proceedings of the Society for Experimental Biology and Medicine·R Kollmar, P J Farnham

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Citations

Sep 17, 1996·Proceedings of the National Academy of Sciences of the United States of America·Y R BoisclairG T Ooi
Jan 5, 2002·DNA Sequence : the Journal of DNA Sequencing and Mapping·F CarrariN D Iusem
Jan 1, 1995·Progress in Growth Factor Research·G T OoiY R Boisclair

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