Four human IFNo: chromosomal genes have been isolated from a newly constructed placental DNA library in XL47. Restriction and sequencing analysis revealed that each gene had been described previously. However, one gene, SMTlll.lA, which encodes a full length IFN, is an allelic varient of a previously characterised pseudogene, thus indicating some degeneracy of the IFNa gene family. A chimaeric gene comprising the MuIFNx1 promoter (-188 to +52) and cat gene coding sequences has been constructed in vitro, enabling promoter function to be examined in mouse cells. Reproducible polyrI.rC mediated induction of CAT expression from the MuIFNx1 promoter has been demonstrated in pools of stably transfected, but not transiently transfected, L929 cells. Monitoring mRNA production revealed the transient accumulation of correctly initiated hybrid gene transcripts which precede optimum CAT production. Aspects of the structure/function relationship of the MuIFNx1 promoter have been investigated by oligonucleofide site directed mutagenesis. Comparative studies of IFNx promoter sequences identified prospective regulatory regions for mutagenesis. Quantitative CAT assays have been employed for promoter assessment. Additionally, the construction of a pseudogene comprising the wildtype MuIFNa1 promoter linked to an internally deleted cat gene has enabled both mutant and wildtype promoters functioning simultaneously in the same cell population to be assessed by S-1 nuclease protection studies using a common probe. Such studies have revealed three distinct cis-acting regions implicated in MuIFNx1 promoter function. Two are located upstream of the TATA box, defined by mutations at -87 and between -66 and -33 respectively. These reduce promoter activity 2 to 5 fold. The third, is defined by a mutation at +14, within the untranslated leader sequence. This enhances activity 2 to 3 fold. A deletion derivative of the MuIFNx1 promoter containing only 94bp of upstream sequence is inactive. Cis-activation by the Mo-MuSV enhancer restores inducibility to this promoter whereas the intact MuIFNx1 promoter is refractory to this element. This suggests that distinct cis-acting sequences dictate the efficiency and regulation of MuIFNx1 gene transcription.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:674161 |
| Date | January 1986 |
| Creators | Bartholomew, C. |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/35240 |
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