Tandemly repeated hexamer sequences within the beta interferon promoter can function as an inducible regulatory element in activation by the adenovirus E1B 19-kilodalton protein.

The expression of the human beta interferon (IFN-beta) gene was activated by the adenovirus E1B-19K protein. The sequence within the IFN-beta promoter which is related to the activation was analyzed by chloramphenicol acetyltransferase (CAT) assay. The repeated hexamer units, present within the region between -109 and -65 relative to the cap site, were required for the activation of the IFN-beta gene by the E1B-19K protein. The (AAGTGA)8 region, as a typical hexamer of the consensus sequences, was tested for function in the activation by the E1B-19K protein. When the hexamer (AAGTGA)4-8 was inserted upstream of several reporter genes (such as p55cat, pdlE1A-CAT, and pE1B-CAT) which were inefficiently stimulated, the CAT activities of these fusion genes were efficiently stimulated by the E1B-19K protein. These results show that the tandemly repeated hexamer sequences within the IFN-beta promoter can function as an inducible regulatory element in the activation by the adenovirus E1B 19K protein.     

Delimitation of a DNA sequence which confers inducibility by glucocorticoid hormones

A chimeric long terminal repeat-thymidine kinase (LTR-tk) gene has been used to define the sequence requirements for glucocorticoid induction of gene expression. The original LTR-tk gene contains an entire mouse mammary tumor virus (MMTV) LTR preceding the tk gene. This gene can be expressed in a hormone-responsive fashion upon transfection into L tk--cells to produce a chimeric LTR-tk mRNA. Stepwise deletion of nucleotide sequences 5' of the viral RNA initiation site revealed that 202 nucleotides upstream of the viral cap site are sufficient for the hormonal regulation. Deletion of 5' sequences up to 59 nucleotides upstream of the viral cap site abolished RNA initiation in the LTR and hormonal induction.     

Tissue-specific regulation of a chicken delta-crystallin gene in mouse cells: involvement of the 5' end region.

A cloned delta-crystallin gene of the chicken is preferentially expressed in lens cells after introduction into various mouse tissues. The level of expression in the lens epithelium is 20 times higher than in fibroblasts. Taking advantage of this system, we attempted to define regulatory regions of the delta-crystallin gene using a variety of deletion and substitution mutants. The results indicate that tissue-specific regulation of the delta-crystallin gene is mediated by the 5' end region of the gene; sequences upstream from -93 are not required for expression and sequences downstream from +58 are not involved in tissue specificity. The high expression in lens cells requires 5' flanking sequences of 80-bp long from the cap site, whereas the low expression in fibroblasts requires an additional 12 bp upstream sequence. Expression of both types is lost in a mutant with only 51 bp of the 5' flanking sequence. Thus, fine deletion analysis demonstrated that expression in lens cells and expression in fibroblasts are distinct not only in level but in regulation.     

A specific domain of the adenovirus EIV promoter is necessary to maintain susceptibility of the integrated promoter to EIA transactivation.

We constructed a series of mutations that delete sequences in the promoter region of the early-region IV (EIV) promoter of adenovirus type 5. We fused these promoter mutations to the coding sequences of either the chloramphenicol acetyltransferase or the dihydrofolate reductase (DHFR) gene and tested the ability of a cotransfected EIa gene to stimulate EIV expression. All of the mutations tested were stimulated in these assays, implying that no specific sequence is required for stimulation. Two mutant promoters, deleted for either the TATA box or the region residing between -39 and -177 upstream from the cap site of EIV mRNA, did show a reduced level of stimulation by the EIa products. To assess the effects of the EIA gene products on expression from an EIV promoter integrated into the chromosome, we isolated CHO cell lines containing EIV-DHFR chimeric genes. After introduction of the EIa gene with a second selectable marker, expression from all mutant EIV-DHFR genes was increased. Surprisingly, one mutant promoter, deleted for sequences between -39 and -177, lost the ability to respond to the EIa region on passage of cells, although deletions in any part of the region still retained this ability. These results demonstrate that multiple elements residing between -39 and -177 in the EIV promoter are necessary to maintain susceptibility of the integrated promoter to regulation.