Cis effect

Abstract: Activity of the murine interleukin-4 (IL-4) promoter was localized to several cis-acting elements present within the first 300 bp from the transcriptional initiation site. Five repeated elements, P0 to P4, that share the common consensus ATTTTCCNNT were located between -40 and -250, and each was shown to interact with the T-cell-specific factor NF(P). These distinct P sites appear functionally interchangeable and cooperatively confer cyclosporin A-sensitive and ionomycin-inducible promoter activity. NF(P) may be closely related to the cytoplasmic component of NF-AT (nuclear factor of activated T cells), a T-cell-specific factor essential for IL-2 gene transcription, as judged from indistinguishable molecular weights and protease fragmentation patterns of UV-photolabeled factors. Also, we identified an element in the IL-4 promoter with homology to the Y box common to all major histocompatibility complex class II gene promoters. Our data show that the IL-4 promoter Y box -114CTGATTGG-107 significantly enhances overall promoter activity, since point mutations within this element diminish promoter activity by 85%. The factor binding this region is indistinguishable from the cloned nuclear factor NF-Y, as judged from interactions with specific anti-NF-Y monoclonal and polyclonal antibodies. Last, we point out the presence of two sites that share sequence identity to the OAP region of the ARRE-1 site within the IL-2 promoter (K. S. Ullman, W. M. Flanagan, C. A. Edwards, and G. R. Crabtree, Science 254:558-562, 1991). These regions, -85GTGTAATA-78 and -245GTGTAATT-238, reside adjacent to the NF(P) binding sites P1 and P4 and bind a distinct nuclear factor.

Abstract: When Escherichia coli cells are transferred from 30 degrees C to 42 degrees C, transcription from specific promoters recognized by RNA polymerase containing sigma 32 (the rpoH gene product) is transiently activated, resulting in induction of heat shock proteins. Transcription from heat shock promoters is activated by an increased cellular concentration of sigma 32 due to enhanced synthesis and stabilization. We have constructed and examined the expression of mutant derivatives (deletions and base substitutions) of rpoH-lacZ gene fusion. Synthesis of a sigma 32-beta-galactosidase fusion protein was found to be regulated at the translational level involving two distinct 5'-proximal rpoH coding regions. A small region immediately downstream of the initiation codon is required for potentially high-level expression, whereas a much larger internal region is required for thermal regulation--namely, repression at low temperature or nonstress conditions. The two mRNA regions act as positive and negative cis elements, respectively, in controlling rpoH translation. We propose that an interplay between these RNA regions involving secondary structure formation is important in regulating translation initiation and that transient disruption of secondary structure represents a primary step of the heat shock response.

Abstract: ′′-UTR) of the DM protein kinase gene (DMPK), but the mechanism(s) of pathogenesis remain unknown. Studies using DM patient materials have often produced confusing results. Therefore, to study the effects of the DM mutation in a controlled environment, we have established a cell culture model system using C2C12 mouse myoblasts. By expressing chimeric reporter constructs containing a reporter gene fused to a human DMPK 3′-UTR, we identified both cis and trans effects that are mediated by the DM mutation. Our data show that a mutant DMPK 3′ ′′-UTR, with as few as 57 CTGs, had a negative cis effect on protein expression and resulted in the aggregation of reporter transcripts into discrete nuclear foci. We determined by deletion analysis that an expanded (CTG) n tract alone was sufficient to mediate these cis effects. Furthermore, in contrast to the normal DMPK 3′-UTR mRNA, a mutant DMPK 3′-UTR mRNA with (CUG) 200 selectively inhibited myogenic differentiation of C2C12 myoblasts. Genetic analysis and the Cre-loxP system were used to clearly demonstrate that the myoblast fusion defect could be rescued by eliminating the expression of the mutant DMPK 3′ ′′-UTR transcript. Characterization of spontaneous deletion events mapped the inhibitory effect to the (CTG) n expansion and/or the 3′ end of theDMPK 3′ ′′-UTR. These results provide evidence that the DM mutation acts in cis to reduce protein production (consistent with DMPK haploinsufficiency) and in trans as a ‘riboregulator’ to inhibit myogenesis.