The human secretin receptor gene: genomic organization and promoter characterization.

Secretin is the most potent regulator of pancreatic bicarbonate, electrolyte and volume secretion. In this report, the organization of the human secretin receptor (hSR) gene was characterized by overlapping genomic phage clones. The hSR gene consists of 13 exons and 12 introns with all the splice donor and acceptor sites conforming to the canonical GT/AG rule. By transient reporter gene assays, the wild-type promoter, containing 3.0 kb of the hSR gene 5' flanking region, was able to drive 5.8 +/- 0.6 and 6.6 +/- 0.2-fold (P < 0.01) increases in luciferase activities in pancreatic ductule-derived PANC-1 and BPD-1 cells, respectively. By subsequent 5' and 3' deletion analysis, a promoter element was identified within -408 to -158, relative to the ATG codon. This promoter element was found to be cell-specific since it could drive reporter gene expression in PANC-1 and BPD-1 cells but not in Hs 262.St, Hs 746T and alphaT3-1 cells. The study of the transcriptional control of human secretin and its receptor should shed light on the pathological developments of pancreatic cancer and autism in the future.

Pediatric sensorineural hearing loss after temporal bone radiation.

In adults, sensorineural hearing loss (SNHL) resulting from megavoltage temporal bone radiation therapy (RT) is dose dependent. A review of the literature reveals only two cases of pediatric SNHL after RT without chemotherapy. This report presents four cases of late onset, asymmetric SNHL after RT in children treated for posterior fossa tumors. The children were treated with surgical resection of the tumor, followed by RT of 50 to 54 gray and no chemotherapy. These children, without evidence of tumor recurrence, developed severe SNHL 18 to 36 months after the completion of RT. Although no definite audiometric pattern was seen, most of the patients had a predominately high frequency SNHL in the poorer hearing ear, and fluctuant hearing in the better ear, without evidence of osteoradionecrosis or otitic infection. A review of the literature suggests that injury to the outer hair cells and cochlear neurons of the basal turn of the cochlea are possible sources for the hearing loss. Pretreatment audiologic evaluation as well as a minimum of 3 years of evaluations following RT would be prudent.

Direct and indirect effects of mutations in lambda PRM on open complex formation at the divergent PR promoter.

A detailed kinetic analysis demonstrates that, in vitro, mutations in the PRM promoter of bacteriophage lambda can increase the rate of open complex formation at the divergent, lytic promoter PR in either of two ways. (1) PRM- mutations, typified by PRMKM11, indirectly stimulate PR by eliminating interference from RNA polymerase (RNAP) molecules bound at wild-type PRM. This effect can be observed only when PR is itself mutated because open complexes normally form so rapidly at wild-type PR that they are unaffected by PRM. It has been shown previously that PR and PRM can be occupied simultaneously by RNAP, suggesting that interference from PRM is mediated at a step subsequent to binding of RNAP to PR. This conclusion is supported by kinetic data, which indicate that inactivating PRM affects PRx3 by increasing kf, the rate of isomerization of closed to open complexes, four- to fivefold. (2) In addition to its indirect effect, the mutation PRM116, which is located at -33 with respect to PRM and -50 with respect to PR, directly increases the intrinsic strength of PR. PRM116 increases from 11 to 12 the number of A:T or T:A base-pairs in a 12 bp AT-rich sequence located between 47 and 58 bp upstream from PR; we suggest that this upstream sequence contributes directly to PR promoter strength. We also show that the PRx3 mutation causes a 100-fold decrease in kf. This result indicates that the -35 consensus region plays a major role in the isomerization of closed to open complexes at PR.

A cryptic promoter in the O(R) region of bacteriophage lambda.

A cryptic promoter, designated P alpha, initiates transcription within the O(R) region of bacteriophage lambda. Transcription from P alpha proceeds in the direction of the cI repressor gene from sites 46 and 48 bp preceding the PRM transcription start site. P alpha is likely to compete with both PR and PRM for formation of open complexes, since it is only active when PR is mutated and can be suppressed by mutations that increase PRM activity. In addition, transcription initiation at P alpha is blocked by lambda repressor. Kinetic analysis of relative abundance of the products of in vitro transcription indicated that P alpha was approximately 1/3 as strong as PRM. However, a P alpha mutation had little effect on KBkf (the association rate constant) for PRM. These observations can be explained by the finding that open complexes formed at P alpha are relatively unstable (half-life = 20 to 25 min). Dissociation of RNA polymerase from P alpha allows additional open complexes to form at PR or PRM, and thus the apparent strength of P alpha decreases with increasing preincubation times.

Modulation of P(RM) activity by the lambda PR promoter in both the presence and absence of repressor.

When the transcription startsites of the phage lambda promoters PRM and PR are separated by 82 bp (the wild-type spacing), mutating PR increases the rate of open complex formation at PRM at all RNA polymerase (RNAP) concentrations tested in vitro. This is reflected in a fourfold increase in kappa f (the rate constant for isomerization of closed to open complexes) and a threefold decrease in KB (the equilibrium constant for formation of closed complexes). These effects of mutating PR resemble qualitatively those we observed when the separation between the two promoters was decreased by a single base-pair, but are quantitatively less dramatic. Although mutating PR has the additional effect of uncovering a weak promoter, P alpha, which overlaps both PRM and PR, the presence of P alpha does not account for the effects of PR mutations on open complex formation at PRM. In fixed-time assays at a single RNAP concentration, repressor stimulated PRM approximately threefold on a PR- template, indicating that activation is mediated substantially by a direct interaction between repressor and RNAP. That is, activation of PRM is not merely an indirect consequence of repressing PR. Kinetic data confirm this conclusion. In a PR- genetic background, repressor increased kappa f six- to eightfold and decreased KB approximately twofold. Similar results were obtained when OR3 was mutated, indicating that the effect on KB is not due to repressor binding to OR3. Thus, repressor causes a significant increase in the rate of open complex formation at PRM even when PR is inactive. On a PR+ template, 75 nM repressor stimulated PRM by increasing kappa f eightfold, with no effect on KB, which agrees with previous results. However, increased repressor concentrations stimulated kappa f by an additional factor of two to four, indicating that previous experiments underestimated the effect of repressor on kappa f. At the same time, increasing the repressor concentration decreased KB for PRM on a wild-type template. At the highest repressor concentration tested (275 nM), KB decreased 15-fold, presumably due to OR3-mediated repression of PRM. However, at an intermediate repressor concentration (170 nM) values of kappa f and KB for PRM on a PR+ template were in close agreement with the corresponding parameters obtained on a PR- template. These data lead us to suggest that repressor causes a decrease in KB for PRM on both a PR+ and a PR- template independent of its ability to bind to OR3.

Effects of a single base-pair deletion in the bacteriophage lambda PRM promoter. Repression of PRM by repressor bound at OR2 and by RNA polymerase bound at PR.

We have deleted a single base-pair in the -35 region of the bacteriophage lambda PRM promoter. The deletion (PRM delta 34) creates a better match of PRM to consensus, thereby substantially increasing the activity of the promoter in vitro and in vivo. Since the mutation also increases the overlap between OR2 and the -35 region of PRM, binding of repressor to OR2 no longer activates, but in fact represses PRM. Finally, the mutation decreases the distance between the PRM and PR transcription start sites from 82 to 81 base-pairs. As a consequence, the interaction of RNA polymerase with either promoter in vitro strongly inhibits open complex formation at the other. Kinetic analyses and DNase I protection assays lead to the surprising result that mutual inhibition is not due to steric occlusion. Both promoters can be occupied by RNA polymerase at the same time. Determination of KB and kf revealed that inhibition of PRM delta 34 by PR was manifest in a 100-fold decrease in the value of kf, but at the same time KB was increased tenfold. These data raise the possibility that RNA polymerase molecules bound at the two promoters contact and mutually stabilize each other and that this interaction subsequently inhibits a substep in the isomerization of closed to open complexes. In footprinting assays, each promoter is characterized by sites of enhanced cleavage when that promoter is occupied alone. These enhancements are substantially diminished when both promoters are occupied, suggesting that complexes of each promoter with RNA polymerase alter the structure of complexes formed at the other promoter. Assays of the effects of the delta 34 mutation in vivo indicate that interference between PRM and PR does not limit the rate of open complex formation at PRM in the cell. Apparently, transcription initiation clears the promoter rapidly enough that neither promoter is occupied a significant fraction of the time.