RNA polymerase II initiation factor interactions and transcription start site selection.

An RNA polymerase II transcription system was resolved and reconstituted from extracts of Schizosaccharomyces pombe. Exchange with components of a Saccharomyces cerevisiae system was undertaken to reveal the factor or factors responsible for the difference in location of the transcription start site, about 30 base pairs and 40 to 120 base pairs downstream of the TATA box in S. pombe and S. cerevisiae, respectively. Two components, counterparts of human transcription factor IIF (TFIIF) and TFIIH, could be exchanged individually between systems without effect on the start site. Three components, counterparts of human TFIIB, TFIIE, and RNA polymerase II, could not be exchanged individually but could be swapped in the pairs TFIIE-TFIIH and TFIIB-RNA polymerase II, which demonstrates that there are functional interactions between these components. Moreover, exchange of the latter pair shifted the starting position, which shows that TFIIB and RNA polymerase II are solely responsible for determining the start site of transcription.

Initiation by yeast RNA polymerase II at the adenoviral major late promoter in vitro.

Transcription of the yeast CYC1 promoter fused to a sequence lacking guanosine residues provided a rapid, sensitive assay of initiation by RNA polymerase II in yeast extracts. Initiation was enhanced by yeast and mammalian activator proteins. The adenoviral major late promoter fused to the G-minus sequence was transcribed in yeast extracts with an efficiency comparable to that observed in HeLa extracts, showing that promoters as well as transcription factors are functionally interchangeable across species. Initiation occurred at different sites, approximately 30 and 63 to 69 base pairs downstream of the TATA element of the adenoviral promoter in HeLa and yeast extracts, respectively, distances characteristic of initiation in the two systems in vivo. A component of the transcription system and not the promoter sequence determines the distance to the initiation site.

Analysis of inhibitors of the site-specific recombination reaction mediated by Tn3 resolvase.

The Tn3-encoded resolvase protein promotes a site-specific recombination reaction between two directly repeated copies of the recombination site res. Several inhibitors that block this event in vitro have been isolated. In this study four of these inhibitors were tested on various steps in the recombination reaction. Two inhibitors. A9387 and A1062, inhibit resolvase binding to the res site. Further, DNase I footprinting revealed that at certain concentrations of A9387 and A1062, resolvase was preferentially bound to site I of res, the site containing the recombinational crossover point. The two other inhibitors, A20812 and A21960, do not affect resolvase binding and bending of the DNA but inhibit synapse formation between resolvase and two directly repeated res sites.

Tuberculosis of the larynx in a lepromatous patient.

Within the black population of South Africa tuberculosis and leprosy are endemic. There is also a significant incidence of laryngeal carcinoma. A patient who presented in acute respiratory stridor to a rural hospital with limited resources is reported. The differential diagnosis and management is discussed in the light of the available literature.

Cervical node metastases presenting with features of branchial cysts.

For many years it has been recognized that seemingly benign neck cysts may contain carcinoma. The true incidence is unknown. This paper investigated nine out of 270 patients presenting with a neck mass--which proved to contain a squamous carcinoma. Records (from a 30-year period) of over 3400 patients with squamous carcinoma of the head and neck, were examined. The histology slides were reviewed, the number of cystic lesions was noted and also the clinical outcome. Out of the 270 patients nine presented with a cystic lesion and these were studied. Six cystic lesions were originally diagnosed as branchial cysts although the youngest age was 39 years. All patients underwent a simple excision. In six cases the tonsil was the primary site, in one the primary was in the base of tongue and in two the primary remained occult. One-third of the patients had died of their disease by the time this report was written and the maximum follow-up time for the remaining patients was 18 months. Therefore 16 per cent of branchial cysts in this series represented metastases from squamous cell carcinoma. At the Royal Liverpool University Hospital only 25 patients had branchial cysts excised between 1988 and 1993: out of these only four contained squamous carcinoma. In patients over 40 years of age panendoscopy and ipsilateral tonsillectomy is mandatory prior to cyst excision.

Nasopharyngeal Corynebacterium ulcerans: a different diphtheria.

A case of toxigenic Corynebacterium ulcerans infection is presented. The diagnosis was delayed and no anti-toxin administered. A nasopharyngeal biopsy was complicated by severe haemorrhage necessitating a post nasal pack. A brief review of the pathology and treatment of Corynebacterium ulcerans is given.

Surfers' awareness of the preventability of 'surfer's ear' and use of water precautions.

OBJECTIVE: To establish surfers' knowledge of the preventability of external auditory canal exostoses ('surfer's ear'), and their use of water precautions. METHOD: Survey of surfers conducted between December 2009 and March 2010 at beaches in Cornwall, UK. RESULTS: Ninety-two surfers were included (78 males and 14 females, mean age 27 years, standard deviation 7.9 years). Participants were grouped according to their awareness of the preventability of surfer's ear (55 aware, 37 unaware). These groups were comparable in age, surfing history and gender mix (p > 0.05). Surfers aware of the preventability of exostoses (66 per cent) were more likely to use water precautions than those who were not (38 per cent) (p < 0.01). Two surfers used water precautions regularly and 48 used them occasionally. Sixty-one of the 76 surfers who did not use water precautions (ear plugs) suggested they would consider doing so in the future. CONCLUSION: Awareness of the preventability of surfer's ear was associated with greater use of water precautions. Further research should explore reasons for the low uptake of such precautions. Most surfers not already using ear plugs would consider doing so in the future.

A novel mediator between activator proteins and the RNA polymerase II transcription apparatus.

One gene activator protein may interfere with the effects of another in eukaryotic cells. We report here that a hybrid yeast-herpes gene activator protein inhibits transcriptional activation by a thymidine-rich DNA element in yeast. This example of activator interference can be faithfully reproduced in vitro. Interference is reversed by a partially purified yeast component, but not by RNA polymerase II or various polymerase II transcription factors. We conclude that the partially purified yeast component is a novel factor, and we suggest this factor mediates the transcriptional activation process.

Neural and hormonal control of membrane conductance in the pig pancreatic acinar cell.

Intracellular microelectrode recordings from superfused segments of pig pancreas have shown the resting acinar cell membrane potentials to range widely, with a mean value of -30.5 +/- 1.3 mV. Electrical field stimulation (FS) of the intrinsic pancreatic nerves induced frequency-dependent membrane hyperpolarization (10-15 mV) accompanied by a concomitant reduction in input resistance. Similar effects could be evoked by the superfusion or electroionophoresis of acetylcholine, amphibian or mammalian bombesin [gastrin-releasing peptide (GRP)], and pentagastrin. In normal Ca2+-containing solutions sustained secretagogue superfusion resulted in sustained hyperpolarization. In the absence of external Ca2+, similar stimulation caused only a transient hyperpolarizing response, with subsequent periods of secretagogue application having no effect. Atropine completely abolished the FS-evoked hyperpolarizations but had no effect on the responses evoked by bombesin, GRP, and pentagastrin. The present findings support the contention that neural and hormonal stimulation of the pig pancreas evokes Ca2+-dependent acinar cell hyperpolarization by causing a selective increase in membrane K+ permeability. A hypothesis is proposed that cellular K+ release through the opened conductance pathway promotes a K+-Na+-Cl- cotransport into the cell that serves a key function in the generation of acinar salt secretion.

Inhibitor analysis of synapsis by resolvase.

Previously, we isolated several inhibitors that block the site-specific recombination reaction mediated by the Tn3-encoded resolvase protein. One class of inhibitors blocks resolvase binding to the recombination (res) sitc, and a second class inhibits synapse formation between resolvase and two directly repeated res sites. In this report, we identify an inhibitor, A20832, that does not inhibit resolvase binding to res, as measured by filter binding, or synapse formation. Inhibition of resolvase-promoted site-specific recombination by A20832 occurs postsynaptically at strand cleavage. DNase I analysis in the presence of A20832 indicates that only site I of res is bound by resolvase.

Simple derivation of TFIID-dependent RNA polymerase II transcription systems from Schizosaccharomyces pombe and other organisms, and factors required for transcriptional activation.

Resolution of whole cell extract through two chromatographic steps yields a single protein fraction requiring only the addition of TFIID for the initiation of transcription at RNA polymerase II promoters. This approach allows the convenient generation of RNA polymerase II transcription systems from Saccharomyces cerevisiae, human lymphocytes, and Schizosaccharomyces pombe. TFIIDs from all three organisms are interchangeable among all three systems. The S. cerevisiae and Sch. pombe systems support effects of acidic activator proteins, provided a further protein fraction from S. cerevisiae is supplied. This further fraction is distinct from the mediator of transcriptional activation described previously and represents a second component in addition to general initiation factors that may facilitate a response to acidic activators.

Yeast RNA polymerase II initiation factor e: isolation and identification as the functional counterpart of human transcription factor IIB.

Yeast RNA polymerase II initiation factor e was purified to homogeneity and identified by biochemical criteria as the counterpart of human transcription factor IIB. Factor e was essential for initiation of transcription from yeast and mammalian promoters in a reconstituted yeast transcription system. Activity resided in a single polypeptide of approximately 41 kDa, identified by peptide sequence analysis as the product of the SUA7 gene. Factor e interacted specifically with RNA polymerase II, consistent with a proposed role in determining the start site of transcription.

A mediator required for activation of RNA polymerase II transcription in vitro.

Activator proteins bind to enhancer DNA elements and stimulate the initiation of transcription. It has been proposed that activators contact general initiation factors at a promoter, and evidence for such direct interaction has been obtained. Studies of transcription in vitro, however, have suggested that activators might function through an intermediary molecule(s) distinct from the general factors. In the first of these studies, we exploited the finding that one activator could inhibit transcription stimulated by a second activator (activator interference or 'squelching'). This inhibition, which is attributed to competition between the activators for a common target factor, could not be relieved by addition of a large excess of general initiation factors, suggesting that the target for which activators compete is distinct from these factors. Similar conclusions came from the observation that TFIID's expressed from cloned genes fail to replace partially purified 'natural' TFIID fractions in supporting activation, evidently because they lacked some component present in the impure fractions. While these lines of evidence for a novel 'mediator' of activation were negative, we also showed that a partially purified fraction from yeast would reverse activator interference. This positive effect of a presumptive mediator provided an assay for its activity, but its role in activation was still only inferred. We now present direct evidence for a mediator which is required for stimulation of transcription in vitro by the activators GAL4-VP16 and GCN4, but which has no effect on transcription in the absence of activator protein.

Yeast and human TFIIDs are interchangeable for the response to acidic transcriptional activators in vitro.

Previous work showed that human TFIID fails to support yeast cell growth, although it is nearly identical to yeast TFIID in a carboxy-terminal region of the molecule that suffices for basal, TATA-element-dependent transcription in vitro. These and other findings raised the possibility that TFIID participates in species-specific interactions, possibly with mediator factors, required for activated transcription. Here, we report that human TFIID and amino-terminally truncated derivatives of yeast TFIID are fully functional in support of both basal transcription and the response to acidic activator proteins in a yeast in vitro transcription system. Conversely, and in contrast to previously published results, yeast TFIID supports both basal and activated transcription in reactions reconstituted with human components. This functional interchangeability of yeast and human TFIIDs argues strongly against species specificity with regard to TFIID function in basal transcription and the response to acidic activator proteins. In addition, our results suggest that any intermediary factors between acidic activators and TFIID are conserved from yeast to man.

Resolution of factors required for the initiation of transcription by yeast RNA polymerase II.

Fractionation of a yeast nuclear extract reveals at least four factors required in addition to RNA polymerase II for accurate initiation of transcription. One of these factors can be replaced by HeLa transcription factor IID or by its yeast counterpart expressed in Escherichia coli. Each of the remaining three factors can be replaced by a fraction from yeast whole cell extract, facilitating further purification of the factors.

Hearing aids and glue ear.

This study was designed to determine the acceptance and effectiveness of a hearing aid in the management of children with persistent hearing loss due to glue ear (otitis media with effusion [OME]). Forty-eight children with OME, in whom the main symptom was deafness, were treated with a hearing aid instead of surgery and reviewed at 3-monthly intervals over 6-12 months. Seventy-one per cent reported unequivocal satisfaction with the aid. Sixty-five per cent used the aid continuously throughout the day whilst 35% used it only at specific times of need. Ninety-eight per cent noticed a definite improvement in their hearing whilst using the aid and this was confirmed audiometrically in 100%. Disability was considered in terms of speech development and educational achievement. In 66% there had been a subjective decline in these parameters prior to aid prescription. In all but one child significant improvement was made sufficient to alleviate parental and teachers' concern. No children reported significant symptoms due to OME other than deafness and there were no complications of hearing aid usage. At follow-up, however, 13% of children continued to use a hearing aid in an ear in which the OME had resolved. This study has shown that in this preselected group of children with persistent OME and the predominant symptom of deafness, a hearing aid was an effective treatment for their deafness with high acceptance and compliance.

Effects of activation-defective TBP mutations on transcription initiation in yeast.

Transcription initiation by RNA polymerase II is effected by an ordered series of general factor interactions with core promoter elements (leading to basal activity) and further regulated by gene-specific factors acting from distal elements. Both the general factor TFIID (refs 2,3), including the constituent TBP (TATA-binding polypeptide) and associated factors, and the interacting factor TFIIB (refs 9-11) have been implicated as targets for various activators. Towards an understanding of the basis for activator function, including the multiplicity of TBP interactions, we have now identified mutations in yeast TBP that selectively block activator (GAL4-VP16)-dependent but not basal transcription. We further show an effect of GAL4-VP16 on TFIIB recruitment to early preinitiation complexes, and that recruitment is disrupted by TBP mutations that impair its interactions with VP16 (L114K), TFIIB (L189K) or an unidentified component (K211L). Thus, GAL4-VP16 function seems to involve both direct interactions with TBP and a corresponding induction (or stabilization) of an activation-specific TBP-TFIIB-promoter complex.