Biomedical subjects
Publications and source records attributed to S D Bell.
Studies of the DNA-replication machinery of Archaea have revealed striking similarities to that of eukaryotes. Indeed, it appears that in most cases Archaea possess a simplified version of the eukaryotic replication apparatus. Studies of Archaea are therefore shedding light on the fundamental processes of DNA replication in both domains of life.
Archaea share many similarities with eukarya in their information processing pathways and have proven to be a useful model for studies of DNA replication and transcription, but DNA repair pathways are not well understood in archaea. Nucleotide Excision Repair (NER) deals with many bulky DNA lesions and involves over 30 proteins in eukarya. Archaeal NER has not been characterized biochemically, but homologues of the human repair nucleases XPF and XPG have been identified by homology searches. Crenarchaeal XPF proteins have a simplified domain structure, consisting of the C-terminal nuclease domain conserved in XPF and Mus81 but lacking the N-terminal 'helicase' domain that is found in eukaryal and euryarchaeal sequences. Unexpectedly, Sulfolobus XPF is only active in the presence of the sliding clamp PCNA, which is a heterotrimer in this organism. Interactions with two of the three subunits of PCNA are mediated via a C-terminal interaction motif. The PCNA-XPF complex acts as a structure-specific nuclease on a similar range of DNA flap, bubble and junction substrates as the human protein, suggesting a fundamental conservation through billions of years of evolution.
Eukaryotic DNA is packaged into nucleosomes that regulate the accessibility of the genome to replication, transcription and repair factors. Chromatin accessibility is controlled by histone modifications including acetylation and methylation. Archaea possess eukary otic-like machineries for DNA replication, transcription and information processing. The conserved archaeal DNA binding protein Alba (formerly Sso10b) interacts with the silencing protein Sir2, which regulates Alba's DNA binding affinity by deacetylation of a lysine residue. We present the crystal structure of Alba from Sulfolobus solfataricus at 2.6 A resolution (PDB code 1h0x). The fold is reminiscent of the N-terminal DNA binding domain of DNase I and the C-terminal domain of initiation factor IF3. The Alba dimer has two extended beta-hairpins flanking a central body containing the acetylated lysine, Lys16, suggesting three main points of contact with the DNA. Fluorescence, calorimetry and electrophoresis data suggest a final binding stoichiometry of approximately 5 bp DNA per Alba dimer. We present a model for the Alba-DNA interaction consistent with the available structural, biophysical and electron microscopy data.
Archaea possess two general transcription factors that are required to recruit RNA polymerase (RNAP) to promoters in vitro. These are TBP, the TATA-box-binding protein and TFB, the archaeal homologue of TFIIB. Thus, the archaeal and eucaryal transcription machineries are fundamentally related. In both RNAP II and archaeal transcription systems, direct contacts between TFB/TFIIB and the RNAP have been demonstrated to mediate recruitment of the polymerase to the promoter. However the subunit(s) directly contacted by these factors has not been identified. Using systematic yeast two-hybrid and biochemical analyses we have identified an interaction between the N-terminal domain of TFB and an evolutionarily conserved subunit of the RNA polymerase, RpoK. Intriguingly, homologues of RpoK are found in all three nuclear RNA polymerases (Rpb6) and also in the bacterial RNA polymerase (omega-subunit).
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PURPOSE: To report data collected by the Canadian Registry of the Günther Tulip Retrievable Filter (GTF). MATERIALS AND METHODS: Between February 1998 and December 2000, 90 patients at eight hospitals underwent implantation of 91 GTFs. There were 45 male patients and 45 female patients, age 17-88 years, with a mean age of 49 years. Indications for filter placement were pulmonary embolism (PE) or deep vein thrombosis (DVT) with a contraindication to anticoagulation in 83 patients, prophylaxis after massive PE in one, prophylaxis for proximal free-floating thrombus in one, and prophylaxis with no DVT or PE in six patients (major trauma, n = 4; high preoperative risk, n = 2). GTF retrieval was attempted in selected patients from a right internal jugular vein approach. RESULTS: One GTF was inadvertently placed in the right iliac vein and could not be retrieved. There were no other major placement complications. GTF retrieval was attempted in 52 patients (53 GTFs); 52 GTFs were successfully retrieved from 51 patients. Implantation times were 2-25 days (mean, 9 d). Of these 51 patients, 37 underwent follow-up for 5-420 days (mean, 103 d) after filter retrieval. Four patients (8% of retrieved GTFs) required reinsertion of a permanent filter 17-167 days (mean, 78 d) after GTF retrieval as a result of bleeding from anticoagulation (n = 2) or because the patient required further surgery (n = 2). One other patient had recurrent DVT 230 days after retrieval; no PE or other complication was documented in the retrieval group. GTFs were not retrieved from 39 patients for various reasons. Of these 39 patients, 25 underwent follow-up 7-420 days (mean, 85 d) after filter placement. Two patients developed filter occlusion (5%); no other complications were documented. CONCLUSION: The GTF has a broad range of utility: it can be used as a permanent filter or retrieved after implantation periods of 15 days and possibly longer. However, indications for retrieval require further study, as does the maximum implantation time.
The archaeal basal transcription machinery resembles the core components of the eucaryal RNA polymerase II apparatus. Thus, studies of the archaeal basal machinery over the last few years have shed light on fundamentally conserved aspects of the mechanisms of transcription pre-initiation complex assembly in both eucarya and archaea. Intriguingly, it has become increasingly apparent that regulators of archaeal transcription resemble regulators initially identified in bacteria. The presence of these shared bacterial-archaeal regulators has given insight into the evolution of gene regulatory processes in all three domains of life.
The basal transcription machinery of Archaea is fundamentally related to the eucaryal RNA polymerase (RNAP) II apparatus. In addition to a 12-subunit RNAP, Archaea possess two general transcription factors, the activities of which are required for accurate and efficient in vitro transcription. These factors, TBP and TFB, are homologues of the eucaryal TATA-box binding protein and TFIIB respectively. Archaea also possess TFE, a homologue of the eucaryal RNAP II general transcription factor TFIIE. Although not absolutely required for transcription in vitro, TFE nonetheless plays a stimulatory role under conditions where promoter recognition by TBP is sub-optimal. The basal transcription apparatus of Archaea is closely related to that of Eucarya but archaeal transcriptional regulators resemble those of bacteria. The mode of action of two such regulators has been characterized to determine how these 'bacterial-like' regulators impinge on the 'eucaryal-like' basal machinery.
Transcription from many archaeal promoters can be reconstituted in vitro using recombinant TATA-box binding protein (TBP) and transcription factor B (TFB)--homologues of eukaryal TBP and TFIIB--together with purified RNA polymerase (RNAP). However, all archaeal genomes sequenced to date reveal the presence of TFE, a homologue of the alpha-subunit of the eukaryal general transcription factor, TFIIE. We show that, while TFE is not absolutely required for transcription in the reconstituted in vitro system, it nonetheless plays a stimulatory role on some promoters and under certain conditions. Mutagenesis of the TATA box or reduction of TBP concentration in transcription reactions sensitizes a promoter to TFE addition. Conversely, saturating reactions with TBP de-sensitizes promoters to TFE. These results suggest that TFE facilitates or stabilizes interactions between TBP and the TATA box.
The basal transcription machinery of archaea corresponds to the core components of the eucaryal RNA polymerase II apparatus. Thus, archaea possess a complex multi-subunit RNA polymerase, a TATA box-binding protein and a protein termed transcription factor B (TFB), which is a homologue of eucaryal transcription factor IIB (TFIIB). Intriguingly, archaeal genome sequencing projects have revealed the existence of homologues of bacterial transcriptional regulators. To investigate the mechanism of transcriptional regulation in archaea we have studied one such molecule, Lrs14, a Sulfolobus solfataricus P2 homologue of the bacterial leucine-responsive regulatory protein, Lrp. We find that purified Lrs14 specifically represses the transcription of its own gene in a reconstituted in vitro transcription system. Furthermore, we show that Lrs14 binding sites overlap the basal promoter elements of the Lrs14 promoter and reveal that binding of Lrs14 to these sites prevents promoter recognition by TATA box-binding protein and TFB.
Mechanisms of transcription initiation appear to be remarkably conserved between archaea and eucaryotes. For instance, there is homology between archaeal and eucaryotic basal transcription factors. Also, the archaeal RNA polymerase (RNAP) resembles eucaryotic nuclear RNAPs in subunit composition and at the amino acid sequence level. Here, we examine the role of transcription factor B, the archaeal homologue of eucaryotic transcription factor IIB, in transcription initiation. We show that the N-terminal region of transcription factor B is required for RNAP recruitment. Furthermore, we reveal that mutation of a conserved residue immediately C-terminal of the N-terminal zinc ribbon motif abrogates transcription on certain promoters. Finally, we identify the promoter sequences responsive to this mutation and demonstrate that the effect of the mutation is to block a late stage in transcription initiation, following formation of the promoter open complex.
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Percutaneous interstitial microwave thermoablation of locally recurrent prostate carcinoma was continually guided with magnetic resonance (MR) imaging. Phase images and data were obtained with a rapid gradient-echo technique and were used to derive tissue temperature change on the basis of proton-resonance shift. Thermally devitalized regions correlated well with the phase image findings. MR imaging-derived temperatures were linearly related to the fluoroptic tissue temperatures. MR imaging can be used to guide thermoablation.
The basal transcription machinery of Archaea corresponds to the minimal subset of factors required for RNA polymerase II transcription in eukaryotes. Using just two factors, Archaea recruit the RNA polymerase to promoters and define the direction of transcription. Notably, the principal determinant for the orientation of transcription is not the recognition of the TATA box by the TATA-box-binding protein. Instead, transcriptional polarity is governed by the interaction of the archaeal TFIIB homologue with a conserved motif immediately upstream of the TATA box. This interaction yields an archaeal preinitiation complex with the same orientation as the analogous eukaryal complex.
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The basal transcription apparatus of Archaea corresponds to the core machinery of the eucaryal RNA polymerase II system. However, it is not yet known how regulation of archaeal transcription is achieved. Examination of complete archaeal genome sequences reveals homologs of bacterial transcriptional regulators. We have studied one such molecule, MDR1, an A. fulgidus homolog of the bacterial metal-dependent transcriptional repressor, DtxR. We find that in vivo expression of the MDR1-containing operon is regulated by metal ion availability. In vitro analyses show that MDR1 recognizes three operator elements in its own promoter in a metal-dependent manner. MDR1 negatively regulates transcription of its own gene in a reconstituted in vitro system, not by abrogating the binding of TBP or TFB to the promoter but by preventing RNA polymerase recruitment.
OBJECTIVE: To assess the safety of performing iliac arterial stenting as an outpatient procedure. METHODS: Retrospective analysis of safety including all patients referred for elective iliac arterial stenting over a 1-year period. Sources of data for the analysis included pre- and post-stenting vascular surgical consultation records, hospital case notes, diagnostic and interventional angiography reports, computerized laboratory data, nursing records from our angiography holding area, and the results of routine post-stenting telephone follow-up. RESULTS: There were 29 outpatient iliac stenting procedures in 28 patients (19 men and 9 women, age range 41.0 to 79.8 years, mean age 66.1 years). Of these 29 procedures, 17 involved unilateral iliac stenting, and 12 involved bilateral iliac stenting. Adjunctive renal artery angioplasty was performed in 1 patient and internal iliac angioplasty and stenting were performed in 2 patients. A total of 51 stents were deployed through 42 femoral punctures via introducer sheaths ranging in size from 6 to 8 French. Percutaneous hemostatic closing devices were used in 6 punctures. Two patients required overnight inpatient observation for moderate-size hematomas; these had no clinical sequelae. All others were discharged safely 5 to 6 hours after sheath removal. No clinically significant sequelae were identified in any patient. CONCLUSION: Arterial stenting can be performed safely on an outpatient basis.
Although Archaea are prokaryotic and resemble Bacteria morphologically, their transcription apparatus is remarkably similar to those of eukaryotic cell nuclei. Because some Archaea exist in environments with temperatures of around 100 degreesC, they are likely to have evolved unique strategies for transcriptional control. Here, we investigate the effects of temperature and DNA template topology in a thermophilic archaeal transcription system. Significantly, and in marked contrast with characterized eucaryal systems, archaeal DNA template topology has negligible effect on transcription levels at physiological temperatures using highly purified polymerase and recombinant transcription factors. Furthermore, archaeal transcription does not require hydrolysis of the beta-gamma phosphoanhydride bond of ATP. However, at lower temperatures, negatively supercoiled templates are transcribed more highly than those that are positively supercoiled. Notably, the block to transcription on positively supercoiled templates at lowered temperatures is at the level of polymerase binding and promoter opening. These data imply that Archaea do not possess a functional homologue of transcription factor TFIIH, and that for the promoters studied, transcription is mediated by TATA box-binding protein, transcription factor TFB, and RNA polymerase alone. Furthermore, they suggest that the reduction of plasmid linking number by hyperthermophilic Archaea in vivo in response to cold shock is a mechanism to maintain gene expression under these adverse circumstances.