Biomedical subjects
Publications and source records attributed to C Condon.
Hereditary benign telangiectasia is an idiopathic condition that causes considerable cosmetic embarrassment but has eluded successful treatment due to its extensive nature. We report a case of hereditary benign telangiectasia of the lower limbs in which the challenge of treating an extensive vascular lesion is met by using an Intense pulsed light (IPL) source.
Escherichia coli ribosomal RNA (rRNA) operons contain antitermination motifs necessary for forming terminator-resistant transcription complexes. In preliminary work, we isolated 'antiterminating' transcription complexes and identified four new proteins potentially involved in rRNA transcription antitermination: ribosomal (r-) proteins S4, L3, L4 and L13. We show here that these r-proteins and Nus factors lead to an 11-fold increase in terminator read-through in in vitro transcription reactions. A significant portion of the effect was a result of r-protein S4. We show that S4 acted as a general antitermination factor, with properties very similar to NusA. It retarded termination and increased read-through at Rho-dependent terminators, even in the absence of the rRNA antiterminator motif. High concentrations of NusG showed reduced antitermination by S4. Like rrn antitermination, S4 selectively antiterminated at Rho-dependent terminators. Lastly, S4 tightly bound RNA polymerase in vivo. Our results suggest that, like NusA, S4 is a general transcription antitermination factor that associates with RNA polymerase during normal transcription and is also involved in rRNA operon antitermination. A model for key r-proteins playing a regulatory role in rRNA synthesis is presented.
Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598-600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along gram-negative and gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored.
The B cell Ag receptor (BCR) has two important functions: first, it binds and takes up Ag for presentation to T lymphocytes; and second, it transmits signals that regulate B cell development. Normal expression of the BCR requires the association of the Ag binding subunit, membrane IgM (mIgM), with the signaling component, the Ig-alpha beta heterodimer. After assembly in the endoplasmic reticulum, the intact BCR travels through the secretory pathway to the cell surface. In this paper, we report two variants of the B lymphoma cell lines, WEHI 279 and WEHI 231, that have both lost the ability to express mu heavy chain and consequently do not express mIgM. However, these variants do express the Ig-alpha beta heterodimer. In one variant, WEHI 279*, the Ig-alpha beta remained trapped intracellularly in the absence of mIgM. The other variant, 303.1.5.LM, expressed an aberrantly glycosylated Ig-alpha beta on the cell surface that was capable of signaling after cross-linking with anti-Ig-beta Abs. Further characterization uncovered a point mutation in the 303.1.5.LM mb1 gene that would change a proline for a leucine in the extracellular domain of Ig-alpha. The 303.1.5.LM Ig-alpha beta could not associate with a wild-type mIgM after mu heavy chain was reconstituted by DNA transfection. Thus, this mutation could define a region of the Ig-alpha polypeptide that is important for recognition by the endoplasmic reticulum quality control system, for association with glycosylating enzymes, and for the association of Ig-alpha beta subunits with mIgM subunits to create a complete BCR complex.
The aetiology of hepatic dysfunction in patients with HIV infection is multifactorial. Re-activation of hepatitis C infection, drug toxicity, and opportunistic infections are all potential causes. Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used as part of combination antiretroviral therapy for the treatment of HIV infection. It is associated with a significant incidence of hepatotoxicity, usually occurring in the initial month of therapy. We report the case of a 49-year-old man who developed NVP-induced prolonged hepatotoxicity 5 months after commencing antiretroviral therapy.
The Escherichia coli genome carries seven rRNA (rrn) operons, each containing three rRNA genes. The presence of multiple operons has been an obstacle to many studies of rRNA because the effect of mutations in one operon is diluted by the six remaining wild-type copies. To create a tool useful for manipulating rRNA, we sequentially inactivated from one to all seven of these operons with deletions spanning the 16S and 23S rRNA genes. In the final strain, carrying no intact rRNA operon on the chromosome, rRNA molecules were expressed from a multicopy plasmid containing a single rRNA operon (prrn). Characterization of these rrn deletion strains revealed that deletion of two operons was required to observe a reduction in the growth rate and rRNA/protein ratio. When the number of deletions was extended from three to six, the decrease in the growth rate was slightly more than the decrease in the rRNA/protein ratio, suggesting that ribosome efficiency was reduced. This reduction was most pronounced in the Delta7 prrn strain, in which the growth rate, unlike the rRNA/protein ratio, was not completely restored to wild-type levels by a cloned rRNA operon. The decreases in growth rate and rRNA/protein ratio were surprisingly moderate in the rrn deletion strains; the presence of even a single operon on the chromosome was able to produce as much as 56% of wild-type levels of rRNA. We discuss possible applications of these strains in rRNA studies.
The Bacillus subtilis thrS gene is a member of the T-box gene family in Gram-positive organisms whose expression is regulated by a tRNA-mediated transcriptional antitermination mechanism involving a direct tRNA:mRNA interaction. The complex leader sequences of these genes share only short stretches of primary sequence homology, but a common secondary structure has been proposed by comparing the leaders of many genes of this family. The proposed mechanism forthe tRNA:mRNA interaction depends heavily on the secondary structure model, but is so far only supported by genetic evidence. We have studied the structure of the B.subtilis thrS leader in solution, in protection experiments using both chemical and enzymatic probes. The thrS leader structure was also probed in vivo using dimethylsulphate and the in vitro and in vivo data are in good accordance. We have organized the thrS leader into three major domains comprising six separate stem-loops. All but one of the short sequences conserved in this gene family are present in loop structures. The ACC specifier codon proposed to interact with the tRNAThrGGUisoacceptor is present in a bulge and probably exists in a stacking conformation. The proposed antiterminator structure is not visible in transcripts containing the terminator, but was probed using a transcript with the 3'-half of the terminator deleted and its folding appears consistent with the regulatory model. The leader sequences, and in particular the specifier domains, of the other genes of this family can be folded similarly to the experimentally solved thrS structure.
We have recently reported that processing occurs in the untranslated leader region of several members of a family of Gram-positive genes regulated by tRNA-mediated antitermination. We showed that cleavage at this site plays an important role in the induction of Bacillus subtilis thrS gene expression, following threonine starvation, by stabilising the downstream mRNA. Here we show that, when transferred on a plasmid, processing of the B. subtilis thrS leader can occur at the same site in Escherichia coli. Cleavage at this site is dependent on the E. coli endoribonuclease E, both in vivo and in vitro, suggesting that a functional homologue of RNase E is responsible for thrS processing in B. subtilis.
Explore the source record for details and available documents.
Twenty-eight cardiac rehabilitation patients who were smokers were followed for three years to measure smoking outcomes after an inpatient smoking cessation intervention. Participants were interviewed at three weeks, six months, one, two, and three year intervals to determine their smoking practice. Thirty-six percent (N = 10) of the smokers were not smoking at three years post education. The results suggest that smoking intervention can have a positive impact on long term smoking cessation outcomes.
The threonyl-tRNA synthetase gene, thrS, is a member of a family of Gram-positive genes that are induced following starvation for the corresponding amino acid by a transcriptional antitermination mechanism involving the cognate uncharged tRNA. Here we show that an additional level of complexity exists in the control of the thrS gene with the mapping of an mRNA processing site just upstream of the transcription terminator in the thrS leader region. The processed RNA is significantly more stable than the full-length transcript. Under nonstarvation conditions, or following starvation for an amino acid other than threonine, the full-length thrS mRNA is more abundant than the processed transcript. However, following starvation for threonine, the thrS mRNA exists primarily in its cleaved form. This can partly be attributed to an increased processing efficiency following threonine starvation, and partly to a further, nonspecific increase in the stability of the processed transcript under starvation conditions. The increased stability of the processed RNA contributes significantly to the levels of functional RNA observed under threonine starvation conditions, previously attributed solely to antitermination. Finally, we show that processing is likely to occur upstream of the terminator in the leader regions of at least four other genes of this family, suggesting a widespread conservation of this phenomenon in their control.
In this review, we summarize progress on the regulation of the aminoacyl-tRNA synthetase genes in Bacillus subtilis. Most of the genes encoding this set of enzymes in B subtilis are members of a large family of Gram-positive genes and operons controlled by a novel antitermination mechanism that uses their cognate uncharged tRNA as the effector. A subset of these genes is, in addition, likely to be controlled at the level of mRNA processing and degradation. We describe the key experiments leading to these conclusions.
Delivery of antigen in a manner that induces effective, antigen-specific immunity is a critical challenge in vaccine design. Optimal antigen presentation is mediated by professional antigen-presenting cells (APCs) capable of taking up, processing and presenting antigen to T cells in the context of costimulatory signals required for T-cell activation. Developing immunization strategies to optimize antigen presentation by dendritic cells, the most potent APCs, is a rational approach to vaccine design. Here we show that cutaneous genetic immunization with naked DNA results in potent, antigen-specific, cytotoxic T lymphocyte-mediated protective tumor immunity. This method of immunization results in the transfection of skin-derived dendritic cells, which localize in the draining lymph nodes. These observations provide a basis for further development of DNA-based vaccines and demonstrate the feasibility of genetically engineering dendritic cells in vivo.
The thrS gene in Bacillus subtilis is specifically induced by starvation for threonine and is, in addition, autorepressed by the overproduction of its own gene product, the threonyl-tRNA synthetase. Both methods of regulation employ an antitermination mechanism at a factor-independent transcription terminator that occurs just upstream of the start codon. The effector of the induction mechanism is thought to be the uncharged tRNA(Thr), which has been proposed to base pair in two places with the leader mRNA to induce antitermination. Here we show that the autoregulation by synthetase overproduction is likely to utilize a mechanism similar to that characterized for induction by amino acid starvation, that is by altering the levels of tRNA charging in the cell. We also demonstrate that the base pairing interaction at the two proposed contact points between the tRNA and the leader are necessary but not always sufficient for either form of regulation. Finally, we present evidence that the thrS gene is expressed in direct proportion to the growth rate. This method of regulation is also at the level of antitermination but is independent of the interaction of the tRNA with the leader region.
We have introduced point mutations into the leader boxA of a plasmid-encoded Escherichia coli rrnB operon to study the in vivo role of this regulatory element in the natural context of rRNA synthesis. The same mutations were previously shown to cause severe antitermination defects in vitro and in the context of a reporter gene assay. The plasmid-encoded rrnB mutant constructs studied here also contained point mutations in the 16S and 23S rRNA genes, which were used to distinguish rRNAs derived from plasmid and chromosomal rrn operons by primer extension analysis. Point mutations in boxA reduced the fraction of plasmid-derived rRNA in the cell from 75% to about 50%. The reduction was similar for both 30S and 50S subunits as well as 70S ribosomes, suggesting that no transcriptional polarity occurred between the expression of the 16S and 23S rRNA genes in plasmid rrnB operons carrying a mutant boxA. The boxA mutations do not affect the amount of transcription initiation, suggesting that a suboptimal leader boxA causes premature transcription termination at an early stage of transcription. Our results are consistent with a role for antitermination in the completion of full-length rrn transcripts but give no indications of posttranscriptional boxA functions.
Here we present evidence that only five of the seven rRNA operons present in Escherichia coli are necessary to support near-optimal growth on complex media. Seven rrn operons are necessary, however, for rapid adaptation to nutrient and temperature changes, suggesting it is the ability to adapt quickly to changing environmental conditions that has provided the selective pressure for the persistence of seven rrn operons in E. coli. We have also found that one consequence of rrn operon inactivation is a miscoordination of the concentrations of initiation factor IF3 and ribosomes.
The control of rRNA synthesis in response to both extra- and intracellular signals has been a subject of interest to microbial physiologists for nearly four decades, beginning with the observations that Salmonella typhimurium cells grown on rich medium are larger and contain more RNA than those grown on poor medium. This was followed shortly by the discovery of the stringent response in Escherichia coli, which has continued to be the organism of choice for the study of rRNA synthesis. In this review, we summarize four general areas of E. coli rRNA transcription control: stringent control, growth rate regulation, upstream activation, and anti-termination. We also cite similar mechanisms in other bacteria and eukaryotes. The separation of growth rate-dependent control of rRNA synthesis from stringent control continues to be a subject of controversy. One model holds that the nucleotide ppGpp is the key effector for both mechanisms, while another school holds that it is unlikely that ppGpp or any other single effector is solely responsible for growth rate-dependent control. Recent studies on activation of rRNA synthesis by cis-acting upstream sequences has led to the discovery of a new class of promoters that make contact with RNA polymerase at a third position, called the UP element, in addition to the well-known -10 and -35 regions. Lastly, clues as to the role of antitermination in rRNA operons have begun to appear. Transcription complexes modified at the antiterminator site appear to elongate faster and are resistant to the inhibitory effects of ppGpp during the stringent response.
Over a 5 year period, from January 1988 to January 1993, 23 patients were admitted with extensive unstable psoriasis, 21 of whom had full case notes available. Over the study period these 21 patients required 45 admissions, the mean duration of each admission being 30.54 days. Those patients living more than 30 miles from our unit required almost twice as many admissions as the city group. In 77% of cases, a precipitating factor for the acute flare was identified, and in 53% of these the precipitating factor was iatrogenic related. These patients are prone to develop complications of which infection is the most common. The complexity of medical management was reflected in the number of cross consultations requested. These findings have implications for the optimal delivery of care to this important sub-group of psoriatics.