ABSTRACT
AIMS: To develop a semi-high-throughput ex vivo mucosal model for determining efficacy and toxicity of antiseptics. METHODS AND RESULTS: Explants (5 mm) from freshly excised, porcine vaginal mucosa were infected with methicillin-sensitive Staphylococcus aureus (1 × 10(6) CFU) at the epithelial surface for 2 h. Haematoxylin and eosin staining revealed healthy uninfected tissue and only minor disruptions in tissue infected with methicillin susceptible Staph. aureus (MSSA), which remained in outer epithelial cell layers. After 2 h infection, 10 µl of chlorhexidine digluconate (CHG, 3%), povidone-iodine (PI, 7·5%), octenidine dihydrochloride (OCT, 0·1%) or polyhexamethylene biguanide (PHMB, 0·1%) was applied. Antiseptics significantly reduced MSSA (1-4 log10 CFU/explants) after 0·25 h to 4 h. CHG, PHMB and OCT exhibited persistence at 24 h. In broth culture, CHG 0·012% and PI 0·625% achieved >6 log10 reductions at 2 h. PI-based formulations were more efficacious than unformulated PI. PI-based formulations exhibited no significant cytotoxicity on explants using an MTT assay. CONCLUSIONS: All antiseptics tested in the mucosal MSSA infection model reduced MSSA. CHG and PI were more potent in broth culture. SIGNIFICANCE AND IMPACT OF THE STUDY: We developed a semi-high-throughput mucosal model that can identify compounds or formulations with promising antimicrobial and limited cytotoxic properties.
Subject(s)
Anti-Infective Agents, Local/pharmacology , Microbial Sensitivity Tests , Models, Biological , Mucous Membrane/microbiology , Animals , Anti-Infective Agents, Local/toxicity , Biguanides/pharmacology , Biguanides/toxicity , Chlorhexidine/analogs & derivatives , Chlorhexidine/pharmacology , Chlorhexidine/toxicity , Female , Mucous Membrane/anatomy & histology , Povidone-Iodine/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/isolation & purification , Swine , Tissue Culture Techniques , Vagina/anatomy & histology , Vagina/microbiologyABSTRACT
Mechanical properties of arena surfaces are extrinsic factors for musculoskeletal injury. Vertical impact forces of harrowed and compacted cushion were measured at five locations on 12 arena surfaces (five dirt, seven synthetic [dirt and fiber]). Eight variables related to impact force, displacement, and acceleration were calculated. Surface temperature, cushion depth and moisture content were also measured. The effects of surface material type (dirt/synthetic) and cushion compaction (harrowed/compacted) on vertical impact properties were assessed using an analysis of variance. Relationships of manageable surface properties with vertical impact forces were examined through correlations. Compacted cushion exhibited markedly higher vertical impact force and deceleration with lower vertical displacement than harrowed cushion (P < 0.001), and the effect was greater on dirt than synthetic surfaces (P = 0.039). Vertical displacement (P = 0.021) and soil rebound (P = 0.005) were the only variables affected by surface type. Surface compaction (harrowed, compacted) had a significantly greater effect on vertical impact forces than surface type (dirt, synthetic). By reducing surface compaction through harrowing, extrinsic factors related to musculoskeletal injury risk are reduced. These benefits were more pronounced on dirt than synthetic surfaces. These results indicate that arena owners should regularly harrow surfaces, particularly dirt surfaces.
Subject(s)
Running , Animals , Surface PropertiesABSTRACT
Shear forces at the surface-hoof interface affect hoof slide, surface grip, forces transferred to the limb, and injury risk. However, the variation in shear forces among surfaces with different compositions have not been quantified. Shear ground reaction forces were measured on five dirt and seven synthetic arena surfaces. Cohesion/adhesion and angle of internal friction/coefficient of friction were calculated. Surface composition, surface temperature, cushion depth, and moisture content were also measured. The effects of surface material (dirt/synthetic) on shear properties were assessed using analysis of variance (ANOVA; P < 0.05). The relationships between surface composition or management properties and shear properties were analyzed using linear correlation. Shear properties were not different between dirt and synthetic surface categories; however, surface fiber content was correlated with adhesion and coefficient of friction. These correlations predict that more fiber will decrease soil adhesion (r = -0.75; P < 0.01) and increase the coefficient of friction (r = 0.81; P < 0.01). Furthermore, maximum shear force was significantly correlated with cushion depth (r = 0.61; P < 0.01) and moisture content (r = 0.57; P < 0.01), where shear force was greater on surfaces with thicker cushion layers or higher moisture content. The findings suggest that shear mechanical behavior is more dependent on surface composition than surface material categories (dirt/synthetic) and also indicate that arena owners can influence shear forces by adjusting either surface composition or management.
Subject(s)
Hoof and Claw , Horses , Animals , Surface Properties , Shear StrengthABSTRACT
It is well known that transport of nutrients and wastes as solute in bone fluid plays an important role in bone remodeling and damage healing. This work presents a chemo-poroelastic model for fluid and solute transport in the lacunar-canalicular network of an osteonal cortical bone under cyclic axial mechanical loading or vascular pressure. Analytical solutions are obtained for the pore fluid pressure, and fluid and solute velocities. Numerical results for fluid and calcium transport indicate that under a cyclic stress of 20 MPa, the magnitudes of the fluid and calcium velocities increase with an increase in the loading frequency for the frequency range considered (≤ 3 Hz) and peak at the inner boundary. The peak magnitude of calcium velocity reaches 18.9 µm/s for an osteon with a permeability of 1.5 × 10-19 m2 under a 3 Hz loading frequency. The magnitude of calcium velocity under a vascular pressure of 50 mmHg is found to be two orders of magnitude smaller than that under the mechanical load. These results have the potential to be important in understanding fundamental aspects of cortical bone remodeling as transport characteristics of calcium and other nutrients at the osteon scale influence bone metabolism.
Subject(s)
Haversian System/physiology , Models, Biological , Calcium , Elasticity , Extracellular Fluid , Porosity , Solvents , Stress, MechanicalABSTRACT
REASONS FOR PERFORMING STUDY: Differences in racing times have been noted on synthetic track surfaces that appear to depend on the temperature of the track. No published study to date has considered this effect in a systematic manner. OBJECTIVES: To investigate the relationship between temperature of track and speed of horses racing on a synthetic surface. Potential changes in the wax component of the synthetic track were investigated as one possible cause of changes in the track speed at the temperatures observed. METHODS: At Del Mar racetrack (California, USA), the air, surface and subsurface temperatures at 4 depths in the synthetic race surface were measured periodically throughout the day over a 42 day period. The 6 furlong (1.2 km) race (afternoon) and fast training 'work' (morning) times were also compiled. Samples of the track were obtained and the wax separated using a solvent separation technique. Differential scanning calorimetry was used to determine the range of temperatures at which the wax from the track underwent softening and other material changes. Transformation temperatures were compared to temperatures acquired from the track to evaluate the likelihood of changes in the wax properties during racing. RESULTS: Average air, surface and subsurface temperatures changed significantly throughout the day. Temperatures were higher during the afternoon race sessions and race times were significantly slower compared to morning work times. Temperatures at which some of the components of the wax began to soften were found to be within the range of temperature measured during track operation. CONCLUSIONS: A correlation was found between temperature of the synthetic track and speed of horse. Wax separated from the track showed that the temperatures experienced in the surface during normal operation exceed the temperatures at which the wax begins to experience thermal transformation. It is therefore hypothesised that the wax may be a cause of the observed changes in the track performance. POTENTIAL RELEVANCE: Future work should include a study of components of the synthetic track responsible for the change and epidemiological association of risk of injury.
Subject(s)
Horses/physiology , Running/physiology , Sports , Temperature , AnimalsABSTRACT
Permeability of a porous material with a fluid interface is shown to be related to the propagation of the slow longitudinal wave. The propagation threshold of the slow longitudinal wave occurs when the wave number is higher than the critical wave number, k(cr). Measuring k(cr) can provide the intrinsic permeability of a porous interface. The ability to detect a change in k(cr) due to differences in pore size and fluid viscosity is demonstrated. This measurement is demonstrated in a model material with two pore sizes and two fluid viscosities. The results suggest opportunities to extend the method to a range of materials of scientific and technical importance.
ABSTRACT
Subject(s)
Extensively Drug-Resistant Tuberculosis/epidemiology , Tuberculosis, Pulmonary/epidemiology , Adult , Cities , Cross-Sectional Studies , Demography , Extensively Drug-Resistant Tuberculosis/etiology , Female , Geographic Information Systems , Humans , Incidence , Male , Socioeconomic Factors , South Africa/epidemiology , Tuberculosis, Pulmonary/etiologyABSTRACT
When Thoroughbred racehorses experience catastrophic injuries, the track surface is often discussed as a factor. The present study investigated the mechanical properties of the surface and found that significant changes in a track occur during routine maintenance. Questions regarding the relative importance of track variability and hardness require further investigation.
Subject(s)
Horse Diseases/prevention & control , Horses/injuries , Lameness, Animal/prevention & control , Physical Conditioning, Animal/physiology , Soil , Wounds and Injuries/veterinary , Animals , Biomechanical Phenomena/physiology , Sports/standards , Wounds and Injuries/prevention & controlABSTRACT
In several biosynthetic pathways of eukaryotes, multiple steps are catalyzed by enzymes physically linked as domains of multi-enzymatic proteins. The same steps in prokaryotes are frequently carried out by mono-enzymatic proteins. If genes encoding mono-enzymatic proteins are the precursors to those genes encoding multi-enzymatic proteins, how these genes fused remains an open question. However, the recent discovery of a cleavage-polyadenylation signal within an intron of the GART gene provides clues to this process and might also have more general implications for the origin of genes that contain alternative RNA processing reactions at their 5' or 3' ends.
Subject(s)
Enzymes/genetics , Evolution, Molecular , Genes/genetics , Amino Acid Sequence , Animals , Humans , Molecular Sequence Data , RNA SplicingABSTRACT
Alternative RNA processing of the heavy-chain immunoglobulin mu gene is regulated during B-cell maturation and requires competition between splice and cleavage-polyadenylation reactions that have balanced efficiencies. Studies with modified mu genes have failed to identify gene-specific sequences required for regulation. Thus, the only important feature for regulation may be the balanced competing splice and cleavage-polyadenylation reactions themselves. If this is so, then alternative RNA processing from any gene with similar competitive RNA processing pathways should also be regulated when expression is compared between B cells and plasma cells. To test this prediction, two nonimmunoglobulin genes engineered to have competing splice and cleavage-polyadenylation reactions were expressed in B cells and plasma cells. The ratios of alternative RNAs produced from both genes are different in the two cell types; like the mu gene, relatively more spliced RNA is produced in B cells than in plasma cells. Also, in a survey of mu gene expression in nine non-B-cell lines, only a T-cell line had an expression pattern similar to that of B cells; the expression patterns of all other lines resembled that of the plasma cells. Therefore, regulated mu RNA processing must be mediated by changes in general processing factors whose activity or abundance is regulated, most likely, in B cells.
Subject(s)
B-Lymphocytes/metabolism , Plasma Cells/metabolism , RNA Processing, Post-Transcriptional , Alternative Splicing , Animals , Cell Line , Gene Expression Regulation , Genes, Immunoglobulin , Genes, MHC Class I , HeLa Cells , Humans , Immunoglobulin mu-Chains/genetics , In Vitro Techniques , Introns , Membrane Glycoproteins/genetics , Mice , Poly A/metabolism , Receptors, Antigen, B-Cell/geneticsABSTRACT
The relative abundance of the mRNAs encoding the membrane (mu m) and secreted (mu s) forms of immunoglobulin mu heavy chain is regulated during B-cell maturation by a change in the mode of RNA processing. Current models to explain this regulation involve either competition between cleavage-polyadenylation at the proximal (mu s) poly(A) site and cleavage-polyadenylation at the distal (mu m) poly(A) site [poly(A) site model] or competition between cleavage-polyadenylation at the mu s poly(A) site and splicing of the C mu 4 and M1 exons, which eliminates the mu s site (mu s site-splice model). To test certain predictions of these models and to determine whether there is a unique structural feature of the mu s poly(A) site that is essential for regulation, we constructed modified mu genes in which the mu s or mu m poly(A) site was replaced by other poly(A) sites and then studied the transient expression of these genes in cells representative of both early- and late-stage lymphocytes. Substitutions at the mu s site dramatically altered the relative usage of this site and caused corresponding reciprocal changes in the usage of the mu m site. Despite these changes, use of the proximal site was still usually higher in plasmacytomas than in pre-B cells, indicating that regulation does not depend on a unique feature of the mu s poly(A) site. Replacement of the distal (mu m) site had no detectable effect on the usage of the mu s site in either plasmacytomas or pre-B cells. These findings are inconsistent with the poly(A) site model. In addition, we noted that in a wide variety of organisms, the sequence at the 5' splice junction of the C mu 4-to-M1 intron is significantly different from the consensus 5' splice junction sequence and is therefore suboptimal with respect to its complementary base pairing with U1 small nuclear RNA. When we mutated this suboptimal sequence into the consensus sequence, the mu mRNA production in plasmacytoma cells was shifted from predominantly mu s to exclusively mu m. This result unequivocally demonstrated that splicing of the C mu 4-to-M1 exon is in competition with usage of the mu s poly(A) site. A key feature of this regulatory phenomenon appears to be the appropriately balanced efficiencies of these two processing reactions. Consistent with predictions of the mu s site-splice model, B cells were found to contain mu m precursor RNA that had undergone the C mu 4-to-M1 splice but had not yet been polyadenylated at the mu m site.
Subject(s)
Immunoglobulin mu-Chains/genetics , RNA, Messenger/biosynthesis , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Base Sequence , Cell Line , Chimera , Gene Expression Regulation , Models, Biological , Mutation , Poly A/genetics , Poly A/metabolism , RNA Processing, Post-Transcriptional , RNA Splicing , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
To determine whether there are any developmental changes in the efficiencies of cleavage-polyadenylation or splicing reactions that could affect the usage of weak (suboptimal) processing signals and thus provide a basis for the regulated production of mu m versus mu s mRNA during B-lymphocyte maturation, we studied the expression of transfected mu genes in which the natural competition between cleavage-polyadenylation and splicing was replaced by alternative usage of tandem weak and strong poly(A) sites or by competition between suboptimal and optimal 5' splice junctions. Our results indicate that there is a 50 to 100% increase in cleavage-polyadenylation efficiency but no measurable change in splicing efficiency as maturation proceeds from the B-cell to plasma cell stage.
Subject(s)
Cell Membrane/metabolism , Immunoglobulin mu-Chains/genetics , Poly A/metabolism , RNA Splicing , Adenovirus Early Proteins , Animals , Binding, Competitive , Genes , Mice , Oncogene Proteins, Viral/genetics , Plasmacytoma , RNA, Messenger/metabolism , Transfection , Tumor Cells, CulturedABSTRACT
The alternative RNA processing of microseconds and microns mRNAs from a single primary transcript depends on competition between a cleavage-polyadenylation reaction to produce microseconds mRNA and a splicing reaction to produce microns mRNA. The ratio of microseconds to microns mRNA is regulated during B-cell maturation; relatively more spliced microns mRNA is made in B cells than in plasma cells. The balance between the efficiencies of splicing and cleavage-polyadenylation is critical to the regulation. The mu gene can be modified to either reduce or improve the efficiency of each reaction and thus alter the ratio of the two RNAs produced. However, as long as neither reaction is so strong that it totally dominates, expression of the modified mu genes is regulated in B cells and plasma cells. The current experiments reveal a relationship between the C mu 4 exon size and the microseconds/microns expression ratio. The shorter the distance between the C mu 4 5' splice site and the nearest upstream 3' splice site, the more spliced microns mRNA was produced. Conversely, when this exon was expanded, more microseconds mRNA was produced. Expression from these mu genes with altered exon sizes were regulated between B cells and plasma cells. Since RNA processing in the mu gene can be considered a competition between defining the C mu 4 exon as an internal exon (in microns mRNA) versus a terminal exon (in microseconds mRNA), exon size may affect the competition among factors interacting with this exon.
Subject(s)
Genes, Immunoglobulin , Immunoglobulin mu-Chains/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Animals , B-Lymphocytes/cytology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Differentiation , Exons , Gene Expression , Mice , Plasma Cells/cytology , Plasma Cells/immunology , Plasma Cells/metabolism , Poly A/genetics , Poly A/metabolism , RNA Splicing , Sequence Deletion , TransfectionABSTRACT
The immunoglobulin (Ig) genes have been extensively studied as model systems for developmentally regulated alternative RNA processing. Transcripts from these genes are alternatively processed at their 3' ends to yield a transcript that is either cleaved and polyadenylated at a site within an intron or spliced to remove the poly(A) site and subsequently cleaved and polyadenylated at a downstream site. Results obtained from expressing modified genes in established tissue culture cell lines that represent different stages of B-lymphocyte maturation have suggested that the only requirement for regulation is that a pre-mRNA contain competing cleavage-polyadenylation and splice reactions whose efficiencies are balanced. Since several non-Ig genes modified to have an Ig gene-like structure are regulated in cell lines, Ig-specific sequences are not essential for this control. This strongly implies that changes in the amounts or activities of general RNA processing components mediate the processing regulation. Despite numerous studies in cell lines, this model of Ig gene regulation has never been tested in vivo during normal lymphocyte maturation. We have now introduced a non-Ig gene with an Ig gene-like structure into the mouse germ line and demonstrate that RNA from the transgene is alternatively processed and regulated in murine splenic B cells. This establishes that the balance and arrangement of competing cleavage-polyadenylation reactions are sufficient for RNA processing regulation during normal B-lymphocyte development. These experiments also validate the use of tissue culture cell lines for studies of Ig processing regulation. This is the first transgenic mouse produced to test a specific model for regulated mRNA processing.
Subject(s)
Alternative Splicing , B-Lymphocytes/cytology , Gene Expression Regulation , Immunoglobulin mu-Chains/genetics , RNA, Messenger/genetics , Transgenes , Animals , B-Lymphocytes/drug effects , Lipopolysaccharides/pharmacology , Mice , Mice, TransgenicABSTRACT
The development of safety and quality standards for equestrian surfaces needs to be based on objective, repeatable measurements which allow comparisons between surfaces. These measurements should incorporate the assessment of surface performance by riders. This study provides data from objective and subjective assessment of functional properties of high-level show jumping competition and warm-up arenas. Twenty-five arenas in nine international show jumping events were evaluated by mechanical in-situ testing with a surface tester, rider assessments using visual analogue scales (198 riders provided 749 arena evaluations), descriptions of arena constructions and by laboratory tests of surface material. Mixed models were used to present subjective evaluation of rider perception of the functional properties for each arena while controlling for rider and event. The association between objective and subjective assessments were also explored creating mixed models, controlling for rider and event. Mechanical measurements of impact firmness, and to a lesser extent cushioning and grip, had a significant positive association with the riders' perception. Responsiveness as assessed by the Orono biomechanical surface tester (OBST) was negatively associated with the riders' perceptions, which suggests riders and the OBST had different concepts of this functional property and that further developments of the OBST might be necessary. Objectively measured uniformity showed no useful association with riders' perception. Even though arena assessments were made by top level riders, a substantial inter-rider variation was demonstrated.
Subject(s)
Animal Welfare , Horses , Sports , Animal Welfare/standards , Animals , Biomechanical Phenomena , Humans , Surveys and QuestionnairesABSTRACT
Vertebrate internal exons are usually between 50 and 400 nt long; exons outside this size range may require additional exonic and/or intronic sequences to be spliced into the mature mRNA. The mouse polymeric immunoglobulin receptor gene has a 654 nt exon that is efficiently spliced into the mRNA. We have examined this exon to identify features that contribute to its efficient splicing despite its large size; a large constitutive exon has not been studied previously. We found that a strong 5' splice site is necessary for this exon to be spliced intact, but the splice sites alone were not sufficient to efficiently splice a large exon. At least two exonic sequences and one evolutionarily conserved intronic sequence also contribute to recognition of this exon. However, these elements have redundant activities as they could only be detected in conjunction with other mutations that reduced splicing efficiency. Several mutations activated cryptic 5' splice sites that created smaller exons. Thus, the balance between use of these potential sites and the authentic 5' splice site must be modulated by sequences that repress or enhance use of these sites, respectively. Also, sequences that enhance cryptic splice site use must be absent from this large exon.
Subject(s)
Alternative Splicing/genetics , Exons/genetics , Animals , Base Composition , Base Sequence , Conserved Sequence , Enhancer Elements, Genetic/genetics , Evolution, Molecular , Humans , Mice , Molecular Sequence Data , Mutation , Plasmids/genetics , RNA/genetics , RNA/metabolism , Receptors, Immunologic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Deletion , Sequence Homology, Nucleic Acid , Transfection , Tumor Cells, CulturedABSTRACT
The fourth exon of the mouse polymeric immuno-globulin receptor (pIgR) is 654 nt long and, despite being surrounded by large introns, is constitutively spliced into the mRNA. Deletion of an 84 nt sequence from this exon strongly activated both cryptic 5' and 3' splice sites surrounding a 78 nt cryptic intron. The 84 nt deletion is just upstream of the cryptic 3' splice site; the cryptic 3' splice site was likely activated because the deletion created a better 3' splice site. However, the cryptic 5' splice site was also required to activate the cryptic splice reaction; point mutations in either of the cryptic splice sites that decreased their match to the consensus splice site sequence inactivated the cryptic splice reaction. The activation and inactivation of these cryptic splice sites as a pair suggests that they are being co-recognized by the splicing machinery. Interestingly, the large fourth exon of the pIgR gene encodes two immunoglobulin-like extracellular protein domains; the cryptic 3' splice site coincides with the junction between these protein domains. The cryptic 5' splice site is located between protein subdomains where an intron is found in another gene of the immunoglobulin superfamily.
Subject(s)
Exons , Introns , RNA Splicing/genetics , Receptors, Immunologic/genetics , Animals , Base Sequence , Gene Deletion , Gene Expression Regulation , Humans , Immunoglobulin A/metabolism , Mice , Models, Genetic , Molecular Sequence Data , Plasmids , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Transfection , Tumor Cells, CulturedABSTRACT
REASONS FOR PERFORMING STUDY: Different horseshoe designs have been developed in an attempt to optimise footing for equine athletes. Horseshoe performance is assumed to be dependent on the surface and gait, but there are limited data on horseshoe performance on different surfaces, independent of gait variation. OBJECTIVES: To quantify the dynamic loading for 3 aluminium racing shoe designs on Thoroughbred racetrack surface materials, using a biomechanical surface tester. STUDY DESIGN: A flat racing plate, a serrated V-Grip and a shoe with a 6 mm toe grab and 10 mm heel calks were tested on synthetic and dirt surfaces under typical operating conditions of temperature and moisture content for the respective material samples. METHODS: Samples were tested under laboratory conditions, replicating a track surface by compacting material into a latex-lined mould surrounded by silica sand for representative boundary conditions. Peak loading and loading rates were measured vertically and horizontally (craniocaudal), simulating aspects of primary and secondary impacts of the hoof in a galloping horse. RESULTS: Maximum vertical and shear loads and loading rates were not significantly different between shoe types, with the exception of a reduced craniocaudal loading rate for the V-Grip shoe on the synthetic surface. All other statistical significance was related to the surface material. CONCLUSIONS: These 3 different Thoroughbred racing shoes do not have a significant impact on loading and loading rate, with the exception of the V-Grip shoe on a synthetic surface. Although the V-Grip may reduce craniocaudal peak load rates in a synthetic material with relatively high wax and/or low oil content, the reduction in load rate is less than the difference found between materials. This study indicates that shoeing has little effect, and that a track's surface material and its preparation have a significant effect on the dynamic loading during the impact phase of the stance.
Subject(s)
Construction Materials , Horses , Shoes , Surface Properties , Animals , Sports , Stress, MechanicalABSTRACT
The Escherichia coli lac promoter has been shown to contain an RNA polymerase binding site (P2) that overlaps with, and is shifted 22 base-pairs upstream from the normal lac promoter (P1). In this paper, we provide RNA polymerase protection data obtained in vitro that show that, in the absence of CAP-cAMP, in vitro P2 is the preferred polymerase binding site on the P+ template. In the presence of CAP-cAMP, polymerase binding to P2 is reduced and more polymerase is bound at P1. Two lac P1 "-35 region" mutations, L157 and 4, which increase the homology between this region and the consensus "-10 region" sequence, are both shown to have an increased affinity for polymerase binding at P2. CAP-cAMP is also able to decrease the amount of polymerase bound to P2 and to increase the amount bound to P1 on these mutant promoter fragments. P2 does not initiate transcription efficiently in vivo. Nuclease S1 mapping experiments detect only a low level of transcription from one of the P2 "up" mutations, but no beta-galactosidase synthesis is directed by this mutant. Mutations such as L157 and 4, which alter the P2-10 region, also alter lac P sensitivity to CAP-cAMP in vivo, suggesting that the P2 sequence plays a role in CAP-cAMP regulation of lac P. Possible roles for P2 in vivo are discussed.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Lac Operon , Base Sequence , Binding Sites , Cyclic AMP/metabolism , DNA, Bacterial , Exodeoxyribonucleases/metabolism , Mutation , Promoter Regions, Genetic , Receptors, Cyclic AMP/metabolism , Transcription, Genetic , beta-Galactosidase/metabolismABSTRACT
The Escherichia coli lac promoter mutation Pr115, an A X T to T X A transversion at +1 (the transcription initiation site of the lac wild-type and lac UV5 promoters), creates a new "-10 region"-like sequence starting at +1. We show that this mutation activates a new RNA polymerase binding site (P115) that overlaps with, and is shifted 12 base-pairs downstream from, the wild-type RNA polymerase binding site (P1). Nuclease S1 mapping studies and RNA polymerase protection experiments in vitro indicate that, in the absence of CAP-cAMP, this new site is used preferentially over the P1 site. In vivo, beta-galactosidase assays of the Pr115 mutation in combination with mutations of the P1 "-35 region" demonstrate that the P1 -35 region sequences are not involved in the interaction between RNA polymerase and P115 in the absence of CAP-cAMP; therefore P115 is an independent binding site. The presence of CAP-cAMP in vivo stimulates polymerase binding and initiation at P1, which serves to block polymerase from binding at P115.