RESUMEN
Unrelated clades of aquatic tetrapod have evolved a similar range of skull shapes, varying from longirostrine (elongate and narrow rostrum) to brevirostrine (short rostrum). However, it is unclear which aspects of organismal performance are associated with this convergence in the range of skull shapes. Furthermore, it is not known how fundamental anatomical differences between groups influence these relationships. Here we address this by examining the load bearing capabilities of the skulls of two of the most diverse groups of living aquatic tetrapod: crocodilians and odontocetes. We use finite element analysis to examine the abilities of different cranial morphologies to resist a range of biologically relevant feeding loads including biting, shaking and twisting. The results allow for form/function relationships to be compared and contrasted between the two groups. We find that cranial shape has similar influences on performance during biting, shaking or twisting load cases at the anterior tooth positions, e.g. brevirostrine species experienced less strain than longirostrine species. The pattern of this form/function relationship is similar for both crocodilians and odontocetes, despite their fundamentally different anatomies. However, when loading teeth at the posterior end or middle of the tooth row the results do not follow the same pattern. Behavioural differences in bite location plays a key role in determining functional abilities in aquatic tetrapod taxa.
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Caimanes y Cocodrilos , Cráneo/anatomía & histología , Cráneo/fisiología , Ballenas , Caimanes y Cocodrilos/anatomía & histología , Caimanes y Cocodrilos/fisiología , Animales , Fenómenos Biomecánicos , Fuerza de la Mordida , Análisis de Elementos Finitos , Diente/fisiología , Ballenas/anatomía & histología , Ballenas/fisiologíaRESUMEN
Biological invasions can induce rapid evolutionary change. As cane toads (Rhinella marina) have spread across tropical Australia over an 80-year period, their rate of invasion has increased from around 15 to 60 km per annum. Toads at the invasion front disperse much faster and further than conspecifics from range-core areas, and their offspring inherit that rapid dispersal rate. We investigated morphological changes that have accompanied this dramatic acceleration, by conducting three-dimensional morphometric analyses of toads from both range-core and invasion-front populations. Morphology of heads, limbs, pectoral girdles and pelvic girdles differed significantly between toads from the two areas, ranging from 0.5% to 16.5% difference in mean bone dimensions between populations, with invasion-front toads exhibiting wider forelimbs, narrower hindlimbs and more compact skulls. Those changes plausibly reflect an increased reliance on bounding (multiple short hops in quick succession) rather than separate large leaps. Within an 80-year period, invasive cane toads have converted the basic anuran body plan - which evolved for occasional large leaps to evade predators - into a morphotype better-suited to sustained long-distance travel.
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SUMMARY: This study examined the effect of a controlled dose of vibration upon bone density and architecture in people with spinal cord injury (who eventually develop severe osteoporosis). Very sensitive computed tomography (CT) imaging revealed no effect of vibration after 12 months, but other doses of vibration may still be useful to test. INTRODUCTION: The purposes of this report were to determine the effect of a controlled dose of vibratory mechanical input upon individual trabecular bone regions in people with chronic spinal cord injury (SCI) and to examine the longitudinal bone architecture changes in both the acute and chronic state of SCI. METHODS: Participants with SCI received unilateral vibration of the constrained lower limb segment while sitting in a wheelchair (0.6g, 30 Hz, 20 min, three times weekly). The opposite limb served as a control. Bone mineral density (BMD) and trabecular micro-architecture were measured with high-resolution multi-detector CT. For comparison, one participant was studied from the acute (0.14 year) to the chronic state (2.7 years). RESULTS: Twelve months of vibration training did not yield adaptations of BMD or trabecular micro-architecture for the distal tibia or the distal femur. BMD and trabecular network length continued to decline at several distal femur sub-regions, contrary to previous reports suggesting a "steady state" of bone in chronic SCI. In the participant followed from acute to chronic SCI, BMD and architecture decline varied systematically across different anatomical segments of the tibia and femur. CONCLUSIONS: This study supports that vibration training, using this study's dose parameters, is not an effective anti-osteoporosis intervention for people with chronic SCI. Using a high-spatial-resolution CT methodology and segmental analysis, we illustrate novel longitudinal changes in bone that occur after spinal cord injury.
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Osteoporosis/prevención & control , Traumatismos de la Médula Espinal/complicaciones , Vibración/uso terapéutico , Adolescente , Adulto , Densidad Ósea/fisiología , Hueso Esponjoso/diagnóstico por imagen , Niño , Preescolar , Femenino , Fémur/diagnóstico por imagen , Fémur/fisiopatología , Humanos , Lactante , Estudios Longitudinales , Extremidad Inferior , Masculino , Osteoporosis/diagnóstico por imagen , Osteoporosis/etiología , Osteoporosis/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Tibia/diagnóstico por imagen , Tibia/fisiopatología , Tomografía Computarizada por Rayos X/métodos , Adulto JovenRESUMEN
BACKGROUND: The mechanisms by which viruses induce asthma exacerbations are not well understood. OBJECTIVE: We characterized fluctuations in nasal aspirate cytokines during naturally occurring respiratory viral infections in children with asthma. METHODS: Sixteen children underwent home collections of nasal aspirates when they were without cold symptoms and again during self-reported respiratory illnesses. The presence of viral infection was ascertained by multiplex PCR. Cytokines were measured using multiplex immune assay. mRNA expression for selected markers of viral infection was measured using RT-PCR. A cumulative respiratory symptom score was calculated for each day of measurement. Generalized estimated equations were used to evaluate associations between viral infection and marker elevation, and between marker elevation and symptom score. RESULTS: The 16 patients completed a total of 37 weeks of assessment (15 'well' weeks; 22 self-assessed 'sick' weeks). Viral infections were detected in 3 of the 'well' weeks and 17 of the 'sick' weeks (10 rhinovirus, three coronavirus, two influenza A, two influenza B, two respiratory syncytial virus, one parainfluenza). Compared to virus-negative well weeks, nasal aspirate IFN-γ, CXCL8/IL-8, CXCL10/IP-10, CCL5/RANTES, CCL11/eotaxin-1, CCL2/MCP-1, CCL4/MIP-1ß, CCL7/MCP-3, and CCL20/MIP3α protein levels increased during virus-positive sick weeks. Only a subset of cytokines (IFN-γ, CXCL8, CCL2, CCL4, CCL5, and CCL20) correlated with self-reported respiratory tract symptoms. While many aspirates were dilute and showed no mRNA signal, viral infection significantly increased the number of samples that were positive for IFN-λ1, IFN-λ2/3, TLR3, RIG-I, and IRF7 mRNA. CONCLUSIONS AND CLINICAL RELEVANCE: We conclude that in children with asthma, naturally occurring viral infections apparently induce a robust innate immune response including expression of specific chemokines, IFNs, and IFN-responsive genes.
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Asma , Citocinas/metabolismo , Cavidad Nasal/inmunología , Infecciones del Sistema Respiratorio/inmunología , Virosis/inmunología , Virus/inmunología , Adolescente , Asma/inmunología , Asma/virología , Quimiocinas/inmunología , Quimiocinas/metabolismo , Niño , Citocinas/genética , Citocinas/inmunología , Femenino , Humanos , Inmunidad Innata , Interferones/inmunología , Interferones/metabolismo , Masculino , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/fisiopatología , Infecciones del Sistema Respiratorio/virología , Virosis/diagnóstico , Virosis/fisiopatología , Virosis/virología , Virus/clasificación , Virus/genética , Virus/aislamiento & purificaciónRESUMEN
The ability to warp three-dimensional (3D) meshes from known biological morphology to fit other known, predicted or hypothetical morphologies has a range of potential applications in functional morphology and biomechanics. One of the most challenging of these applications is Finite Element Analysis (FEA), a potentially powerful non-destructive tool in the prediction of mechanical behaviour. Geometric morphometrics is another typically computer-based approach commonly applied in morphological studies that allows for shape differences between specimens to be quantified and analysed. There has been some integration of these two fields in recent years. Although a number of shape warping approaches have been developed previously, none are easily accessible. Here we present an easily accessed method for warping meshes based on freely available software and test the effectiveness of the approach in FEA using the varanoid lizard mandible as a model. We further present new statistical approaches, strain frequency plots and landmark point strains, to analyse FEA results quantitatively and further integrate FEA with geometric morphometrics. Using strain frequency plots, strain field, bending displacements and landmark point strain data we demonstrate that the mechanical behaviour of warped specimens reproduces that of targets without significant error. The influence of including internal cavity morphology in FEA models was also examined and shown to increase bending displacements and strain magnitudes in FE models. The warping approaches presented here will be useful in a range of applications including the generation and analysis of virtual reconstructions, generic models that approximate species means, hypothetical morphologies and evolutionary intermediaries.
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Lagartos/anatomía & histología , Mandíbula/anatomía & histología , Modelos Anatómicos , Animales , Fenómenos Biomecánicos , Biometría/métodos , Biología Computacional/métodos , Análisis de Elementos Finitos , Imagenología Tridimensional/métodos , Filogenia , Estrés MecánicoRESUMEN
The aim of this study is to review the experience of the clonidine suppression test in a regional endocrine centre and to compare the diagnostic sensitivity and specificity using various previous published criteria. The design used is retrospective study. The subjects include 56 patients in whom clonidine suppression tests had been performed from 1995 to 2000: 15 with phaeochromocytoma and 41 patients in whom the diagnosis was excluded using a combination of biochemical testing, abdominal computed tomography scanning and clinical follow-up. Plasma catecholamines were measured by high pressure liquid chromatography on basal samples and at hourly intervals for 3 h after the administration of clonidine 300 µg orally and the following diagnostic criteria were applied: plasma noradrenaline+adrenaline>2.96 nmol l(-1) at 3 h post-clonidine or a baseline plasma adrenaline plus noradrenaline>11.82 nmol l(-1); plasma noradrenaline>2.96 nmol l(-1) at 3 h post-clonidine and plasma noradrenaline>2.96 nmol l(-1) and <50% fall in noradrenaline at 3 h post-clonidine. The results obtained is that mean plasma noradrenaline plus adrenaline fell across the test in 40/41 patients in the non-phaeochromocytoma patients and was lowest at 3 h (basal 2.28 ± 0.14 vs 1.36 ± 0.11 nmol l(-1), P<0.001). In the phaeochromocytoma group, clonidine had a variable effect on adrenaline plus noradrenaline levels with increases in 7/15. Using an abnormal result as a 3 h level of noradrenaline plus adrenaline>2.96 mmol l(-1) gave a sensitivity of 93% and specificity of 95%. When a 3 h noradrenaline>2.96 mmol l(-1) was used, sensitivity was 87% and specificity 95%. Using the former criteria, noradrenaline plus adrenaline>2.96 mmol l(-1), 1/15 in the phaeochromocytoma group had a normal result after clonidine suppression testing. Two of 41 in the non-phaeochromocytoma group had a false-positive result. Under carefully controlled conditions, the clonidine suppression test is well tolerated, safe and accurate for use in the investigation of patients with suspected phaeochromocytoma.
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Neoplasias de las Glándulas Suprarrenales/diagnóstico , Clonidina , Pruebas Diagnósticas de Rutina , Feocromocitoma/diagnóstico , Neoplasias de las Glándulas Suprarrenales/sangre , Antihipertensivos , Catecolaminas/sangre , Epinefrina/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Norepinefrina/sangre , Feocromocitoma/sangre , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
Although a strong correlation between jaw mechanics and prey selection has been demonstrated in bony fishes (Osteichthyes), how jaw mechanics influence feeding performance in cartilaginous fishes (Chondrichthyes) remains unknown. Hence, tooth shape has been regarded as a primary predictor of feeding behavior in sharks. Here we apply Finite Element Analysis (FEA) to examine form and function in the jaws of two threatened shark species, the great white (Carcharodon carcharias) and the sandtiger (Carcharias taurus). These species possess characteristic tooth shapes believed to reflect dietary preferences. We show that the jaws of sandtigers and great whites are adapted for rapid closure and generation of maximum bite force, respectively, and that these functional differences are consistent with diet and dentition. Our results suggest that in both taxa, insertion of jaw adductor muscles on a central tendon functions to straighten and sustain muscle fibers to nearly orthogonal insertion angles as the mouth opens. We argue that this jaw muscle arrangement allows high bite forces to be maintained across a wider range of gape angles than observed in mammalian models. Finally, our data suggest that the jaws of sub-adult great whites are mechanically vulnerable when handling large prey. In addition to ontogenetic changes in dentition, further mineralization of the jaws may be required to effectively feed on marine mammals. Our study is the first comparative FEA of the jaws for any fish species. Results highlight the potential of FEA for testing previously intractable questions regarding feeding mechanisms in sharks and other vertebrates.
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Fuerza de la Mordida , Conducta Alimentaria/fisiología , Maxilares/anatomía & histología , Maxilares/fisiología , Masticación/fisiología , Animales , Fenómenos Biomecánicos , Tiburones/anatomía & histología , Tiburones/fisiologíaRESUMEN
The incidence of thyroid cancer has increased dramatically, yet the mortality has remained unchanged. There remain many challenges and "difficult problems" in diagnosing and treating patients with differentiated thyroid cancer (DTC). A significant percentage of patients with DTC will present with a thyroid nodule and an indeterminate fine needle aspiration biopsy underscoring the importance of proper evaluation and management. It is important when considering the extent of thyroidectomy and the management of lymph node metastases to balance the outcome of treatment in terms of recurrence and mortality with the morbidity of the procedure. Total thyroidectomy and lymphadenectomy for macroscopic lymph node metastases appear to be the optimal therapy for most patients with DTC. Preservation of function is a priority in managing patients with DTC invading the recurrent laryngeal nerve, trachea, esophagus, or the larynx.
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Neoplasias de la Tiroides/cirugía , Tiroidectomía/métodos , Humanos , Escisión del Ganglio Linfático , Invasividad Neoplásica , Neoplasias de la Tiroides/patología , Neoplasias de la Tráquea/patologíaRESUMEN
BACKGROUND: Sporadic nodular goiter is a common problem in the United States and significant compressive symptoms may occur with progression to a critical size. METHODS: Potential epidemiological variables associated with the development of large unilateral (> or = 50 g) and bilateral (> or = 100 g) nodular goiter were investigated including: age, gender, race, body mass index (BMI), family history of thyroid disease, pregnancy at time of diagnosis, insurance status, and tobacco or alcohol use. Data were obtained from an IRB-approved thyroid database and retrospective chart review of consecutive patients operated on for nodular goiter from 1990 through 2005. A univariate and multivariate analysis of epidemiological variables in patients with "large" versus "small" nodular goiter was completed. RESULTS: Of the 488 patients operated on for nodular goiter, 113 (23%) were classified as "large," 43 with unilateral (mean 106 +/- 72 g) and 70 with bilateral enlargement (mean 173 +/- 92 g) and 375 (77%) were classified as "small," 179 with unilateral (18 +/- 10 g) and 196 with bilateral (37 +/- 24 g) enlargement. Based on univariate analysis, African-American race, age > or = 40 years, BMI > or = 30 kg/m2, and lack of insurance were associated with an increased risk of large nodular goiter (P < or = 0.001), whereas alcohol use was protective (P = 0.002). A multivariate analysis revealed that African-American race [adjusted odds ratio (adj. OR) 3.3, 95% CI = 2.0-5.4], age > or = 40 years (adj. OR 2.1, 95% CI = 1.2-3.8), and BMI > or = 30 kg/m2 (adj. OR 2.5, 95% CI = 1.5-4.0) were independently associated with large nodular goiter. No significant differences were observed in gender, family history of thyroid disease, pregnancy, or tobacco use (P > 0.1). CONCLUSIONS: African-American race, obesity, and increasing age are independent risk factors for the development of large nodular goiter. These results may be helpful in determining how best to monitor patients with nodular goiter, with earlier intervention to help prevent progressive enlargement and its sequelae.
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Bocio Nodular/epidemiología , Bocio Nodular/patología , Glándula Tiroides/patología , Adulto , Negro o Afroamericano , Índice de Masa Corporal , Femenino , Bocio Nodular/etnología , Humanos , Masculino , Oportunidad Relativa , Tamaño de los Órganos , EmbarazoRESUMEN
Cross-resistance between zidovudine, stavudine, and lamivudine was studied, using purified recombinant reverse transcriptase from a zidovudine-susceptible and -resistant pair of clinical isolates of human immunodeficiency virus type 1. The zidovudine-resistant isolate exhibited low-level cross-resistance to both stavudine and lamivudine in drug susceptibility assays. Enzyme from the resistant isolate demonstrated reduced inhibition by zidovudine triphosphate and stavudine triphosphate and, to a lesser extent, lamivudine triphosphate. These findings provide additional evidence at the viral and enzyme level for cross-resistance between zidovudine and stavudine, and they suggest a possible effect of zidovudine resistance on susceptibility to lamivudine.
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Fármacos Anti-VIH/farmacología , Citidina Trifosfato/análogos & derivados , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Lamivudine/análogos & derivados , Inhibidores de la Transcriptasa Inversa/farmacología , Zidovudina/análogos & derivados , Citidina Trifosfato/farmacología , Didesoxinucleótidos , Relación Dosis-Respuesta a Droga , Farmacorresistencia Microbiana , Escherichia coli/genética , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , Lamivudine/farmacología , ADN Polimerasa Dirigida por ARN/biosíntesis , ADN Polimerasa Dirigida por ARN/genética , Proteínas Recombinantes/antagonistas & inhibidores , Estavudina/farmacología , Nucleótidos de Timina/farmacología , Transfección , Zidovudina/farmacología , Zidovudina/uso terapéuticoRESUMEN
The delta' subunit of the DNA polymerase-III holoenzyme is a key component of the DnaX complex; it is required for loading the beta(2) processivity factor onto a primed template. The x-ray crystal structure of delta' indicates a three domain C-shaped structure (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). In this study, we localized the DnaX-binding domain of delta' to its carboxyl-terminal domain III by quantifying protein-protein interactions using a series of delta' fusion proteins lacking specific domains. The fusion protein corresponding to domain III of delta' bound to DnaX with an affinity approaching that of full-length delta'. In contrast, a construct bearing delta' domains I-II did not bind DnaX at detectable levels. The presence of delta and chi psi strengthened the interaction of DnaX with full-length delta' and delta' domain III. Thus, domain III of delta' not only contains the DnaX-binding site, but also contains the elements required for positive cooperative assembly of the DnaX complex. A domain III-specific anti-delta' monoclonal antibody interfered with DnaX complex formation and abolished the replication activity of DNA polymerase III holoenzyme.
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Proteínas Bacterianas/metabolismo , ADN Polimerasa III/metabolismo , Anticuerpos Monoclonales/metabolismo , ADN Polimerasa III/química , ADN Polimerasa III/genética , Sustancias Macromoleculares , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The delta and delta' subunits are essential components of the DNA polymerase III holoenzyme, required for assembly and function of the DnaX-complex clamp loader (tau2gammadeltadelta'chipsi). The x-ray crystal structure of delta' contains three structural domains (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). In this study, we localize the delta-binding domain of delta' to a carboxyl-terminal domain III by quantifying the interaction of delta with a series of delta' fusion proteins lacking specific domains. Purification and immobilization of the fusion proteins were facilitated by the inclusion of a tag containing hexahistidine and a short biotinylation sequence. Both NH2- and COOH-terminal-tagged full-length delta' were soluble and had specific activities comparable with that of native delta'. delta and delta' form a 1:1 heterodimer with a dissociation constant (K(D)) of 5 x 10(-7) m determined by equilibrium sedimentation. The K(D) determined by surface plasmon resonance was comparable. Domain III alone bound delta at an affinity comparable to that of wild type delta', whereas proteins lacking domain III did not bind delta. Using a panel of domain-specific anti-delta' monoclonal antibodies, we found that two of the domain III-specific monoclonal antibodies interfered with delta-delta' interaction and abolished the replication activity of DNA polymerase-III holoenzyme.
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ADN Polimerasa III/metabolismo , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Cromatografía Liquida , Cristalografía por Rayos X , ADN Polimerasa III/química , ADN Polimerasa III/inmunología , ADN Polimerasa III/aislamiento & purificación , Cartilla de ADN , Replicación del ADN , Electroforesis en Gel de Poliacrilamida , Unión ProteicaRESUMEN
A dominant form of human congenital nightblindness is caused by a gly90-->asp (G90D) mutation in rhodopsin. G90D has been shown to activate the phototransduction cascade in the absence of light in vitro. Such constitutive activity of G90D rhodopsin in vivo would desensitize rod photoreceptors and lead to nightblindness. In contrast, other rhodopsin mutations typically give rise to nightblindness by causing rod cell death. Thus, the proposed desensitization without rod degeneration would be a novel mechanism for this disorder. To explore this possibility, we induced mice to express G90D opsin in their rods and then examined rod function and morphology, after first crossing the transgenic animals with rhodopsin knock-out mice to obtain appropriate levels of opsin expression. The G90D mouse opsin bound the chromophore and formed a bleachable visual pigment with lambda(max) of 492 nm that supported rod photoresponses. (G+/-, R+/-) retinas, heterozygous for both G90D and wild-type (WT) rhodopsin, possessed normal numbers of photoreceptors and had a normal rhodopsin complement but exhibited considerable loss of rod sensitivity as measured electroretinographically. The rod photoresponses were desensitized, and the response time to peak was faster than in (R+/-) animals. An equivalent desensitization resulted by exposing WT retinas to a background light producing 82 photoisomerizations rod(-1) sec(-1), suggesting that G90D rods in darkness act as if they are partially "light-adapted." Adding a second G90D allele gave (G+/+, R+/-) animals that exhibited a further increase of equivalent background light level but had no rod cell loss by 24 weeks of age. (G+/+, R-/-) retinas that express only the mutant rhodopsin develop normal rod outer segments and show minimal rod cell loss even at 1 year of age. We conclude that G90D is constitutively active in mouse rods in vivo but that it does not cause significant rod degeneration. Instead, G90D desensitizes rods by a process equivalent to light adaptation.
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Adaptación Ocular/genética , Ceguera Nocturna/etiología , Ceguera Nocturna/fisiopatología , Células Fotorreceptoras Retinianas Bastones/fisiopatología , Rodopsina/metabolismo , Alelos , Sustitución de Aminoácidos , Animales , Recuento de Células , Modelos Animales de Enfermedad , Relación Dosis-Respuesta en la Radiación , Electrorretinografía , Genes Dominantes , Genotipo , Heterocigoto , Homocigoto , Humanos , Inmunohistoquímica , Luz , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Retina/patología , Retina/fisiopatología , Células Fotorreceptoras Retinianas Bastones/patología , Rodopsina/genética , Rodopsina/efectos de la radiaciónRESUMEN
Although the two alternative Escherichia coli dnaX gene products, tau and gamma, are found co-assembled in purified DNA polymerase III holoenzyme, the pathway of assembly is not well understood. When the 10 subunits of holoenzyme are simultaneously mixed, they rapidly form a nine-subunit assembly containing tau but not gamma. We developed a new assay based on the binding of complexes containing biotin-tagged tau to streptavidin-coated agarose beads to investigate the effects of various DNA polymerase III holoenzyme subunits on the kinetics of co-assembly of gamma and tau into the same complex. Auxiliary proteins in combination with delta' almost completely blocked co-assembly, whereas chipsi or delta' alone slowed the association only moderately compared with the interaction of tau with gamma alone. In contrast, DNA polymerase III core, in the absence of deltadelta' and chipsi, accelerated the co-assembly of tau and gamma, suggesting a role for DNA polymerase III' [tau(2)(pol III core)(2)] in the assembly pathway of holoenzyme.
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Proteínas Bacterianas/química , ADN Polimerasa III/química , Holoenzimas/química , Proteínas Bacterianas/biosíntesis , ADN Polimerasa III/biosíntesis , Subunidades de ProteínaRESUMEN
The tau and gamma proteins of the DNA polymerase III holoenzyme DnaX complex are products of the dnaX gene with gamma being a truncated version of tau arising from ribosomal frameshifting. tau is comprised of five structural domains, the first three of which are shared by gamma (Gao, D., and McHenry, C. (2001) J. Biol. Chem. 276, 4433-4453). In the absence of the other holoenzyme subunits, DnaX exists as a tetramer. Association of delta, delta', chi, and psi with domain III of DnaX(4) results in a DnaX complex with a stoichiometry of DnaX(3)deltadelta'chipsi. To identify which domain facilitates DnaX self-association, we examined the properties of purified biotin-tagged DnaX fusion proteins containing domains I-II or III-V. Unlike domain I-II, treatment of domain III-V, gamma, and tau with the chemical cross-linking reagent BS3 resulted in the appearance of high molecular weight intramolecular cross-linked protein. Gel filtration of domains I-II and III-V demonstrated that domain I-II was monomeric, and domain III-V was an oligomer. Biotin-tagged domain III-V, and not domain I-II, was able to form a mixed DnaX complex by recruiting tau, delta, delta', chi, and psi onto streptavidin-agarose beads. Thus, domain III not only contains the delta, delta', chi, and psi binding interface, but also the region that enables DnaX to oligomerize.
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Proteínas Bacterianas/metabolismo , ADN Polimerasa III/metabolismo , Escherichia coli/metabolismo , Proteínas tau/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Biopolímeros , ADN Polimerasa III/química , ADN Polimerasa III/aislamiento & purificación , Replicación del ADN , Escherichia coli/enzimología , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
delta and delta' are required for assembly of the processivity factor beta(2) onto primed DNA in the DNA polymerase III holoenzyme-catalyzed reaction. We developed protocols for generating highly purified preparations of delta and delta'. In holoenzyme reconstitution assays, delta' could not be replaced by delta, tau, or gamma, even when either of the latter were present at a 10,000-fold molar excess. Likewise, delta could not be replaced by delta', tau, or gamma. Bacterial strains bearing chromosomal knockouts of either the holA(delta) or holB(delta') genes were not viable, demonstrating that both delta and delta' are essential. Western blots of isolated initiation complexes demonstrated the presence of both delta and delta'. However, in the absence of chipsi and single-stranded DNA-binding protein, a stable initiation complex lacking deltadelta' was isolated by gel filtration. Lack of delta-delta' decreased the rate of elongation about 3-fold, and the extent of processive replication was significantly decreased. Adding back delta-delta' but not chipsi, delta, or delta' alone restored the diminished activity, indicating that in addition to being key components required for the beta loading activity of the DnaX complex, deltadelta' is present in initiation complex and is required for processive elongation.
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ADN Polimerasa III/metabolismo , Replicación del ADN , Holoenzimas/metabolismo , Adenosina Trifosfato/metabolismo , ADN Polimerasa III/clasificación , Holoenzimas/clasificación , Subunidades de ProteínaRESUMEN
The DNA Polymerase III holoenzyme forms initiation complexes on primed DNA in an ATP-dependent reaction. We demonstrate that the nonhydrolyzable ATP analog, ATP gamma S, supports the formation of an isolable leading strand complex that loads and replicates the lagging strand only in the presence of ATP, beta, and the single-stranded DNA binding protein. The single endogenous DnaX complex within DNA polymerase III holoenzyme assembles beta onto both the leading and lagging strand polymerases by an ordered mechanism. The dimeric replication complex disassembles in the opposite order from which it assembled. Upon ATP gamma S-induced dissociation, the leading strand polymerase is refractory to disassembly allowing cycling to occur exclusively on the lagging strand. These results establish holoenzyme as an intrinsic asymmetric dimer with distinguishable leading and lagging strand polymerases.