Delphi consensus on the diagnosis and treatment of patients with short stature in Spain: GROW-SENS study.

PURPOSE: To identify consensus aspects related to the diagnosis, monitoring, and treatment of short stature in children to promote excellence in clinical practice. METHODS: Delphi consensus organised in three rounds completed by 36 paediatric endocrinologists. The questionnaire consisted of 26 topics grouped into: (1) diagnosis; (2) monitoring of the small-for-gestational-age (SGA) patient; (3) growth hormone treatment; and (4) treatment adherence. For each topic, different questions or statements were proposed. RESULTS: After three rounds, consensus was reached on 16 of the 26 topics. The main agreements were: (1) diagnosis tests considered as a priority in Primary Care were complete blood count, biochemistry, thyroid profile, and coeliac disease screening. The genetic test with the greatest diagnostic value was karyotyping. The main criterion for initiating a diagnostic study was prediction of adult stature 2 standard deviations below the target height; (2) the main criterion for initiating treatment in SGA patients was the previous growth pattern and mean parental stature; (3) the main criterion for response to treatment was a significant increase in growth velocity and the most important parameter to monitor adverse events was carbohydrate metabolism; (4) the main attitude towards non-responding patients is to check their treatment adherence with recording devices. The most important criterion for choosing the delivery device was its technical characteristics. CONCLUSIONS: This study shows the different degrees of consensus among paediatric endocrinologists in Spain concerning the diagnosis and treatment of short stature, which enables the identification of research areas to optimise the management of such patients.

[Prophylactic thyroidectomy in RET proto-oncogen mutation]. / Tiroidectomía profiláctica en mutación del protooncogén RET.

OBJECTIVE: Analysis of prophylactic thyroidectomy cases carried out in our Center in patients with RET gene mutations. MATERIAL AND METHODS: Retrospective study of 25 patients with RET proto-oncogene mutations subjected to prophylactic thyroidectomy between January 2000 and January 2016. Epidemiologic variables, surgical technique, histological results and follow-up were studied. RESULTS: Our sample consists of 25 patients, 15 males and 10 females. The range of age was from 7 months to 12 years old, with a median of 5 years old. We obtained 21 cases with NEM2A, from which 19 (76%) presented 634 mutation and 2 (8%) presented 611 mutation. Four cases were NEM2B, all with 918 mutation. Microscopical findings showed microcarcinoma, in situ carcinoma or medullary thyroid carcinoma in 16 patients (64%). Eight of them showed hyperplasia (32%) and 1 presented fibrosis (4%). The presence of elevated calcitonin was correlated with histologic alterations in 7 cases (43.7%), without significant differences (χ2 0.3; p 0.6). From 16 patients with carcinoma (13 NEM2A and 3 NEM2B), 10 were 5 years old or less at the moment of the surgery. A total thyroidectomy was performed in all patients. There were no intra or post-surgical complications. During the follow-up of the patients, levels of calcitonin, calcium, parathormone, catecholamines and metanephrines were normal, except from one case. CONCLUSIONS: The study of RET proto-oncogene allows the identification of patients susceptible of performing a prophylactic thyroidectomy, which have to be carried out early, in an experienced centers.

OBJETIVO: Analizar los casos de tiroidectomía profiláctica realizados en nuestro centro en pacientes con mutaciones del gen RET. MATERIAL Y METODOS: Estudio retrospectivo de 25 pacientes con mutación del protooncogén RET a los que se les realizó tiroidectomía profiláctica entre enero del 2000 y enero de 2016. Se estudiaron variables epidemiológicas, técnica quirúrgica, resultados anatomopatológicos y seguimiento. RESULTADOS: Nuestra serie consta de 25 pacientes, 15 varones y 10 mujeres. La mediana de la edad fue de 5 años con un rango de 7 meses a 12 años. Obtuvimos 21 casos con MEN2A de los que 19 (76%) presentaban la mutación 634 y 2 (8%) la mutación 611. Cuatro casos fueron MEN2B, todos con la mutación 918. Los hallazgos microscópicos revelaron microcarcimona, carcinoma in situ o carcinoma medular de tiroides en 16 casos (64%). 8 presentaron hiperplasia (32%) y 1 (4%) fibrosis. La presencia de calcitonina elevada se correlacionó con alteraciones anatomopatológicas en 7 casos (43,7%), pero no mostró diferencias significativas (χ² 0,3; p 0,6). De los 16 pacientes con carcinoma, (13 MEN2A, 3 MEN2B), 10 de ellos (62,5%) tenían 5 años o menos en el momento de la intervención. En todos los casos se realizó tiroidectomía total. No existieron complicaciones intra ni postoperatorias. Durante el seguimiento, los valores de calcitonina, calcio, paratohormona, catecolaminas y metanefrinas se han mantenido normales, excepto en 1 paciente. CONCLUSIONES: El estudio del protooncogén RET permite identificar pacientes susceptibles de realizar tiroidectomía profiláctica, la cual debe ser realizada de forma precoz, y en centros con experiencia.

Carotid artery stenting in patients with symptomatic carotid stenosis: a single-center series.

OBJECTIVES: Carotid angioplasty with stenting (CAS) in patients with carotid stenosis (CS) has become more restricted in France especially since the disclosure of such studies as EVA-3S and Stent-supported percutaneous angioplasty of the carotid artery versus endarterectomy (SPACE). This report is of a series of CS cases contraindicated for endarterectomy that underwent CAS at a French center of interventional neuroradiology. PATIENTS AND METHODS: Fifty-five patients with symptomatic CS more than 60% consecutively submitted to CAS between September 2008 and February 2011. The primary endpoint was either death or stroke within 30 days of the procedure; a secondary goal was to identify any possible factors that might have influenced the success and outcome of the intervention. RESULTS: The overall periprocedural stroke/death rate at 30 days was 5.4% (three out of 55 patients), with three non-disabling strokes and no deaths. Twenty-seven patients (49.1%) were treated with a cerebral protection device (CPD). Stent placement was achieved in all cases. Open- and closed-cell stents were implanted in 40 (72.7%) and 15 procedures (27.3%), respectively. Neither the use of a CPD, the carotid stent cell design nor any anatomical or technical factors were associated with a lower risk of stroke or death within 30 days of CAS. CONCLUSION: CAS in symptomatic patients with CS contraindicated for endarterectomy in this selected French series proved feasible and safe, with acceptable levels of morbidity. Use of a CPD, type of stent (open- or closed-cell), and anatomical and technical factors had no influence on the success of the procedure or the outcome within 30 days of the operation.

[Management of stroke in sub-Saharan Africa: current issues]. / Prise en charge des accidents vasculaires cérébraux en Afrique subsaharienne.

In sub-Saharan Africa, stroke is likely to present an increasingly important public health problem with a larger relative share of overall morbidity and mortality. Overall, sub-Saharan Health Care is characterized by a lack of human resources, lack of facilities for special investigations, and especially an absence of specific programs addressing the prevention of cardiovascular conditions. Current data on the epidemiology of stroke in sub-Saharan Africa, although sparse and fragmentary, indicate a comparatively high incidence of cerebral hemorrhage associated with high blood pressure, while ischemic stroke in black Africans still appears to be related primarily to small artery disease, HIV infection, and sickle cell disease. With urbanization, the role of large-vessel atherosclerosis is increasing. It is thus essential to coordinate government funding, health care professionals and development agencies to address this rising health problem. Access to health care needs to be better structured, and screening programs should be developed in order to identify and treat vascular risk factors. Improved training of health care professionals is also required in the areas of prevention, diagnosis and management of stroke. Implementation of best-practice recommendations for the management of stroke adapted to the specificities and resources of African countries would help rationalize the scarce resources currently available.

[Complete isosexual precocious puberty: clinical, pelvic ultrasound and laboratory features]. / Pubertad precoz completa isosexual: hallazgos clínicos, de laboratorio y ecografía pélvica.

OBJECTIVE: To determine whether initial presentation varies according to aetiology, whether such differences allow differential diagnosis between idiopathic and organic forms, and whether CNS imaging can be avoided in some patients with central precocious puberty (CPP). PATIENTS AND METHODS: Children referred for evaluation of precocious puberty were evaluated, and the subpopulation of children with CPP was enrolled in this prospective observational study. Clinical, laboratory and ultrasound features of 62 consecutive patients with CPP (5 boys and 57 girls) were recorded. We compared the characteristics of idiopathic (3 boys, 49 girls) and organic (2 boys, 8 girls) CPP. RESULTS: There were no differences in pubertal staging, age at puberty onset (7.0 [5.8-7.5] vs. 7.3 [5.1-8.3] years), bone age/chronological age ratio (1.26 [1.2-1.3] vs. 1.23 [1.1-1.3]), maternal menarche (11.7+/-0.2 vs. 11.7+/-0.6 years) between idiopathic and organic CPP, respectively. Organic CPP patients had a poorer height SD (0.35+/-0.4 vs. 1.6+/-0.1; p<0.01), predicted adult height, growth rate and growth rate SD (0.8+/-0.9 vs. 3.7+/-0.7). Girls with organic CPP had significantly higher oestradiol levels (47.5 [25-68] vs. 27 [14-43] pg/ml) than girls with idiopathic CPP. Pelvic ultrasound at the time of diagnosis revealed the presence of pubertal changes in internal genitalia in 43.9% of girls (37.2% idiopathic versus 62.5% organic CPP subpopulation; p=0.18). CONCLUSIONS: There is a clinical-ultrasound overlap between idiopathic and organic CPP. Imaging remains necessary in all cases of central precocious puberty, and ultrasound data should not be replaced by other diagnostic investigations.

Reciprocal modulatory interaction between human immunodeficiency virus type 1 Tat and transcription factor NFAT1.

Human immunodeficiency virus type 1 (HIV-1) gene expression is regulated by interactions between both viral and host factors. These interactions are also responsible for changes in the expression of many host cell genes, including cytokines and other immune regulators, which may account for the state of immunological dysregulation that characterizes HIV-1 infection. We have investigated the role of a host cell protein, the transcription factor NFAT1, in HIV-1 pathogenesis. We show that NFAT1 interacts with Tat and that this interaction, which involves the major transactivation domain of NFAT1 and the amino-terminal region of Tat, results in a reciprocal modulatory interplay between the proteins: whereas Tat enhances NFAT1-driven transcription in Jurkat T cells, NFAT1 represses Tat-mediated transactivation of the HIV-1 long terminal repeat (LTR). Moreover, NFAT1 binds to the kappaB sites on the viral LTR and negatively regulates NF-kappaB-mediated activation of HIV-1 transcription, by competing with NF-kappaB1 for its binding sites on the HIV-1 LTR. Tat-mediated enhancement of NFAT1 transactivation may explain the upregulation of interleukin 2 and other cytokines that occurs during HIV-1 infection. We discuss the potentially opposing roles of NFAT1 and another family member, NFAT2, in regulating gene transcription of HIV-1 and endogenous cytokine genes.

Partners in transcription: NFAT and AP-1.

Combinatorial regulation is a powerful mechanism that enables tight control of gene expression, via integration of multiple signaling pathways that induce different transcription factors required for enhanceosome assembly. The four calcium-regulated transcription factors of the NFAT family act synergistically with AP-1 (Fos/Jun) proteins on composite DNA elements which contain adjacent NFAT and AP-1 binding sites, where they form highly stable ternary complexes to regulate the expression of diverse inducible genes. Concomitant induction of NFAT and AP-1 requires concerted activation of two different signaling pathways: calcium/calcineurin, which promotes NFAT dephosphorylation, nuclear translocation and activation; and protein kinase C (PKC)/Ras, which promotes the synthesis, phosphorylation and activation of members of the Fos and Jun families of transcription factors. A fifth member of the NFAT family, NFAT5, controls the cellular response to osmotic stress, by a mechanism that requires dimer formation and is independent of calcineurin or of interaction with AP-1. Pharmacological interference with theNFAT:AP-1 interaction may be useful in selective manipulation of the immune response. Balanced activation of NFAT and AP-1 is known to be required for productive immune responses, but the role of NFAT:AP-1 interactions in other cell types and biological processes remains to be understood.

An improved vector system for constructing transcriptional lacZ fusions: analysis of regulation of the dnaA, dnaN, recF and gyrB genes of Escherichia coli.

We describe a new vector system for the in vitro construction of transcriptional fusions to the lacZ gene, which is expressed from the translational start signals of galK. The galK ribosome-binding site (RBS) and its natural preceding region ensure a constant efficiency for lacZ translation and, thus, the beta-galactosidase (beta Gal) production of a given fusion is directly proportional to the in vivo transcriptional activity of the inserted DNA fragment. Single-copy lambda prophage versions of multicopy constructs can be made by in vivo recombination. We use this system to compare the transcriptional activities of the promoters present in the dnaA-dnaN-recF-gyrB cluster. The order of strength of these promoters is gyrB > dnaA > recF > dnaN. It is assumed that gyrB belongs to the dnaA-dnaN-recF operon, because the short recF-gyrB intercistronic region does not contain a terminator. By using this new vector system, we have detected strong termination signals within recF that are functional even when recF is translated at its normal rate. The low level of transcription coming to the end of recF, and the highest activity of the gyrB promoter, as well as results obtained with several gyrB::lacZ translational fusions, support the conclusion that gyrB is predominantly expressed from its own promoter under standard growth conditions. Finally, we have found that transcription from the dnaA promoters is constant at different growth rates. This supports the idea that autoregulation of the dnaA gene is responsible for the coupling of the DnaA protein synthesis to cell mass increase, and accumulation of DnaA protein governs the initiation of chromosome replication.

Positive and negative regulatory elements in the dnaA-dnaN-recF operon of Escherichia coli.

The recF gene of E coli lies within a cluster of genes which play essential roles in DNA replication; the gene order is dnaA dnaN recF gyrB. Each of these genes has its own promoters which, with the exception of dnaA promoters, reside entirely within the translated region of the respective preceding gene. In this report, we analyze the effect of the dnaA and dnaN promoters on recF expression by translational fusions between recF and the lacZ reporter gene. Our results indicate that recF is a distal gene of the dnaA operon, and support the previous proposal that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters. They also suggest that dnaA, dnaN and recF are predominantly expressed from the same mRNA although transcriptional and/or post-transcriptional mechanisms should be specifically involved in lowering expression of the recF gene. Recently, we have localized 3 tandem transcription termination sites in the second half of the dnaN gene, downstream from the recF promoters. Neither of them shows the typical features of simple terminators and apparently they do not work in a minimal system of in vitro transcription. In this report, we present evidence that only one of them is dependent on the Rho protein. Although the operon structure allows coordinate expression of dnaA, dnaN and recF, the presence of internal promoters (the dnaN and recF promoters), which appear to be inducible by DNA damage, and intracistronic terminators, whose activity is inversely proportional to the efficiency of translation, permits expression of individual genes to be independently regulated in response to altered growth conditions.

Nerve conduction velocity decrease and synaptic transmission alterations in caffeine-treated rats.

The action of caffeine on peripheral neuromuscular function was studied by means of in vivo determinations of electrophysiological parameters, i.e., amplitude of extracellularly recorded muscle action potentials and nerve conduction velocity in the dorsal skeletal muscle and caudal nerve of the rat tail, respectively. Repeated exposure of the rats was carried out by adding caffeine to the drinking water for 10 days. Here we report the novel finding that motor nerve conduction velocity showed a significant decrease in caffeine-treated animals, whereas no change was observed in the amplitude of indirectly evoked extracellular muscle action potentials. The physiological recovery of the amplitude of the compound muscle action potential observed in nonintoxicated rats after high-frequency stimulation (10 Hz) was not observed in intoxicated animals and is also discussed.

Temperature dependence of the toxic effects of phenytoin on peripheral neuromuscular function of the rat tail.

We studied the acute effects of a single dose of phenytoin (250 mg/kg) on peripheral neuromuscular function. The evoked muscle action potentials of the dorsal segmental muscles in the rat tail, and the conduction velocity of the dorsal nerve trunk which innervates them, were measured before and after the intraperitoneal injection of phenytoin. The experiments were performed at different temperatures, 27 (physiological tail temperature), 36 and 37 degrees C (physiological central temperature) in different groups of animals. The amplitudes of the evoked muscle action potentials in the treated groups showed no significant modifications at 27 degrees C, at 36 degrees C a small nonsignificant decrease could be observed, and a complete block occurred at 37 degrees C. The mean blocking time was approximately one hour. No significant variations of conduction velocity were observed at 27 and 36 degrees C, whereas it decreased significantly after 30 minutes at 37 degrees C. The results presented confirm phenytoin toxicity. How far these results, especially the decrease of nerve conduction velocity observed at 37 degrees C, confirm a previous hypothesis which supported that peripheral and central nervous system are affected by phenytoin by similar mechanisms, is discussed.

[Chronic inflammatory demyelinating neuropathies and their variants] / Polyradiculonévrites chroniques et leurs frontières.

The Chronic Inflammatory Demyelinating Polyradiculoneuropathies (CIDP) constitute a syndrome whose incidence is difficult to evaluate, and is probably underestimated. In the course of this presentation, we deliberately restricted discussion to issues raised in recent years concerning the extent of this syndrome. We discuss diagnostic criteria, especially electrophysiological ones. As the criteria proposed by the ad hoc committee of the American Academy of Neurology in 1991 have been questioned due to lack of sensitivity, new ones have been proposed recently. We briefly discuss the different types of chronic dysimmune demyelinating neuropathy: not only the CIDP, but also the Lewis and Sumner syndrome or multifocal inflammatory demyelinating neuropathy and the multiple conduction block neuropathies. At last, we point out the consistent finding of axonal involvement in the course of a chronic demyelinating neuropathy; over time, it can become predominant, which may make diagnosis difficult by suggesting a chronic axonal neuropathy that may be assumed to be primary. Consideration of these points may help clinicians recognize more chronic dysimmune neuropathies, for which immunosuppressive therapy has been found to be effective.

[Chronic polyradiculoneuritis and its frontiers]. / Polyradiculonévrites chroniques et leurs frontières.

The Chronic Inflammatory Demyelinating Polyradiculoneuropathies (CIDP) constitute a syndrome whose incidence is difficult to evaluate, and is probably underestimated. In the course of this presentation, we deliberately restricted discussion to issues raised in recent years concerning the extent of this syndrome. We discuss diagnostic criteria, especially electrophysiological ones. As the criteria proposed by the ad hoc committee of the American Academy of Neurology in 1991 have been questioned due to lack of sensitivity, new ones have been proposed recently. We briefly discuss the different types of chronic dysimmune demyelinating neuropathy: not only the CIDP, but also the Lewis and Sumner syndrome or multifocal inflammatory demyelinating neuropathy and the multiple conduction block neuropathies. At last, we point out the consistent finding of axonal involvement in the course of a chronic demyelinating neuropathy; over time, it can become pre-dominant, which may make diagnosis difficult by suggesting a chronic axonal neuropathy that may be assumed to be primary. Consideration of these points may help clinicians recognize more chronic dysimmune neuropathies, for which immunosuppressive therapy has been found to be effective.

Vital and functional outcomes of the first-ever hemispheric stroke, epidemiological comparative study between Kunming (China) and Limoges (France).

BACKGROUND: Clinical outcomes and socioeconomic consequences after a stroke may differ between regions. METHODS: One cohort was established prospectively in Kunming (China) to compare with a cohort of 156 stroke patients included in Limoges (France). During 1 year, patients hospitalized within 48 hours for a first-ever hemispheric stroke were included. Demographic data and neurocardiovascular risk factors were registered. Hemiplegia was evaluated. Functional outcome was assessed using the Barthel Index (BI) after 3 months. RESULTS: One hundred and eighteen patients were included in Kunming. Patients of Kunming were younger (61.4 ± 13.4 vs 72.3 ± 14.6 years in Limoges, P<0.0001), more involved in professional activity (36.4% vs 12.8%, P<0.0001). Survival analysis indicated that mortality did not differ between cohorts, but independently predicted by coma at the 2nd day (HR=9.33, 95% CI [4.39, 19.78]) and age>70 years (HR=6.29, 95% CI [2.36, 16.59]). Despite a better baseline BI for patients of Kunming (50.0 ± 34.9 vs 37.4 ± 34.2, P=0.0031), after adjustment for confusing, patients in Limoges had a 2.11 OR 95% CI [1.03, 4.31]) to reach a BI>80 at 3 months. CONCLUSIONS: Functional recovery for patients of Kunming was not as good as expected. The socioeconomic consequences of stroke in Kunming are significant as they involved younger subjects who were still in work.

Transcription termination in the Escherichia coli dnaA gene is not mediated by the internal DnaA box.

DnaA protein is a DNA-binding protein which recognizes a 9-bp consensus sequence called the DnaA box. By binding to DnaA boxes, DnaA protein regulates initiation of chromosomal replication and transcription of several genes. The dnaA gene contains two DnaA boxes, one located in the regulatory region and one within the structural gene. In this paper, we explore the role of the internal DnaA box in dnaA expression because it has been proposed that the DnaA box-DnaA protein complex can block transcribing RNA polymerase. Firstly, we analyzed the degree of derepression of the dnaA gene, measured as beta-galactosidase activity of a dnaA-lacZ fusion inserted onto the bacterial chromosome, produced by an extra copy number of the dnaA DnaA boxes carried by multicopy plasmids. Secondly, we analyzed repression produced by elevated levels of DnaA protein on single-copy dnaA-lacZ fusions containing or not containing the internal DnaA box. Our results indicate that the internal DnaA box does not play a regulatory role in dnaA expression.

Gene expression elicited by NFAT in the presence or absence of cooperative recruitment of Fos and Jun.

Cooperation between nuclear factor of activated T cells (NFAT) and AP-1 (Fos-Jun) proteins on composite NFAT-AP-1 DNA elements constitutes a powerful mechanism for signal integration of the calcium and protein kinase C/Ras pathways in the regulation of gene expression. Here we report that NFAT can induce expression of certain genes in T cells without the need for cooperative recruitment of Fos and Jun. Using NFAT1 mutant proteins that are unable to interact with Fos-Jun dimers but are unaffected in DNA binding or transcriptional activity, we show that expression of interleukin (IL)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, IL-4, MIP1alpha and Fas ligand mRNAs is absolutely dependent on cooperation between NFAT and Fos-Jun; in contrast, NFAT induces tumor necrosis factor alpha (TNFalpha) mRNA and IL-13 promoter activity without any necessity to recruit Fos and Jun. Furthermore, we show that NFAT-Fos-Jun cooperation is also essential to elicit the NFAT-dependent program of activation-induced cell death. Our results support the hypothesis that even in a single cell type, NFAT activation can evoke two distinct biological programs of gene expression, dependent or independent of NFAT-AP-1 cooperation.

Stationary phase induction of dnaN and recF, two genes of Escherichia coli involved in DNA replication and repair.

The beta subunit of DNA polymerase III holoenzyme, the Escherichia coli chromosomal replicase, is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity. The gene encoding beta, dnaN, maps between dnaA and recF, which are involved in initiation of DNA replication at oriC and resumption of DNA replication at disrupted replication forks, respectively. In exponentially growing cells, dnaN and recF are expressed predominantly from the dnaA promoters. However, we have found that stationary phase induction of the dnaN promoters drastically changes the expression pattern of the dnaA operon genes. As a striking consequence, synthesis of the beta subunit and RecF protein increases when cell metabolism is slowing down. Such an induction is dependent on the stationary phase sigma factor, RpoS, although the accumulation of this factor alone is not sufficient to activate the dnaN promoters. These promoters are located in DNA regions without static bending, and the -35 hexamer element is essential for their RpoS-dependent induction. Our results suggest that stationary phase-dependent mechanisms have evolved in order to coordinate expression of dnaN and recF independently of the dnaA regulatory region. These mechanisms might be part of a developmental programme aimed at maintaining DNA integrity under stress conditions.

Cervical artery dissection: An atypical presentation with Ehlers-Danlos-like collagen pathology?

The authors took skin biopsies of the macroscopically normal skin of seven consecutive patients with spontaneous cervical artery dissection (SCAD). Histologically, alterations of the collagen and elastic fiber networks were found in six patients. In five, the histologic, immunohistochemical, and ultrastructural changes were similar to those usually found in Ehlers-Danlos syndrome (EDS). This suggests that SCAD is frequently associated with the dermal alterations seen in EDS.

Tandem transcription termination sites in the dnaN gene of Escherichia coli.

The dnaN gene of Escherichia coli encodes the beta-subunit of DNA polymerase III and maps between the dnaA and recF genes. We demonstrated previously that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters. However, the recF gene has its own promoter region located in the middle of the dnaN structural gene. In this report, we use S1 mapping of mRNAs, transcriptional and translational fusions to the galK and lacZ genes, and in vitro mutagenesis to identify and characterize three tandem transcription termination sites responsible for transcriptional polarity in the dnaN-recF operon. These sites are located in the dnaN gene, downstream from the recF promoter region. Cumulatively, they terminate about 80% of the untranslated transcripts started at the recF promoters. As expected, they do not reduce transcription coming from the dnaN promoters unless dnaN translation was prematurely disrupted by the presence of a nonsense codon. The particular arrangement of regulatory elements (promoters and terminators) in the dnaN-recF region provides an exceptional in vivo system to confirm the latent termination site model of transcriptional polarity. In addition, our results contribute to the understanding of the complex regulation of the dnaA, dnaN, and recF genes. We propose that these three genes constitute an operon and that the terminators described in this work could be used to reduce expression of the distal genes of the operon under circumstances in which the dnaN translation happens to be slowed down.

The Escherichia coli trmE (mnmE) gene, involved in tRNA modification, codes for an evolutionarily conserved GTPase with unusual biochemical properties.

The evolutionarily conserved 50K protein of Escherichia coli, encoded by o454, contains a consensus GTP-binding motif. Here we show that 50K is a GTPase that differs extensively from regulatory GTPases such as p21. Thus, 50K exhibits a very high intrinsic GTPase hydrolysis rate, rather low affinity for GTP, and extremely low affinity for GDP. Moreover, it can form self-assemblies. Strikingly, the 17 kDa GTPase domain of 50K conserves the guanine nucleotide-binding and GTPase activities of the intact 50K molecule. Therefore, the structural requirements for GTP binding and GTP hydrolysis by 50K are without precedent and justify a separate classification in the GTPase superfamily. Immunoelectron microscopy reveals that 50K is a cytoplasmic protein partially associated with the inner membrane. We prove that o454 is allelic with trmE, a gene involved in the biosynthesis of the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, which is found in the wobble position of some tRNAs. Our results demonstrate that 50K is essential for viability depending on the genetic background. We propose that combination of mutations affecting the decoding process, which separately do not reveal an obvious defect in growth, can give rise to lethal phenotypes, most likely due to synergism.