Minimum information about a marker gene sequence (MIMARKS) and minimum information about any (x) sequence (MIxS) specifications

Here we present a standard developed by the Genomic Standards Consortium (GSC) for reporting marker gene sequences--the minimum information about a marker gene sequence (MIMARKS). We also introduce a system for describing the environment from which a biological sample originates. The 'environmental packages' apply to any genome sequence of known origin and can be used in combination with MIMARKS and other GSC checklists. Finally, to establish a unified standard for describing sequence data and to provide a single point of entry for the scientific community to access and learn about GSC checklists, we present the minimum information about any (x) sequence (MIxS). Adoption of MIxS will enhance our ability to analyze natural genetic diversity documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.     

Comparison of Effect on Tobacco Consumption and Carbon Monoxide Absorption of Changing to High and Low Nicotine Cigarettes

In 10 sedentary workers, smoking as they felt inclined over a five-hour period in the middle of a typical working day, changing to low nicotine cigarettes (<0·3 mg) caused an increase in the number and weight of cigarettes smoked, while changing to high nicotine cigarettes (3·2 mg) caused a decrease (P < 0·01). The average number and weight smoked in five hours for usual, low, and high nicotine brands were respectively 10·6 (6·00 g), 12·5 (6·52 g), and 6·7 (4·19 g). When smoking the usual brand the average blood carboxyhaemoglobin (COHb) increased 1·78% (from 6·38% to 8·16%). But on changing to either high or low nicotine cigarettes the COHb levels instead of increasing, tended to fall (P < 0·01). The average fall of 0·34% while smoking low nicotine cigarettes was due to the low carbon monoxide (CO) yield of these cigarettes, while the fall of 1·04% when smoking high nicotine cigarettes was attributable to reduced consumption. The findings support the view that smoking behaviour is modified to regulate nicotine intake. Besides having low tar and CO yields, the least harmful cigarettes for heavy smokers may be those with a high, rather than low, nicotine yield.     

Energetics of SecA dimerization

Transport of many proteins to extracytoplasmic locations occurs via the general secretion (Sec) pathway. In Escherichia coli, this pathway is composed of the SecYEG protein-conducting channel and the SecA ATPase. SecA plays a central role in binding the signal peptide region of preproteins, directing preproteins to membrane-bound SecYEG and promoting translocation coupled with ATP hydrolysis. Although it is well established that SecA is crucial for preprotein transport and thus cell viability, its oligomeric state during different stages of transport remains ill defined. We have characterized the energetics of SecA dimerization as a function of salt concentration and temperature and defined the linkage of SecA dimerization and signal peptide binding using analytical ultracentrifugation. The use of a new fluorescence detector permitted an analysis of SecA dimerization down to concentrations as low as 50 nM. The dimer dissociation constants are strongly dependent on salt. Linkage analysis indicates that SecA dimerization is coupled to the release of about five ions, demonstrating that electrostatic interactions play an important role in stabilizing the SecA dimer interface. Binding of signal peptide reduces SecA dimerization affinity, such that K_d increases about 9-fold from 0.28 μM in the absence of peptide to 2.68 μM in the presence of peptide. The weakening of the SecA dimer that accompanies signal peptide binding may poise the SecA dimer to dissociate upon binding to SecYEG.     

Mutations Affecting the Ability of the Escherichia coli UmuD′ Protein To Participate in SOS Mutagenesis

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli, a process that results from a translesion synthesis mechanism. The UmuD protein is activated for its role in mutagenesis by a RecA-facilitated autodigestion that removes the N-terminal 24 amino acids. A previous genetic screen for nonmutable umuD mutants had resulted in the isolation of a set of missense mutants that produced UmuD proteins that were deficient in RecA-mediated cleavage (J. R. Battista, T. Ohta, T. Nohmi, W. Sun, and G. C. Walker, Proc. Natl. Acad. Sci. USA 87:7190–7194, 1990). To identify elements of the UmuD′ protein necessary for its role in translesion synthesis, we began with umuD′, a modified form of the umuD gene that directly encodes the UmuD′ protein, and obtained missense umuD′ mutants deficient in UV and methyl methanesulfonate mutagenesis. The D39G, L40R, and T51I mutations affect residues located at the UmuD′₂ homodimer interface and interfere with homodimer formation in vivo. The D75A mutation affects a highly conserved residue located at one end of the central strand in a three-stranded β-sheet and appears to interfere with UmuD′₂ homodimer formation indirectly by affecting the structure of the UmuD′ monomer. When expressed from a multicopy plasmid, the L40R umuD′ mutant gene exhibited a dominant negative effect on a chromosomal umuD⁺ gene with respect to UV mutagenesis, suggesting that the mutation has an effect on UmuD′ function that goes beyond its impairment of homodimer formation. The G129D mutation affects a highly conserved residue that lies at the end of the long C-terminal β-strand and results in a mutant UmuD′ protein that exhibits a strongly dominant negative effect on UV mutagenesis in a umuD⁺ strain. The A30V and E35K mutations alter residues in the N-terminal arms of the UmuD′₂ homodimer, which are mobile in solution.     

Heritability of hyperphagic eating behavior and appetite-related hormones among Hispanic children

Objective: Eating in the absence of hunger (EAH) may be a genetically influenced phenotype of overweight children, but evidence is limited. This research evaluated the heritability (h²) of EAH and its association with overweight among Hispanic children 5 to 18 years old. Genetic and environmental associations of EAH with overweight, fat mass, and key hormonal regulators of food intake were also evaluated. Research Methods and Procedures: A family design was used to study 801 children from 300 Hispanic families. Weighed food intakes were used to measure EAH after an ad libitum dinner providing 50% of estimated energy needs. Fasting ghrelin, amylin, insulin, and leptin were measured by immunoassays. Measured heights, weights, and fat mass (using DXA) were obtained. Total energy expenditure (TEE) was measured by room respiration calorimetry. Results: On average, children consumed 41% of TEE at the dinner meal, followed by an additional 19% of TEE in the absence of hunger. Overweight children consumed 6.5% more energy at dinner (p < 0.001) and 14% more energy in the absence of hunger (p < 0.001) than non‐overweight children. Significant heritabilities were seen for EAH (h² = 0.51) and dinner intake (h² = 0.52) and for fasting levels of ghrelin (h² = 0.67), amylin (h² = 0.37), insulin (h² = 0.37), and leptin (h² = 0.34). Genetic correlations were seen between eating behavior and fasting hormones, suggesting common underlying genes affecting their expression. Discussion: This research provides new evidence that overweight Hispanic children exhibit elevated levels of hyperphagic eating behaviors that are influenced by genetic endowment.     

Fluorochroming Nuclei of Gold Chloride-Stained Motor Endings

Four millimeter cubes of muscle were soaked in 1% citric acid, 10 min; stained 60 min in 1% Au Cl₃,; then reduced 24 hr in 20% formic acid. Small pieces of muscle were teased on a slide and flooded with a single fluorescent dye or combination of fluorochromes. Single saturated solutions of berberine, acridine orange, coriphosphine, and entozon granulate gave adequate results. Berberine in a dilution of 1:5; acridine orange, berberine, coriphosphine and rhodamine B in a dilution of 1:10 were suitable. Combinations of acridine orange and berberine, berberine and coriphosphine, rhodamine B and berberine, entoion granulate and berberine, primulin and berberine and a combination of berberine, coriphosphine and rhodamine B in dilutions of 1:10 in equal parts were successful. Dilutions were volumetric measurements from saturated solutions. After the application of one or more fluorochromes, the preparations were washed with distilled water and observed with a fluorescence microscope. The plasmodesmata of the motor endings and the axon appeared dark purple or black when stained with gold chloride alone or in combination. The nuclei appeared as light bodies throughout the field and the color varied according to the dyes used. There was no color differentation between connective tissue, nerve or muscle nuclei. The method quickly and consistently permits identification of the cellular elements of the motor end plate, connective tissue and muscle fibers.     

Phase I randomised clinical trial of an HIV-1(CN54), clade C, trimeric envelope vaccine candidate delivered vaginally

We conducted a phase 1 double-blind randomised controlled trial (RCT) of a HIV-1 envelope protein (CN54 gp140) candidate vaccine delivered vaginally to assess immunogenicity and safety. It was hypothesised that repeated delivery of gp140 may facilitate antigen uptake and presentation at this mucosal surface. Twenty two healthy female volunteers aged 18-45 years were entered into the trial, the first receiving open-label active product. Subsequently, 16 women were randomised to receive 9 doses of 100 μg of gp140 in 3 ml of a Carbopol 974P based gel, 5 were randomised to placebo solution in the same gel, delivered vaginally via an applicator. Participants delivered the vaccine three times a week over three weeks during one menstrual cycle, and were followed up for two further months. There were no serious adverse events, and the vaccine was well tolerated. No sustained systemic or local IgG, IgA, or T cell responses to the gp140 were detected following vaginal immunisations. Repeated vaginal immunisation with a HIV-1 envelope protein alone formulated in Carbopol gel was safe, but did not induce local or systemic immune responses in healthy women. TRIAL REGISTRATION: ClinicalTrials.gov NCT00637962.     

The role of the genesnrf EFG andccmFH in cytochromec biosynthesis inEscherichia coli

It has been suggested that two groups ofEscherichia coli genes, theccm genes located in the 47-min region and thenrfEFG genes in the 92-min region of the chromosome, are involved in cytochromec biosynthesis during anaerobic growth. The involvement of the products of these genes in cytochromec synthesis, assembly and secretion has now been investigated. Despite their similarity to other bacterial cytochromec assembly proteins, NrfE, F and G were found not to be required for the biosynthesis of any of thec-type cytochromes inE. coli. Furthermore, these proteins were not required for the secretion of the periplasmic cytochromes, cytochromec ₅₅₀ and cytochromec ₅₅₂, or for the correct targeting of the NapC and NrfB cytochromes to the cytoplasmic membrane. NrfE and NrfG are required for formate-dependent nitrite reduction (the Nrf pathway), which involves at least twoc-type cytochromes, cytochromec ₅₅₂ and NrfB, but NrfF is not essential for this pathway. Genes similar tonrfE, nrfF andnrfG are present in theE. coli nap-ccm locus at minute 47. CcmF is similar to NrfE, the N-terminal region of CcmH is similar to NrfF and the C-terminal portion of CcmH is similar to NrfG. In contrast to NrfF, the N-terminal, NrfF-like portion of CcmH is essential for the synthesis of allc-type cytochromes. Conversely, the NrfG-like C-terminal region of CcmH is not essential for cytochromec biosynthesis. The data are consistent with proposals from this and other laboratories that CcmF and CcmH form part of a haem lyase complex required to attach haemc to C-X-X-C-H haem-binding domains. In contrast, NrfE and NrfG are proposed to fulfill a more specialised role in the assembly of the formate-dependent nitrite reductase.     

biparous:A Novel bHLH Gene Expressed in Neuronal and Glial Precursors inDrosophila

Genetic studies have uncovered many genes that are involved in the first steps of neuronal development inDrosophila.Less is known about the intermediate steps during which individual precursor cells follow either the neuronal pathway or the glial pathway. We report the identification of a novel bHLH gene,biparous,expressed in neuronal and glial precursors inDrosophila.Unlike most bHLH genes,biparousexpression continues to the final stages of neurogenesis in the embryo. Expression ofbiparousis not observed in end stage postmitotic neurons and precedes the expression ofrepo,a gene activated in later stages of glial differentiation. The bHLH domain is sufficiently different from previously described bHLH domains to imply a novel function.