High-Rate Nitrification at Low pH in Suspended- and Attached-Biomass Reactors

This article reports on high-rate nitrification at low pH in biofilm and suspended-biomass reactors by known chemolithotrophic bacteria. In the biofilm reactor, at low pH (4.3 ± 0.1) and low bulk ammonium concentrations (9.3 ± 3.3 mg·liter⁻¹), a very high nitrification rate of 5.6 g of N oxidized·liter⁻¹·day⁻¹ was achieved. The specific nitrification rate (0.55 g of N·g of biomass⁻¹·day⁻¹) was similar to values reported for nitrifying reactors at optimal pH. In the suspended-biomass reactor, the average pH was significantly lower than that in the biofilm reactor (pH 3.8 ± 0.3), and values as low as pH 3.2 were found. In addition, measurements in the suspended-biomass reactor, using isotope-labeled ammonium (¹⁵N), showed that in spite of the very low pH, biomass growth occurred with a yield of 0.1 g of biomass·g of N oxidized⁻¹. Fluorescence in situ hybridization using existing rRNA-targeted oligonucleotide probes showed that the nitrifying bacteria were from the monophyletic genus Nitrosomonas, suggesting that autotrophic nitrification at low pH is more widespread than previously thought. The results presented in this paper clearly show that autotrophic nitrifying bacteria have the ability to nitrify at a high rate at low pH and in the presence of only a negligible free ammonia concentration, suggesting the presence of an efficient ammonium uptake system and the means to cope with low pH.     

Application of a Specific and Sensitive Radiometric Assay for Microbial Lipase Activities in Marine Water Samples from the Lagoon of Nouméa

Marine microbiologists commonly assay lipase activities by using a synthetic fluorescent analog, 4-methylumbelliferyl (MUF)-oleate. The technique is convenient, but it is considered to be unspecific because of the structure of this analog. This study reports the design of a new specific and sensitive lipase assay based on the use of a radiolabeled triglyceride, [³H]triolein. Free fatty acids (FFA) resulting from its hydrolysis are isolated as a function of time in a one-step liquid-liquid extraction and then radioassayed. MUF-oleate and [³H]triolein techniques were compared by measuring lipase activities at similar substrate concentrations along a trophic gradient in the Southwest Lagoon of New Caledonia, near Nouméa. Hydrolysis rates decreased from the nearshore station to the offshore station and showed similar trends regardless of the technique used. Rates decreased from 5.83 to 0.88 nmol of FFA·liter⁻¹·h⁻¹ and from 0.76 to 0.23 nmol of ³H-FFA·liter⁻¹·h⁻¹, respectively. These results appeared to be consistent with bacterial production results, which also decreased similarly (from 0.59 to 0.26 μg of C·liter⁻¹·h⁻¹). However, the ratio of MUF-oleate activities to [³H]triolein activities, which was constant at the offshore stations (3.8 ± 0.1), gradually increased at the nearshore stations (from 4.1 to 7.6). This result shows that the two assays respond in different ways to changes in environmental conditions and validates the need to set up more specific enzymatic assays.     

Saturation Mutagenesis of Toluene ortho-Monooxygenase of Burkholderia cepacia G4 for Enhanced 1-Naphthol Synthesis and Chloroform Degradation

Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase α-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (V_max of 9.3 versus 4.5 nmol·min⁻¹·mg of protein⁻¹ and unchanged K_m), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 ± 0.07 versus 1.30 ± 0.06 nmol·min⁻¹·mg of protein⁻¹ [mean ± standard deviation]) at 91 μM and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (V_max of 2.61 versus 0.95 nmol·min⁻¹·mg of protein⁻¹ and unchanged K_m) and chloride release (0.034 ± 0.002 versus 0.012 ± 0.001 nmol·min⁻¹·mg of protein⁻¹). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 ± 0.3 versus 1.30 ± 0.06 nmol·min⁻¹·mg of protein⁻¹). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.     

Chlorine Disinfection of Atypical Mycobacteria Isolated from a Water Distribution System

We studied the resistance of various mycobacteria isolated from a water distribution system to chlorine. Chlorine disinfection efficiency is expressed as the coefficient of lethality (liters per minute per milligram) as follows: Mycobacterium fortuitum (0.02) > M. chelonae (0.03) > M. gordonae (0.09) > M. aurum (0.19). For a C·t value (product of the disinfectant concentration and contact time) of 60 mg·min·liter⁻¹, frequently used in water treatment lines, chlorine disinfection inactivates over 4 log units of M. gordonae and 1.5 log units of M. fortuitum or M. chelonae. C·t values determined under similar conditions show that even the most susceptible species, M. aurum and M. gordonae, are 100 and 330 times more resistant to chlorine than Escherichia coli. We also investigated the effects of different parameters (medium, pH, and temperature) on chlorine disinfection in a chlorine-resistant M. gordonae model. Our experimental results follow the Arrhenius equation, allowing the inactivation rate to be predicted at different temperatures. Our results show that M. gordonae is more resistant to chlorine in low-nutrient media, such as those encountered in water, and that an increase in temperature (from 4°C to 25°C) and a decrease in pH result in better inactivation.     

Decreasing the Level of Ethyl Acetate in Ethanolic Fermentation Broths of Escherichia coli KO11 by Expression of Pseudomonas putida estZ Esterase

During the fermentation of sugars to ethanol relatively high levels of an undesirable coproduct, ethyl acetate, are also produced. With ethanologenic Escherichia coli strain KO11 as the biocatalyst, the level of ethyl acetate in beer containing 4.8% ethanol was 192 mg liter⁻¹. Although the E. coli genome encodes several proteins with esterase activity, neither wild-type strains nor KO11 contained significant ethyl acetate esterase activity. A simple method was developed to rapidly screen bacterial colonies for the presence of esterases which hydrolyze ethyl acetate based on pH change. This method allowed identification of Pseudomonas putida NRRL B-18435 as a source of this activity and the cloning of a new esterase gene, estZ. Recombinant EstZ esterase was purified to near homogeneity and characterized. It belongs to family IV of lipolytic enzymes and contains the conserved catalytic triad of serine, aspartic acid, and histidine. As expected, this serine esterase was inhibited by phenylmethylsulfonyl fluoride and the histidine reagent diethylpyrocarbonate. The native and subunit molecular weights of the recombinant protein were 36,000, indicating that the enzyme exists as a monomer. By using α-naphthyl acetate as a model substrate, optimal activity was observed at pH 7.5 and 40°C. The K_m and V_max for α-naphthyl acetate were 18 μM and 48.1 μmol·min⁻¹·mg of protein⁻¹, respectively. Among the aliphatic esters tested, the highest activity was obtained with propyl acetate (96 μmol·min⁻¹·mg of protein⁻¹), followed by ethyl acetate (66 μmol·min⁻¹·mg of protein⁻¹). Expression of estZ in E. coli KO11 reduced the concentration of ethyl acetate in fermentation broth (4.8% ethanol) to less than 20 mg liter⁻¹.     

Exocyclic amino groups of flanking guanines govern sequence-dependent adduct conformations and local structural distortions for minor groove-aligned benzo[a]pyrenyl-guanine lesions in a GG mutation hotspot context

The environmental carcinogen benzo[a]pyrene (BP) is metabolized to reactive diol epoxides that bind to cellular DNA by predominantly forming N²-guanine adducts (G*). Mutation hotspots for these adducts are frequently found in 5′-···GG··· dinucleotide sequences, but their origins are poorly understood. Here we used high resolution NMR and molecular dynamics simulations to investigate differences in G* adduct conformations in 5′-···CG*GC··· and 5′-···CGG*C··· sequence contexts in otherwise identical 12-mer duplexes. The BP rings are positioned 5′ along the modified strand in the minor groove in both cases. However, subtle orientational differences cause strong distinctions in structural distortions of the DNA duplexes, because the exocyclic amino groups of flanking guanines on both strands compete for space with the BP rings in the minor groove, acting as guideposts for placement of the BP. In the 5′-···CGG*C··· case, the 5′-flanking G · C base pair is severely untwisted, concomitant with a bend deduced from electrophoretic mobility. In the 5′-···CG*GC··· context, there is no untwisting, but there is significant destabilization of the 5′-flanking Watson–Crick base pair. The minor groove width opens near the lesion in both cases, but more for 5′-···CGG*C···. Differential sequence-dependent removal rates of this lesion result and may contribute to the mutation hotspot phenomenon.     

Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine and Its Mononitroso Derivative Hexahydro-1-Nitroso-3,5-Dinitro-1,3,5-Triazine by Klebsiella pneumoniae Strain SCZ-1 Isolated from an Anaerobic Sludge

In previous work, we found that an anaerobic sludge efficiently degraded hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), but the role of isolates in the degradation process was unknown. Recently, we isolated a facultatively anaerobic bacterium, identified as Klebsiella pneumoniae strain SCZ-1, using MIDI and the 16S rRNA method from this sludge and employed it to degrade RDX. Strain SCZ-1 degraded RDX to formaldehyde (HCHO), methanol (CH₃OH) (12% of total C), carbon dioxide (CO₂) (72% of total C), and nitrous oxide (N₂O) (60% of total N) through intermediary formation of methylenedinitramine (O₂NNHCH₂NHNO₂). Likewise, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) was degraded to HCHO, CH₃OH, and N₂O (16.5%) with a removal rate (0.39 μmol·h⁻¹·g [dry weight] of cells⁻¹) similar to that of RDX (0.41 μmol·h⁻¹·g [dry weight] of cells⁻¹) (biomass, 0.91 g [dry weight] of cells·liter⁻¹). These findings suggested the possible involvement of a common initial reaction, possibly denitration, followed by ring cleavage and decomposition in water. The trace amounts of MNX detected during RDX degradation and the trace amounts of hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine detected during MNX degradation suggested that another minor degradation pathway was also present that reduced —NO₂ groups to the corresponding —NO groups.     

Synthesis and characterization of 2′-modified-4′-thioRNA: a comprehensive comparison of nuclease stability

We report herein the synthesis and physical and physiological characterization of fully modified 2′-modified-4′-thioRNAs, i.e. 2′-fluoro-4′-thioRNA (F-SRNA) and 2′-O-Me-4′-thioRNA (Me-SRNA), which can be considered as a hybrid chemical modification based on 2′-modified oligonucleotides (ONs) and 4′-thioRNA (SRNA). In its hybridization with a complementary RNA, F-SRNA (15mer) showed the highest T_m value (+16°C relative to the natural RNA duplex). In addition, both F-SRNA and Me-SRNA preferred RNA as a complementary partner rather than DNA in duplex formation. The results of a comprehensive comparison of nuclease stability of single-stranded F-SRNA and Me-SRNA along with 2′-fluoroRNA (FRNA), 2′-O-MeRNA (MeRNA), SRNA, and natural RNA and DNA, revealed that Me-SRNA had the highest stability with t_1/2 values of>24h against S1 nuclease (an endonuclease) and 79.2min against SVPD (a 3′-exonuclease). Moreover, the stability of Me-SRNA was significantly improved in 50% human plasma (t_1/2=1631min) compared with FRNA (t_1/2=53.2min) and MeRNA (t_1/2=187min), whose modifications are currently used as components of therapeutic aptamers. The results presented in this article will, it is hoped, contribute to the development of 2′-modified-4′-thioRNAs, especially Me-SRNA, as a new RNA molecule for therapeutic applications.     

Meta-Analysis of Genome-Wide Scans for Human Adult Stature Identifies Novel Loci and Associations with Measures of Skeletal Frame Size

Recent genome-wide (GW) scans have identified several independent loci affecting human stature, but their contribution through the different skeletal components of height is still poorly understood. We carried out a genome-wide scan in 12,611 participants, followed by replication in an additional 7,187 individuals, and identified 17 genomic regions with GW-significant association with height. Of these, two are entirely novel (rs11809207 in CATSPER4, combined P-value=6.1×10⁻⁸ and rs910316 in TMED10, P-value=1.4×10⁻⁷) and two had previously been described with weak statistical support (rs10472828 in NPR3, P-value=3×10⁻⁷ and rs849141 in JAZF1, P-value=3.2×10⁻¹¹). One locus (rs1182188 at GNA12) identifies the first height eQTL. We also assessed the contribution of height loci to the upper- (trunk) and lower-body (hip axis and femur) skeletal components of height. We find evidence for several loci associated with trunk length (including rs6570507 in GPR126, P-value=4×10⁻⁵ and rs6817306 in LCORL, P-value=4×10⁻⁴), hip axis length (including rs6830062 at LCORL, P-value=4.8×10⁻⁴ and rs4911494 at UQCC, P-value=1.9×10⁻⁴), and femur length (including rs710841 at PRKG2, P-value=2.4×10⁻⁵ and rs10946808 at HIST1H1D, P-value=6.4×10⁻⁶). Finally, we used conditional analyses to explore a possible differential contribution of the height loci to these different skeletal size measurements. In addition to validating four novel loci controlling adult stature, our study represents the first effort to assess the contribution of genetic loci to three skeletal components of height. Further statistical tests in larger numbers of individuals will be required to verify if the height loci affect height preferentially through these subcomponents of height.     

Admixture Mapping of 15,280 African Americans Identifies Obesity Susceptibility Loci on Chromosomes 5 and X

The prevalence of obesity (body mass index (BMI) ≥30 kg/m²) is higher in African Americans than in European Americans, even after adjustment for socioeconomic factors, suggesting that genetic factors may explain some of the difference. To identify genetic loci influencing BMI, we carried out a pooled analysis of genome-wide admixture mapping scans in 15,280 African Americans from 14 epidemiologic studies. Samples were genotyped at a median of 1,411 ancestry-informative markers. After adjusting for age, sex, and study, BMI was analyzed both as a dichotomized (top 20% versus bottom 20%) and a continuous trait. We found that a higher percentage of European ancestry was significantly correlated with lower BMI (ρ=−0.042, P=1.6×10⁻⁷). In the dichotomized analysis, we detected two loci on chromosome X as associated with increased African ancestry: the first at Xq25 (locus-specific LOD=5.94; genome-wide score=3.22; case-control Z=−3.94); and the second at Xq13.1 (locus-specific LOD=2.22; case-control Z=−4.62). Quantitative analysis identified a third locus at 5q13.3 where higher BMI was highly significantly associated with greater European ancestry (locus-specific LOD=6.27; genome-wide score=3.46). Further mapping studies with dense sets of markers will be necessary to identify the alleles in these regions of chromosomes X and 5 that may be associated with variation in BMI.     

NRXN3 Is a Novel Locus for Waist Circumference: A Genome-Wide Association Study from the CHARGE Consortium

Central abdominal fat is a strong risk factor for diabetes and cardiovascular disease. To identify common variants influencing central abdominal fat, we conducted a two-stage genome-wide association analysis for waist circumference (WC). In total, three loci reached genome-wide significance. In stage 1, 31,373 individuals of Caucasian descent from eight cohort studies confirmed the role of FTO and MC4R and identified one novel locus associated with WC in the neurexin 3 gene [NRXN3 (rs10146997, p=6.4×10⁻⁷)]. The association with NRXN3 was confirmed in stage 2 by combining stage 1 results with those from 38,641 participants in the GIANT consortium (p=0.009 in GIANT only, p=5.3×10⁻⁸ for combined analysis, n=70,014). Mean WC increase per copy of the G allele was 0.0498 z-score units (0.65 cm). This SNP was also associated with body mass index (BMI) [p=7.4×10⁻⁶, 0.024 z-score units (0.10 kg/m²) per copy of the G allele] and the risk of obesity (odds ratio 1.13, 95% CI 1.07–1.19; p=3.2×10⁻⁵ per copy of the G allele). The NRXN3 gene has been previously implicated in addiction and reward behavior, lending further evidence that common forms of obesity may be a central nervous system-mediated disorder. Our findings establish that common variants in NRXN3 are associated with WC, BMI, and obesity.     

High-yield production of short GpppA- and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2′O positions

Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, ^7MeG^5′-ppp^5′-A_2′OMe. The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and ^7MeGpppA-capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppAC_n and ^7MeGpppAC_n (1≤n≤9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1≤n≤7) in quantities of up to 100nmol starting from 200nmol cap analog. The capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2′-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.     

Rapid Acyl-Homoserine Lactone Quorum Signal Biodegradation in Diverse Soils

Signal degradation impacts all communications. Although acyl-homoserine lactone (acyl-HSL) quorum-sensing signals are known to be degraded by defined laboratory cultures, little is known about their stability in nature. Here, we show that acyl-HSLs are biodegraded in soils sampled from diverse U.S. sites and by termite hindgut contents. When amended to samples at physiologically relevant concentrations, ¹⁴C-labeled acyl-HSLs were mineralized to ¹⁴CO₂ rapidly and, at most sites examined, without lag. A lag-free turf soil activity was characterized in further detail. Heating or irradiation of the soil prior to the addition of radiolabel abolished mineralization, whereas protein synthesis inhibitors did not. Mineralization exhibited an apparent K_m of 1.5 μM acyl-HSL, ca. 1,000-fold lower than that reported for a purified acyl-HSL lactonase. Under optimal conditions, acyl-HSL degradation proceeded at a rate of 13.4 nmol·h⁻¹·g of fresh weight soil⁻¹. Bioassays established that the final extent of signal inactivation was greater than for its full conversion to CO₂ but that the two processes were well coupled kinetically. A most probable number of 4.6 × 10⁵ cells·g of turf soil⁻¹ degraded physiologically relevant amounts of hexanoyl-[1-¹⁴C]HSL to ¹⁴CO₂. It would take chemical lactonolysis months to match the level of signal decay achieved in days by the observed biological activity. Rapid decay might serve either to quiet signal cross talk that might otherwise occur between spatially separated microbial aggregates or as a full system reset. Depending on the context, biological signal decay might either promote or complicate cellular communications and the accuracy of population density-based controls on gene expression in species-rich ecosystems.     

Effect of Increasing the Copy Number of Bacteriophage Origins of Replication, in trans, on Incoming-Phage Proliferation

Bacteriophage resistance mechanisms which are derived from a bacteriophage genome are termed Per (phage-encoded resistance). When present in trans in Lactococcus lactis NCK203, Per50, the cloned origin of replication from phage 50, interferes with 50 replication. The per50 fragment was found to afford negligible protection to NCK203 against 50 infection when present in a low-copy-number plasmid, pTRK325. A high-copy-number Per50 construct (pTRK323) dramatically affected 50 infection, reducing the efficiency of plaquing (EOP) to 2.5 × 10^-4 and the plaque size to pinhead proportions. This clone also afforded significant protection against other related small isometric phages. Per31 was cloned from phage 31 and demonstrated to function as an origin of replication by enabling replication of per31-containing plasmids, in NCK203, on 31 infection. A low-copy-number Per31 plasmid (pTRK360) reduced the EOP of 31 on NCK203 to 0.3 and the plaque diameter from 1.5 to 0.5 mm. When this plasmid was cloned in high copy number, the EOP was further reduced to 7.2 × 10^-7 but the plaques were large and contained Per31-resistant phages. Characterization of these “new” phages revealed at least two different types that were similar to 31, except that DNA alterations were noted in the region containing the origin. This novel and powerful abortive phage resistance mechanism should prove useful when directed at specific, problematic phages.     

Distributed control for recruitment, scanning and subunit joining steps of translation initiation

Protein synthesis utilizes a large proportion of the available free energy in the eukaryotic cell and must be precisely controlled, yet up to now there has been no systematic rate control analysis of the in vivo process. We now present a novel study of rate control by eukaryotic translation initiation factors (eIFs) using yeast strains in which chromosomal eIF genes have been placed under the control of the tetO7 promoter system. The results reveal that, contrary to previously published reports, control of the initiation pathway is distributed over all of the eIFs, whereby rate control (the magnitude of their respective component control coefficients) follows the order: eIF4G>eIF1A>eIF4E>eIF5B. The apparent rate control effects of eIFs observed in standard cell-free extract experiments, on the other hand, do not accurately reflect the steady state in vivo data. Overall, this work establishes the first quantitative control framework for the study of in vivo eukaryotic translation.     

Cooccurrence of ScDSP Gene Expression, Cell Death, and DNA Fragmentation in a Marine Diatom, Skeletonema costatum

A novel death-specific gene, ScDSP, was obtained from a death stage subtraction cDNA library of the diatom Skeletonema costatum. The full length of ScDSP cDNA was 921 bp in length, containing a 699-bp open reading frame encoding 232 amino acids and two stretches of 66 and 156 bp in the 5′ and 3′ untranslated regions, respectively. Analysis of the peptide structure revealed that ScDSP contained a signal peptide domain, a transmembrane domain, and a pair of EF-hand motifs. When S. costatum grew exponentially at a rate of 1.3 day⁻¹, the ScDSP mRNA level was at 2 μmol·mole 18S rRNA⁻¹. In contrast, when the culture entered the death phase with a growth rate decreasing to 0.5 day⁻¹, ScDSP mRNA increased dramatically to 668 μmol·mole 18S rRNA⁻¹, and a high degree of DNA fragmentation was simultaneously observed. Under the influence of a light-dark cycle, ScDSP expression in both exponential and stationary phases clearly showed a diel rhythm, but the daily mean mRNA level was significantly higher in the stationary phase. Our results suggest that ScDSP may play a role in the molecular mechanism of self-destructive autolysis in phytoplankton under stress.     

Genome-Wide Association Scan Meta-Analysis Identifies Three Loci Influencing Adiposity and Fat Distribution

To identify genetic loci influencing central obesity and fat distribution, we performed a meta-analysis of 16 genome-wide association studies (GWAS, N=38,580) informative for adult waist circumference (WC) and waist–hip ratio (WHR). We selected 26 SNPs for follow-up, for which the evidence of association with measures of central adiposity (WC and/or WHR) was strong and disproportionate to that for overall adiposity or height. Follow-up studies in a maximum of 70,689 individuals identified two loci strongly associated with measures of central adiposity; these map near TFAP2B (WC, P=1.9×10⁻¹¹) and MSRA (WC, P=8.9×10⁻⁹). A third locus, near LYPLAL1, was associated with WHR in women only (P=2.6×10⁻⁸). The variants near TFAP2B appear to influence central adiposity through an effect on overall obesity/fat-mass, whereas LYPLAL1 displays a strong female-only association with fat distribution. By focusing on anthropometric measures of central obesity and fat distribution, we have identified three loci implicated in the regulation of human adiposity.     

Polarization of Tryptophan Fluorescence from Single Striated Muscle Fibers : A molecular probe of contractile state

Instrumentation has been developed to detect rapidly the polarization of tryptophan fluorescence from single muscle fibers in rigor, relaxation, and contraction. The polarization parameter (P) obtained by exiciting the muscle tryptophans with light polarized perpendicular to the long axis of the muscle fiber had a magnitude P (relaxation) > P (contraction) > P (rigor) for the three types of muscle fibers examined (glycerinated rabbit psoas, glycerinated dorsal longitudinal flight muscle of Lethocerus americanus, and live semitendinosus of Rana pipiens). P from single psoas fibers in rigor was found to increase as the sarcomere length increased but in relaxed fibers P was independent of sarcomere length. After rigor, pyrophosphate produced little or no change in P, but following an adenosine triphosphate (ATP)-containing solution, pyrophosphate produced a value of P that fell between the contraction and relaxation values. Sinusoidal or square wave oscillations of the muscle of amplitude 0.5–2.0% of the sarcomere length and frequency 1, 2, or 5 Hz were applied in rigor when the myosin cross-bridges are considered to be firmly attached to the thin filaments. No significant changes in P were observed in either rigor or relaxation. The preceding results together with our present knowledge of tryptophan distribution in the contractile proteins has led us to the conclusion that the parameter P is a probe of the contractile state of myosin which is probably sensitive to the orientation of the myosin S1 subfragment.     

Common Elements Regulating Gene Expression in Temperate and Lytic Bacteriophages of Lactococcus Species

A phage-inducible middle promoter (P_15A10) from the lytic, lactococcal bacteriophage 31, a member of the P335 species, is located in an 888-base pair fragment near the right cohesive end. Sequence analysis revealed extensive homology (>95%) to the right cohesive ends of two temperate phages of the P335 species, r1t and LC3. Sequencing upstream and downstream of P_15A10 showed that the high degree of homology between 31 and r1t continued beyond the phage promoter. With the exception of one extra open reading frame in 31, the sequences were highly homologous (95 to 98%) between nucleotides 13448 and 16320 of the published r1t sequence. By use of a β-galactosidase (β-Gal) gene under the control of a smaller, more tightly regulated region within the P_15A10 promoter, P_566–888, it was established that mitomycin C induction of a lactococcal strain harboring the prophage r1t induced the P_566–888 promoter, as determined from an increase in β-Gal activity. Hybridization of nine other lactococcal strains with ³²P-labeled P_566–888 showed that the Lactococcus lactis strains C10, ML8, and NCK203 harbored sequences homologous to that of the phage-inducible promoter. Mitomycin C induced the resident prophages in all these strains and concurrently induced the P_566–888 promoter, as determined from an increase in β-Gal activity. DNA restriction analysis revealed that the prophages in C10, ML8, and NCK203 had identical restriction patterns which were different from that of r1t. In addition, DNA sequencing showed that the promoter elements in the three phages were identical to each other and to P_566–888 from the lytic phage 31. These results point to a conserved mechanism in the regulation of gene expression between the lytic phage 31 and at least two temperate bacteriophages and provide further evidence for a link in the evolution of certain temperate phages and lytic phages.     

A genome-wide association study identifies novel alleles associated with hair color and skin pigmentation

We conducted a multi-stage genome-wide association study of natural hair color in more than 10,000 men and women of European ancestry from the United States and Australia. An initial analysis of 528,173 single nucleotide polymorphisms (SNPs) genotyped on 2,287 women identified IRF4 and SLC24A4 as loci highly associated with hair color, along with three other regions encompassing known pigmentation genes. We confirmed these associations in 7,028 individuals from three additional studies. Across these four studies, SLC24A4 rs12896399 and IRF4 rs12203592 showed strong associations with hair color, with p=6.0×10⁻⁶² and p=7.46×10⁻¹²⁷, respectively. The IRF4 SNP was also associated with skin color (p=6.2×10⁻¹⁴), eye color (p=6.1×10⁻¹³), and skin tanning response to sunlight (p=3.9×10⁻⁸⁹). A multivariable analysis pooling data from the initial GWAS and an additional 1,440 individuals suggested that the association between rs12203592 and hair color was independent of rs1540771, a SNP between the IRF4 and EXOC2 genes previously found to be associated with hair color. After adjustment for rs12203592, the association between rs1540771 and hair color was not significant (p=0.52). One variant in the MATP gene was associated with hair color. A variant in the HERC2 gene upstream of the OCA2 gene showed the strongest and independent association with hair color compared with other SNPs in this region, including three previously reported SNPs. The signals detected in a region around the MC1R gene were explained by MC1R red hair color alleles. Our results suggest that the IRF4 and SLC24A4 loci are associated with human hair color and skin pigmentation.