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The detection of antisense RNA is hampered by reverse transcription (RT) non-specific priming, due to the ability of RNA secondary structures to prime RT in the absence of specific primers. The detection of antisense RNA by conventional RT-PCR does not allow assessment of the polarity of the initial RNA template, causing the amplification of non-specific cDNAs. In this study we have developed a modified protocol for the detection of human immunodeficiency virus type 1 (HIV-1) antisense protein (ASP) RNA. Using this approach, we have identified ASP transcripts in CD4+ T cells isolated from five HIV-infected individuals, either untreated or under suppressive therapy. We show that ASP RNA can be detected in stimulated CD4+ T cells from both groups of patients, but not in unstimulated cells. We also show that in untreated patients, the patterns of expression of ASP and env are very similar, with the levels of ASP RNA being markedly lower than those of env. Treatment of cells from one viraemic patient with α-amanitin greatly reduces the rate of ASP RNA synthesis, suggesting that it is associated with RNA polymerase II, the central enzyme in the transcription of protein-coding genes. Our data represent the first nucleotide sequences obtained in patients for ASP, demonstrating that its transcription indeed occurs in those HIV-1 lineages in which the ASP open reading frame is present.

The development of therapeutic and prophylactic HIV vaccines for African countries is urgently needed, but the question of what immunogens to use needs to be answered. One approach is to include HIV envelope immunogens derived from HIV-positive individuals from a geographically concentrated epidemic with more limited viral genetic diversity for a region-based vaccine. To address if there is a basis for a regional selected antibody vaccine, we have screened two regionally separate cohorts from Guinea-Bissau and Denmark for neutralizing antibody activity and antibody-dependent cellular cytotoxicity (ADCC) against local and nonlocal circulating HIV-1 strains. The neutralizing activity did not demonstrate higher potential against local circulating strains according to geography and subtype determination, but the plasma from Danish individuals demonstrated significantly higher inhibitory activity than that from Guinea-Bissau individuals against both local and nonlocal virus strains. Interestingly, an opposite pattern was observed with ADCC activity, where Guinea-Bissau individual plasma demonstrated higher activity than Danish plasma and was specifically against the local circulating subtype. Thus, on basis of samples from these two cohorts, no local-specific neutralizing activity was detected, but a local ADCC response was identified in the Guinea-Bissau samples, suggesting potential use of regional immunogens for an ADCC-inducing vaccine.

Streptococcus pneumoniae is an opportunistic human pathogen that typically colonizes the nasopharyngeal passage and causes lethal disease in other host niches such as the lung or the meninges. How pneumococcal genes are expressed and regulated at the different stages of its life cycle, as commensal or as pathogen, has not been entirely described. To chart the transcriptional responses of S. pneumoniae, we quantified the transcriptome under 22 different infection-relevant conditions. The transcriptomic compendium exposed a high level of dynamic expression and, strikingly, all annotated pneumococcal genomic features were expressed in at least one of the studied conditions. By computing the correlation of gene expression of every two genes across all studied conditions, we created a co-expression matrix that provides valuable information on both operon structure and regulatory processes. The co-expression data is highly consistent with well-characterized operons and regulons, such as the PyrR, ComE and ComX regulons, and has allowed us to identify a new member of the competence regulon. Finally, we created an interactive data center named PneumoExpress (www.veeninglab.com/pneumoexpress) that enables users to access the expression data as well as the co-expression matrix in an intuitive and efficient manner, providing a valuable resource to the pneumococcal research community.

The three-dimensional structure of DNA is highly susceptible to changes by mechanical and biochemical cues in vivo and in vitro. In particular, large increases in base pair spacing compared to regular B-DNA are effected by mechanical (over)stretching and by intercalation of compounds that are widely used in biophysical/chemical assays and drug treatments. We present single-molecule experiments and a three-state statistical mechanical model that provide a quantitative understanding of the interplay between B-DNA, overstretched DNA and intercalated DNA. The predictions of this model include a hitherto unconfirmed hyperstretched state, twice the length of B-DNA. Our force-fluorescence experiments confirm this hyperstretched state and reveal its sequence dependence. These results pin down the physical principles that govern DNA mechanics under the influence of tension and biochemical reactions. A predictive understanding of the possibilities and limitations of DNA extension can guide refined exploitation of DNA in, e.g., programmable soft materials and DNA origami applications., The mechanics and structural transitions of DNA are important to many essential processes inside living cells. Here the authors combine theory and single-molecule experiments to show that intercalator binding stabilises a new structural state of DNA: hyperstretched DNA.

DNA Differences The extent to which genetic variation affects an individual's phenotype has been difficult to predict because the majority of variation lies outside the coding regions of genes. Now, three studies examine the extent to which genetic variation affects the chromatin of individuals with diverse ancestry and genetic variation (see the Perspective by Furey and Sethupathy ). Kasowski et al. (p. 750 , published online 17 October) examined how genetic variation affects differences in chromatin states and their correlation to histone modifications, as well as more general DNA binding factors. Kilpinen et al. (p. 744 , published online 17 October) document how genetic variation is linked to allelic specificity in transcription factor binding, histone modifications, and transcription. McVicker et al. (p. 747 , published online 17 October) identified how quantitative trait loci affect histone modifications in Yoruban individuals and established which specific transcription factors affect such modifications.

Mammalian genomes contain highly conserved sequences that are not functionally transcribed. These sequences are single copy and comprise approximately 1-2% of the human genome. Evolutionary analysis strongly supports their functional conservation, although their potentially diverse, functional attributes remain unknown. It is likely that genomic variation in conserved non-genic sequences is associated with phenotypic variability and human disorders. So how might their function and contribution to human disorders be examined?

Two loci for nonsyndromic recessive deafness located on chromosome 21q22.3 have previously been reported, DFNB8 and DFNB10. Recently a gene which encodes a transmembrane serine protease, TMPRSS3 or ECHOS1, was found to be responsible for both the DFNB8 and DFNB10 phenotypes. To determine the contribution of TMPRSS3 mutations in the general congenital/childhood nonsyndromic deaf population we performed mutation analysis of the TMPRSS3 gene in 448 unrelated deaf patients from Spain, Italy, Greece, and Australia who did not have the common 35delG GJB2 mutation. From the 896 chromosomes studied we identified two novel pathogenic mutations accounting for four mutant alleles and at least 16 nonpathogenic sequence variants. The pathogenic mutations were a 1-bp deletion resulting in a frameshift and an amino acid substitution in the LDLRA domain of TMPRSS3. From this and another study we estimate the frequency of TMPRSS3 mutations in our sample as 0.45%, and approximately 0.38% in the general Caucasian childhood deaf population. However, TMPRSS3 is still an important contributor to genetic deafness in populations with large consanguineous families.

Evidence is presented showing that a neuronal isoform of nitric oxide synthase (NOS) is expressed in rat pancreatic islets and INS-1 cells. Sequencing of the coding region indicated a 99.8% homology with rat neuronal NOS (nNOS) with four mutations, three of them resulting in modifications of the amino acid sequence. Double-immunofluorescence studies demonstrated the presence of nNOS in insulin-secreting beta-cells. Electron microscopy studies showed that nNOS was mainly localized in insulin secretory granules and to a lesser extent in the mitochondria and the nucleus. We also studied the mechanism involved in the dysfunction of the beta-cell response to arginine and glucose after nNOS blockade with N(G)-nitro-L-arginine methyl ester. Our data show that miconazole, an inhibitor of nNOS cytochrome c reductase activity, either alone for the experiments with arginine or combined with sodium nitroprusside for glucose, is able to restore normal secretory patterns in response to the two secretagogues. Furthermore, these results were corroborated by the demonstration of a direct enzyme-substrate interaction between nNOS and cytochrome c, which is strongly reinforced in the presence of the NOS inhibitor. Thus, we provide immunochemical and pharmacological evidence that beta-cell nNOS exerts, like brain nNOS, two catalytic activities: a nitric oxide production and an NOS nonoxidating reductase activity, both of which are essential for normal beta-cell function. In conclusion, we suggest that an imbalance between these activities might be implicated in beta-cell dysregulation involved in certain pathological hyperinsulinic states.

From the cDNAs of two defective RNAs naturally exhibiting a large difference in replication efficiency, a series of Sendai virus RNA chimeras were constructed by reciprocal exchanges of their 3′ end primary sequences. Using a reverse genetics system, the ability of these RNAs to replicate when expressed from cDNAs in the context of the viral proteins N, P, and L, also expressed from plasmids, was analyzed. First the extent of potential RNA 3′/5′ end complementarity was tested by disrupting and restoring the terminal 110-nucleotide complementarity of a copy-back RNA. Alternatively, this base pairing potential was gradually increased from 12 to 57 or to 98 nucleotides by continuous substitutions. In all cases, the restoration or the creation of more extended base pairing potential had no effect on RNA replication. Reciprocal exchanges were then made in order to identifycis-acting sequences that could induce high replication efficiency. It was found that nucleotides 1–31 of the antigenome 3′ end were sufficient to confer a high replication property (more than a 10-fold increase), regardless of the sequence adjacent to these terminal nucleotides. It is concluded that one of the most important features that modulate replication efficiency is contained in the promoter end primary sequence and that this feature is likely to operate independently of the ability to form a potential terminal base pairing.

Background PCR has the potential to detect and precisely quantify specific DNA sequences, but it is not yet often used as a fully quantitative method. A number of data collection and processing strategies have been described for the implementation of quantitative PCR. However, they can be experimentally cumbersome, their relative performances have not been evaluated systematically, and they often remain poorly validated statistically and/or experimentally. In this study, we evaluated the performance of known methods, and compared them with newly developed data processing strategies in terms of resolution, precision and robustness. Results Our results indicate that simple methods that do not rely on the estimation of the efficiency of the PCR amplification may provide reproducible and sensitive data, but that they do not quantify DNA with precision. Other evaluated methods based on sigmoidal or exponential curve fitting were generally of both poor resolution and precision. A statistical analysis of the parameters that influence efficiency indicated that it depends mostly on the selected amplicon and to a lesser extent on the particular biological sample analyzed. Thus, we devised various strategies based on individual or averaged efficiency values, which were used to assess the regulated expression of several genes in response to a growth factor. Conclusion Overall, qPCR data analysis methods differ significantly in their performance, and this analysis identifies methods that provide DNA quantification estimates of high precision, robustness and reliability. These methods allow reliable estimations of relative expression ratio of two-fold or higher, and our analysis provides an estimation of the number of biological samples that have to be analyzed to achieve a given precision.

In this study, we describe a patient with a phenotype of complete hypogonadotropic hypogonadism who presented primary failure of pulsatile GnRH therapy, but responded to exogenous gonadotropin administration. This patient bore a novel point mutation (T for A) at codon 168 of the gene encoding the GnRH receptor (GnRH-R), resulting in a serine to arginine change in the fourth transmembrane domain of the receptor. This novel mutation was present in the homozygous state in the patient, whereas it was in the heterozygous state in both phenotypically normal parents. When introduced into the complementary DNA coding for the GnRH-R, this mutation resulted in the complete loss of the receptor-mediated signaling response to GnRH. In conclusion, we report the first mutation of the GnRH-R gene that can induce a total loss of function of this receptor and is associated with a phenotype of complete hypogonadotropic hypogonadism.

Background Solexa/Illumina short-read ultra-high throughput DNA sequencing technology produces millions of short tags (up to 36 bases) by parallel sequencing-by-synthesis of DNA colonies. The processing and statistical analysis of such high-throughput data poses new challenges; currently a fair proportion of the tags are routinely discarded due to an inability to match them to a reference sequence, thereby reducing the effective throughput of the technology. Results We propose a novel base calling algorithm using model-based clustering and probability theory to identify ambiguous bases and code them with IUPAC symbols. We also select optimal sub-tags using a score based on information content to remove uncertain bases towards the ends of the reads. Conclusion We show that the method improves genome coverage and number of usable tags as compared with Solexa's data processing pipeline by an average of 15%. An R package is provided which allows fast and accurate base calling of Solexa's fluorescence intensity files and the production of informative diagnostic plots.

Urea movement across plasma membranes is modulated by specialized transporter proteins that are products of two genes, termed UT-A and UT-B. These proteins play key roles in the urinary concentrating mechanism and fluid homeostasis. We have isolated and characterized a 1.4-kb cDNA from testes encoding a new isoform (UT-A5) belonging to the UT-A transporter family. For comparison, we also isolated a 2.0-kb cDNA from mouse kidney inner medulla encoding the mouse UT-A3 homologue. The UT-A5 cDNA has a putative open reading frame encoding a 323-amino acid protein, making UT-A5 the smallest UT-A family member in terms of molecular size. Its putative topology is of particular interest, because it calls into question earlier models of UT-A transporter structure. Expression of UT-A5 cRNA in Xenopus oocytes mediates phloretin-inhibitable urea uptake and does not translocate water. The distribution of UT-A5 mRNA is restricted to the peritubular myoid cells forming the outermost layer of the seminiferous tubules within the testes and is not detected in kidney. UT-A5 mRNA levels are coordinated with the stage of testes development and increase 15 days postpartum, commensurate with the start of seminiferous tubule fluid movement.