B-Raf mutation : a key player in molecular biology of cancer

B-Raf is one of the more commonly mutated proto-oncogenes implicated in the development of cancers. In this review, we consider the mechanisms and clinical impacts of B-Raf mutations in cancer and discuss the implications for the patient in melanoma, thyroid cancer and colorectal cancer, where B-Raf mutations are particularly common.

Signet-ring cell carcinoma of colorectum : current perspectives and molecular biology

PURPOSE Colorectal signet-ring cell carcinoma (SRCC) is rare, and very little detailed information on the molecular biology of the disease is available. METHODS The literature on the clinical, pathological and, in particular, the molecular biology of this rare entity was critically reviewed. The reviewed articles take into account a total of 1,817 cases of SRCC, but only 143 cases have molecular data available. The characteristics of two patients with colorectal SRCC were also discussed. RESULTS Colorectal SRCC mostly occurs in younger patients, is larger and has different site predilection compared with conventional colorectal adenocarcinoma. It can occur as one of the synchronous cancers in the colorectum. The cancer is usually diagnosed at advanced stages because of the late manifestation of symptoms, and aggressive treatment strategy is required. Limited reports in the literature have shown that the variant of colorectal cancer demonstrated a different pattern of genetic alterations of common growth kinase-related oncogenes (K-ras, BRAF), tumour suppressor genes (p53, p16), gene methylation and cell adhesion-related genes related to the Wingless signalling pathway (E-cadherin and beta-catenin) from conventional colorectal adenocarcinoma. Colorectal SRCC also showed high expression of mucin-related genes and genes related to the gastrointestinal system. There was also a higher prevalence of microsatellite instability-high tumours and low Cox-2 expression in colorectal SRCC as opposed to conventional adenocarcinoma. CONCLUSIONS Colorectal SRCC has unique molecular pathological features. The unique molecular profiles in SRCC may provide molecular-based improvements to patient management in colorectal SRCC.

Training for performance : insights from molecular biology

In this commentary the authors discuss the molecular basis of the training adaptation and review the role of several key signaling proteins important in the adaptation to endurance and resistance training.

Time dependency of molecular rate estimates and systematic overestimation of recent divergence times

Studies of molecular evolutionary rates have yielded a wide range of rate estimates for various genes and taxa. Recent studies based on population-level and pedigree data have produced remarkably high estimates of mutation rate, which strongly contrast with substitution rates inferred in phylogenetic (species-level) studies. Using Bayesian analysis with a relaxed-clock model, we estimated rates for three groups of mitochondrial data: avian protein-coding genes, primate protein-coding genes, and primate d-loop sequences. In all three cases, we found a measurable transition between the high, short-term (<1–2 Myr) mutation rate and the low, long-term substitution rate. The relationship between the age of the calibration and the rate of change can be described by a vertically translated exponential decay curve, which may be used for correcting molecular date estimates. The phylogenetic substitution rates in mitochondria are approximately 0.5% per million years for avian protein-coding sequences and 1.5% per million years for primate protein-coding and d-loop sequences. Further analyses showed that purifying selection offers the most convincing explanation for the observed relationship between the estimated rate and the depth of the calibration. We rule out the possibility that it is a spurious result arising from sequence errors, and find it unlikely that the apparent decline in rates over time is caused by mutational saturation. Using a rate curve estimated from the d-loop data, several dates for last common ancestors were calculated: modern humans and Neandertals (354 ka; 222–705 ka), Neandertals (108 ka; 70–156 ka), and modern humans (76 ka; 47–110 ka). If the rate curve for a particular taxonomic group can be accurately estimated, it can be a useful tool for correcting divergence date estimates by taking the rate decay into account. Our results show that it is invalid to extrapolate molecular rates of change across different evolutionary timescales, which has important consequences for studies of populations, domestication, conservation genetics, and human evolution.

A functional screen identifies lateral transfer of beta-glucuronidase (gus) from bacteria to fungi

Lateral gene transfer (LGT) from prokaryotes to microbial eukaryotes is usually detected by chance through genome-sequencing projects. Here, we explore a different, hypothesis-driven approach. We show that the fitness advantage associated with the transferred gene, typically invoked only in retrospect, can be used to design a functional screen capable of identifying postulated LGT cases. We hypothesized that beta-glucuronidase (gus) genes may be prone to LGT from bacteria to fungi (thought to lack gus) because this would enable fungi to utilize glucuronides in vertebrate urine as a carbon source. Using an enrichment procedure based on a glucose-releasing glucuronide analog (cellobiouronic acid), we isolated two gus(+) ascomycete fungi from soils (Penicillium canescens and Scopulariopsis sp.). A phylogenetic analysis suggested that their gus genes, as well as the gus genes identified in genomic sequences of the ascomycetes Aspergillus nidulans and Gibberella zeae, had been introgressed laterally from high-GC gram(+) bacteria. Two such bacteria (Arthrobacter spp.), isolated together with the gus(+) fungi, appeared to be the descendants of a bacterial donor organism from which gus had been transferred to fungi. This scenario was independently supported by similar substrate affinities of the encoded beta-glucuronidases, the absence of introns from fungal gus genes, and the similarity between the signal peptide-encoding 5' extensions of some fungal gus genes and the Arthrobacter sequences upstream of gus. Differences in the sequences of the fungal 5' extensions suggested at least two separate introgression events after the divergence of the two main Euascomycete classes. We suggest that deposition of glucuronides on soils as a result of the colonization of land by vertebrates may have favored LGT of gus from bacteria to fungi in soils.

Molecular analysis of the myosin gene family in Arabidopsis thaliana

Myosin is believed to act as the molecular motor for many actin-based motility processes in eukaryotes. It is becoming apparent that a single species may possess multiple myosin isoforms, and at least seven distinct classes of myosin have been identified from studies of animals, fungi, and protozoans. The complexity of the myosin heavy-chain gene family in higher plants was investigated by isolating and characterizing myosin genomic and cDNA clones from Arabidopsis thaliana. Six myosin-like genes were identified from three polymerase chain reaction (PCR) products (PCR1, PCR11, PCR43) and three cDNA clones (ATM2, MYA2, MYA3). Sequence comparisons of the deduced head domains suggest that these myosins are members of two major classes. Analysis of the overall structure of the ATM2 and MYA2 myosins shows that they are similar to the previously-identified ATM1 and MYA1 myosins, respectively. The MYA3 appears to possess a novel tail domain, with five IQ repeats, a six-member imperfect repeat, and a segment of unique sequence. Northern blot analyses indicate that some of the Arabidopsis myosin genes are preferentially expressed in different plant organs. Combined with previous studies, these results show that the Arabidopsis genome contains at least eight myosin-like genes representing two distinct classes.

Analysis of HapMap tag-SNPs in dysbindin (DTNBP1) reveals evidence of consistent association with schizophrenia

Dystrobrevin binding protein 1 (DTNBP1), or dysbindin, is thought to be critical in regulating the glutamatergic system. While the dopamine pathway is known to be important in the aetiology of schizophrenia, it seems likely that glutamatergic dysfunction can lead to the development of schizophrenia. DTNBP1 is widely expressed in brain, levels are reduced in brains of schizophrenia patients and a DTNBP1 polymorphism has been associated with reduced brain expression. Despite numerous genetic studies no DTNBP1 polymorphism has been strongly implicated in schizophrenia aetiology. Using a haplotype block-based gene-tagging approach we genotyped 13 SNPs in DTNBP1 to investigate possible associations with DTNBP1 and schizophrenia. Four polymorphisms were found to be significantly associated with schizophrenia. The strongest association was found with an A/C SNP in intron 7 (rs9370822). Homozygotes for the C allele of rs9370822 were more than two and a half times as likely to have schizophrenia compared to controls. The other polymorphisms showed much weaker association and are less likely to be biologically significant. These results suggest that DTNBP1 is a good candidate for schizophrenia risk and rs9370822 is either functionally important or in disequilibrium with a functional SNP, although our observations should be viewed with caution until they are independently replicated.

Genome-wide identification and expression analysis of the NF-Y family of transcription factors in Triticum aestivum

Nuclear Factor Y (NF-Y) is a trimeric complex that binds to the CCAAT box, a ubiquitous eukaryotic promoter element. The three subunits NF-YA, NF-YB and NF-YC are represented by single genes in yeast and mammals. However, in model plant species (Arabidopsis and rice) multiple genes encode each subunit providing the impetus for the investigation of the NF-Y transcription factor family in wheat. A total of 37 NF-Y and Dr1 genes (10 NF-YA, 11 NF-YB, 14 NF-YC and 2 Dr1) in Triticum aestivum were identified in the global DNA databases by computational analysis in this study. Each of the wheat NF-Y subunit families could be further divided into 4-5 clades based on their conserved core region sequences. Several conserved motifs outside of the NF-Y core regions were also identified by comparison of NF-Y members from wheat, rice and Arabidopsis. Quantitative RT-PCR analysis revealed that some of the wheat NF-Y genes were expressed ubiquitously, while others were expressed in an organ-specific manner. In particular, each TaNF-Y subunit family had members that were expressed predominantly in the endosperm. The expression of nine NF-Y and two Dr1 genes in wheat leaves appeared to be responsive to drought stress. Three of these genes were up-regulated under drought conditions, indicating that these members of the NF-Y and Dr1 families are potentially involved in plant drought adaptation. The combined expression and phylogenetic analyses revealed that members within the same phylogenetic clade generally shared a similar expression profile. Organ-specific expression and differential response to drought indicate a plant-specific biological role for various members of this transcription factor family.

Multiple evolutionary rate classes in animal genome evolution

The proportion of functional sequence in the human genome is currently a subject of debate. The most widely accepted figure is that approximately 5% is under purifying selection. In Drosophila, estimates are an order of magnitude higher, though this corresponds to a similar quantity of sequence. These estimates depend on the difference between the distribution of genomewide evolutionary rates and that observed in a subset of sequences presumed to be neutrally evolving. Motivated by the widening gap between these estimates and experimental evidence of genome function, especially in mammals, we developed a sensitive technique for evaluating such distributions and found that they are much more complex than previously apparent. We found strong evidence for at least nine well-resolved evolutionary rate classes in an alignment of four Drosophila species and at least seven classes in an alignment of four mammals, including human. We also identified at least three rate classes in human ancestral repeats. By positing that the largest of these ancestral repeat classes is neutrally evolving, we estimate that the proportion of nonneutrally evolving sequence is 30% of human ancestral repeats and 45% of the aligned portion of the genome. However, we also question whether any of the classes represent neutrally evolving sequences and argue that a plausible alternative is that they reflect variable structure-function constraints operating throughout the genomes of complex organisms.

Accuracy of rate estimation using relaxed-clock models with a critical focus on the early metazoan radiation

In recent years, a number of phylogenetic methods have been developed for estimating molecular rates and divergence dates under models that relax the molecular clock constraint by allowing rate change throughout the tree. These methods are being used with increasing frequency, but there have been few studies into their accuracy. We tested the accuracy of several relaxed-clock methods (penalized likelihood and Bayesian inference using various models of rate change) using nucleotide sequences simulated on a nine-taxon tree. When the sequences evolved with a constant rate, the methods were able to infer rates accurately, but estimates were more precise when a molecular clock was assumed. When the sequences evolved under a model of autocorrelated rate change, rates were accurately estimated using penalized likelihood and by Bayesian inference using lognormal and exponential models of rate change, while other models did not perform as well. When the sequences evolved under a model of uncorrelated rate change, only Bayesian inference using an exponential rate model performed well. Collectively, the results provide a strong recommendation for using the exponential model of rate change if a conservative approach to divergence time estimation is required. A case study is presented in which we use a simulation-based approach to examine the hypothesis of elevated rates in the Cambrian period, and it is found that these high rate estimates might be an artifact of the rate estimation method. If this bias is present, then the ages of metazoan divergences would be systematically underestimated. The results of this study have implications for studies of molecular rates and divergence dates.

A Bias in ML Estimates of Branch Lengths in the Presence of Multiple Signals

Sequence data often have competing signals that are detected by network programs or Lento plots. Such data can be formed by generating sequences on more than one tree, and combining the results, a mixture model. We report that with such mixture models, the estimates of edge (branch) lengths from maximum likelihood (ML) methods that assume a single tree are biased. Based on the observed number of competing signals in real data, such a bias of ML is expected to occur frequently. Because network methods can recover competing signals more accurately, there is a need for ML methods allowing a network. A fundamental problem is that mixture models can have more parameters than can be recovered from the data, so that some mixtures are not, in principle, identifiable. We recommend that network programs be incorporated into best practice analysis, along with ML and Bayesian trees.

Toward resolving deep neoaves phylogeny : data, signal enhancement, and priors

We report three developments toward resolving the challenge of the apparent basal polytomy of neoavian birds. First, we describe improved conditional down-weighting techniques to reduce noise relative to signal for deeper divergences and find increased agreement between data sets. Second, we present formulae for calculating the probabilities of finding predefined groupings in the optimal tree. Finally, we report a significant increase in data: nine new mitochondrial (mt) genomes (the dollarbird, New Zealand kingfisher, great potoo, Australian owlet-nightjar, white-tailed trogon, barn owl, a roadrunner [a ground cuckoo], New Zealand long-tailed cuckoo, and the peach-faced lovebird) and together they provide data for each of the six main groups of Neoaves proposed by Cracraft J (2001). We use his six main groups of modern birds as priors for evaluation of results. These include passerines, cuckoos, parrots, and three other groups termed “WoodKing” (woodpeckers/rollers/kingfishers), “SCA” (owls/potoos/owlet-nightjars/hummingbirds/swifts), and “Conglomerati.” In general, the support is highly significant with just two exceptions, the owls move from the “SCA” group to the raptors, particularly accipitrids (buzzards/eagles) and the osprey, and the shorebirds may be an independent group from the rest of the “Conglomerati”. Molecular dating mt genomes support a major diversification of at least 12 neoavian lineages in the Late Cretaceous. Our results form a basis for further testing with both nuclear-coding sequences and rare genomic changes.

Comparison of genomic DNA extraction techniques from whole blood samples : a time, cost and quality evaluation study

Genomic DNA obtained from patient whole blood samples is a key element for genomic research. Advantages and disadvantages, in terms of time-efficiency, cost-effectiveness and laboratory requirements, of procedures available to isolate nucleic acids need to be considered before choosing any particular method. These characteristics have not been fully evaluated for some laboratory techniques, such as the salting out method for DNA extraction, which has been excluded from comparison in different studies published to date. We compared three different protocols (a traditional salting out method, a modified salting out method and a commercially available kit method) to determine the most cost-effective and time-efficient method to extract DNA. We extracted genomic DNA from whole blood samples obtained from breast cancer patient volunteers and compared the results of the product obtained in terms of quantity (concentration of DNA extracted and DNA obtained per ml of blood used) and quality (260/280 ratio and polymerase chain reaction product amplification) of the obtained yield. On average, all three methods showed no statistically significant differences between the final result, but when we accounted for time and cost derived for each method, they showed very significant differences. The modified salting out method resulted in a seven- and twofold reduction in cost compared to the commercial kit and traditional salting out method, respectively and reduced time from 3 days to 1 hour compared to the traditional salting out method. This highlights a modified salting out method as a suitable choice to be used in laboratories and research centres, particularly when dealing with a large number of samples.

Induction of antioxidative Nrf2 gene transcription by coffee in humans : depending on genotype?

The Nrf2/ARE pathway is a major cellular defense mechanism that prevents damage by reactive oxygen species through induction of antioxidative phase II enzymes. However, the activity of the Nrf2/ARE system is not uniform with variability in response presumed to be dependent on the Nrf2 genotype. We recently completed a pilot human coffee intervention trial with healthy humans, where large interindividual differences in the antioxidative response to the study coffee were examined. Here, we address the question whether differences in the modulation of Nrf2 gene transcription, assessed as an induction of Nrf2 gene transcription by Q-PCR, might be correlated with specific Nrf2 genotypes. To date, nine single nucleotide polymorphisms (SNPs) have been identified in the Nrf2 (NFE2L2) gene. Two of these, the -617C/A and -651G/A SNPs are located within the promoter region and have previously been reported to influence the activity of the Nrf2/ARE pathway by reducing Nrf2 transcriptional activity. Sequencing of the critical Nrf2 gene promoter region not only confirmed the existence of these SNPs within the participants of the trial at the expected frequency (33% carrying the -617C/A, 17% the -651G/A and 56% the -653A/G SNP) but also indicated reduced Nrf2 gene transcription associated with a normal diet if the SNPs at position -617, -651 or -653 were present. Of note, the data also indicated the study coffee increased Nrf2 gene transcription even in SNP carriers. This further highlights the relevance of genotype-dependent induction of Nrf2 gene transcription that appears to be largely influenced by dietary factors.

A population genomics overview of the neuronal nitric oxide synthase (nNOS) gene and its relationship to migraine susceptibility

The ubiquitous chemical messenger molecule nitric oxide (NO) has been implicated in a diverse range of biological activities including neurotransmission, smooth muscle motility and mediation of nociception. Endogenous synthesis of NO by the neuronal isoform of the nitric oxide synthase gene family has an essential role within the central and peripheral nervous systems in addition to the autonomic innervation of cerebral blood vessels. To investigate the potential role of NO and more specifically the neuronal nitric oxide synthase (nNOS) gene in migraine susceptibility, we investigated two microsatellite repeat variants residing within the 5′ and 3′ regions of the nNOS gene. Population genomic evaluation of the two nNOS repeat variants indicated significant linkage disequilibrium between the two loci. Z-DNA conformational sequence structures within the 5′ region of the nNOS gene have the potential to enhance or repress gene promoter activity. We suggest that genetic analysis of this 5′ repeat variant is the more functional variant expressing gene wide information that could affect endogenous NO synthesis and potentially result in diseased states. However, no association with migraine (with or without aura) was seen in our extensive case-control cohort (n = 579 affected with matched controls), when both the 5′ and 3′ genetic variants were investigated.