383 research outputs found
Mutation and selective constraint in the murid genome
A large proportion of the genome of many higher eukaryotes consists of apparently functionless noncoding DNA, the significance of which is a longstanding puzzle in biology. The aim of this work was to quantify the extent to which both mutation and natural selection have influenced molecular evolution in murid noncoding sequence. In particular, the magnitude of and variation in selective constraint within murid noncoding DNA was investigated. Selective constraint is defined as the proportion of all mutations occurring at a locus or site which are strongly deleterious and therefore removed by natural selection. The approach adopted to estimate selective constraint relies on the assumption that we can quantify the past strength of purifying selection in a DNA sequence by comparison with nearby regions which are assumed to be evolving neutrally. To this end, work in this thesis deals both with mutational variation and bias (Chapters 2 and 3) as well as with selective constraint (Chapters 4 and 5) in noncoding DNA.Chapter 2 is concerned with the differential effects of context-dependent mutation (namely, CpG hypermutability) at fourfold synonymous and noncoding sites. Using simulations it was shown that a common method of assigning ancestral CpG status often introduces a substantial level of bias into the estimation of nucleotide substitution rates. The effects of this bias can easily be misconstrued as the action of purifying selection at synonymous sites.Chapter 3 is concerned with mutational variation in the murid genome. Nucleotide substitution rates in murid transposable elements were estimated. It was assumed that the majority of murid transposable elements were evolving neutrally and, therefore, that their molecular evolutionary rate was dictated by mutation alone. Under this assumption, variation in estimated element substitution rates reflects sampling and mutational variation only. The results indicate that greater mutational variation occurs along the length of a chromosome than between individual chromosomes, although the latter has been the primary focus in the literature. This result illustrates the importance of accounting for mutational variation in studies of selective constraint and sequence conservation.In Chapter 4, the level of constraint in intergenic DNA adjacent to coding sequences and a moderate distance inside first introns was estimated in a sample of 300 mouse-rat gene orthologues. The results suggested that whilst selective constraint in intergenic sequence adjacent to the start and stop codons is moderately high, this becomes statistically indistinguishable from zero within 4kb upstream/downstream of the first/last exon. Selective constraint in the 5' end of the first intron was also found to be moderately high. Taking the contributions from noncoding sequence into account, it was estimated that the number of deleterious mutations occurring in murid noncoding DNA was approximately equal to that in protein-coding sequence.Chapter 5 expands on the work done in Chapter 4. The assumption of neutral evolution in non-first introns was addressed by comparing their evolutionary rates with those in transposable elements. In addition the selective constraint in intergenic DNA immediately adjacent to genes with that found large distances from known genes was compared. The results showed that, when repetitive sequence is removed, the selective constraints in intergenic DNA are significantly different from zero. Furthermore, this constraint does not become indistinguishable from zero, even at large distances ( 50kb) from genic regions. The data also showed that a weak correlation between intron length and nucleotide substitution rate exists in murid non-first introns.
Effect of the assignment of ancestral CpG state on the estimation of nucleotide substitution rates in mammals
<p>Abstract</p> <p>Background</p> <p>Molecular evolutionary studies in mammals often estimate nucleotide substitution rates within and outside CpG dinucleotides separately. Frequently, in alignments of two sequences, the division of sites into CpG and non-CpG classes is based simply on the presence or absence of a CpG dinucleotide in either sequence, a procedure that we refer to as CpG/non-CpG assignment. Although it likely that this procedure is biased, it is generally assumed that the bias is negligible if species are very closely related.</p> <p>Results</p> <p>Using simulations of DNA sequence evolution we show that assignment of the ancestral CpG state based on the simple presence/absence of the CpG dinucleotide can seriously bias estimates of the substitution rate, because many true non-CpG changes are misassigned as CpG. Paradoxically, this bias is most severe between closely related species, because a minimum of two substitutions are required to misassign a true ancestral CpG site as non-CpG whereas only a single substitution is required to misassign a true ancestral non-CpG site as CpG in a two branch tree. We also show that CpG misassignment bias differentially affects fourfold degenerate and noncoding sites due to differences in base composition such that fourfold degenerate sites can appear to be evolving more slowly than noncoding sites. We demonstrate that the effects predicted by our simulations occur in a real evolutionary setting by comparing substitution rates estimated from human-chimp coding and intronic sequence using CpG/non-CpG assignment with estimates derived from a method that is largely free from bias.</p> <p>Conclusion</p> <p>Our study demonstrates that a common method of assigning sites into CpG and non CpG classes in pairwise alignments is seriously biased and recommends against the adoption of <it>ad hoc </it>methods of ancestral state assignment.</p
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Selective Constraints in Experimentally Defined Primate Regulatory Regions
Changes in gene regulation may be important in evolution. However, the evolutionary properties of regulatory mutations are currently poorly understood. This is partly the result of an incomplete annotation of functional regulatory DNA in many species. For example, transcription factor binding sites (TFBSs), a major component of eukaryotic regulatory architecture, are typically short, degenerate, and therefore difficult to differentiate from randomly occurring, nonfunctional sequences. Furthermore, although sites such as TFBSs can be computationally predicted using evolutionary conservation as a criterion, estimates of the true level of selective constraint (defined as the fraction of strongly deleterious mutations occurring at a locus) in regulatory regions will, by definition, be upwardly biased in datasets that are a priori evolutionarily conserved. Here we investigate the fitness effects of regulatory mutations using two complementary datasets of human TFBSs that are likely to be relatively free of ascertainment bias with respect to evolutionary conservation but, importantly, are supported by experimental data. The first is a collection of almost >2,100 human TFBSs drawn from the literature in the TRANSFAC database, and the second is derived from several recent high-throughput chromatin immunoprecipitation coupled with genomic microarray (ChIP-chip) analyses. We also define a set of putative cis-regulatory modules (pCRMs) by spatially clustering multiple TFBSs that regulate the same gene. We find that a relatively high proportion (∼37%) of mutations at TFBSs are strongly deleterious, similar to that at a 2-fold degenerate protein-coding site. However, constraint is significantly reduced in human and chimpanzee pCRMS and ChIP-chip sequences, relative to macaques. We estimate that the fraction of regulatory mutations that have been driven to fixation by positive selection in humans is not significantly different from zero. We also find that the level of selective constraint in our TFBSs, pCRMs, and ChIP-chip sequences is negatively correlated with the expression breadth of the regulated gene, whereas the opposite relationship holds at that gene's nonsynonymous and synonymous sites. Finally, we find that the rate of protein evolution in a transcription factor appears to be positively correlated with the breadth of expression of the gene it regulates. Our study suggests that strongly deleterious regulatory mutations are considerably more likely (1.6-fold) to occur in tissue-specific than in housekeeping genes, implying that there is a fitness cost to increasing “complexity” of gene expression.</p
Cell reprogramming shapes the mitochondrial DNA landscape.
Individual induced pluripotent stem cells (iPSCs) show considerable phenotypic heterogeneity, but the reasons for this are not fully understood. Comprehensively analysing the mitochondrial genome (mtDNA) in 146 iPSC and fibroblast lines from 151 donors, we show that most age-related fibroblast mtDNA mutations are lost during reprogramming. However, iPSC-specific mutations are seen in 76.6% (108/141) of iPSC lines at a mutation rate of 8.62 × 10-5/base pair. The mutations observed in iPSC lines affect a higher proportion of mtDNA molecules, favouring non-synonymous protein-coding and tRNA variants, including known disease-causing mutations. Analysing 11,538 single cells shows stable heteroplasmy in sub-clones derived from the original donor during differentiation, with mtDNA variants influencing the expression of key genes involved in mitochondrial metabolism and epidermal cell differentiation. Thus, the dynamic mtDNA landscape contributes to the heterogeneity of human iPSCs and should be considered when using reprogrammed cells experimentally or as a therapy
Direct-View Multi-Point Two-Component Interferometric Rayleigh Scattering Velocimeter
This paper describes an instantaneous velocity measurement system based on the Doppler shift of elastically scattered laser light from gas molecules (Rayleigh scattering) relative to an incident laser. The system uses a pulsed laser as the light source, direct-viewing optics to collect the scattered light, an interferometer to analyze spectrally the scattered light mixed with the incident laser light, and a CCD camera to capture the resulting interferogram. The system is capable of simultaneous, spatially (approximately 0.2 mm(exp 3)) and temporally (approximately 40 ns) resolved, multiple point measurements of two orthogonal components of flow velocity in the presence of background scattered light, acoustic noise and vibrations, and flow particulates. Measurements in a large-scale axi-symmetric Mach 1.6 H2-air combustion-heated jet running at a flow sensible enthalpy specific to Mach 5.5 hypersonic flight are performed to demonstrate the technique. The measurements are compared with CFD calculations using a finite-volume discretization of the Favre-averaged Navier-Stokes equations (VULCAN code)
Alternative splicing is frequent during early embryonic development in mouse
<p>Abstract</p> <p>Background</p> <p>Alternative splicing is known to increase the complexity of mammalian transcriptomes since nearly all mammalian genes express multiple pre-mRNA isoforms. However, our knowledge of the extent and function of alternative splicing in early embryonic development is based mainly on a few isolated examples. High throughput technologies now allow us to study genome-wide alternative splicing during mouse development.</p> <p>Results</p> <p>A genome-wide analysis of alternative isoform expression in embryonic day 8.5, 9.5 and 11.5 mouse embryos and placenta was carried out using a splicing-sensitive exon microarray. We show that alternative splicing and isoform expression is frequent across developmental stages and tissues, and is comparable in frequency to the variation in whole-transcript expression. The genes that are alternatively spliced across our samples are disproportionately involved in important developmental processes. Finally, we find that a number of RNA binding proteins, including putative splicing factors, are differentially expressed and spliced across our samples suggesting that such proteins may be involved in regulating tissue and temporal variation in isoform expression. Using an example of a well characterized splicing factor, <it>Fox2</it>, we demonstrate that changes in <it>Fox2 </it>expression levels can be used to predict changes in inclusion levels of alternative exons that are flanked by Fox2 binding sites.</p> <p>Conclusions</p> <p>We propose that alternative splicing is an important developmental regulatory mechanism. We further propose that gene expression should routinely be monitored at both the whole transcript and the isoform level in developmental studies</p
Peak event: the rise, crisis and potential decline of the Olympic Games and the World Cup
This paper tracks the growth of two of the largest tourist events: the Olympic Games and the Football World Cup, drawing on a dataset containing all events between 1964 and 2018. Overall, the size of the three events has grown about 60-fold over the past 50 years, thirteen times faster than world GDP. We identify an S-shaped growth curve and four different growth periods, with an emergent crisis phase in the late 2010s that may have brought us to ‘peak event’ – the point at which these events have reached their largest size. Outlining three different scenarios, we argue that the Olympics and the World Cup are at a critical bifurcation point, which also requires new bidding and hosting policies
Perspective: Is It Time for Advocacy Training in Medical Education?
As the modern medical system becomes increasingly complex, a debate has arisen over the place of advocacy efforts within the medical profession. The authors argue that advocacy can help physicians fulfill their social contract. For physicians to become competent in patient-centered, clinical, administrative, or legislative advocacy, they require professional training. Many professional organizations have called for curricular reform to meet society's health needs during the past 30 years, and the inclusion of advocacy training in undergraduate, graduate, and continuing medical education is supported on both pragmatic and ethical grounds. Undergraduate medical education, especially, is an ideal time for this training because a standard competency can be instilled across all specialties. Although the Accreditation Council for Graduate Medical Education includes advocacy training in curricula for residency programs, few medical schools or residency programs have advocacy electives. By understanding the challenges of the health care system and how to change it for the better, physicians can experience increased professional satisfaction and effectiveness in improving patient care, systems-based practice, and public health
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