Novel human pathological mutations

Human Gene Mutations

Novel human pathological mutations     

iMARS--mutation analysis reporting software: an analysis of spontaneous cII mutation spectra

The sensitivity of any mutational assay is determined by the level at which spontaneous mutations occur in the corresponding untreated controls. Establishing the type and frequency at which mutations occur naturally within a test system is essential if one is to draw scientifically sound conclusions regarding chemically induced mutations. Currently, mutation-spectra analysis is laborious and time-consuming. Thus, we have developed iMARS, a comprehensive mutation-spectrum analysis package that utilises routinely used methodologies and visualisation tools. To demonstrate the use and capabilities of iMARS, we have analysed the distribution, types and sequence context of spontaneous base substitutions derived from the cII gene mutation assay in transgenic animals. Analysis of spontaneous mutation spectra revealed variation both within and between the transgenic rodent test systems Big Blue Mouse, MutaMouse and Big Blue Rat. The most common spontaneous base substitutions were G:C-->A:T transitions and G:C-->T:A transversions. All Big Blue Mouse spectra were significantly different from each other by distribution and nearly all by mutation type, whereas the converse was true for the other test systems. Twenty-eight mutation hotspots were observed across all spectra generally occurring in CG, GA/TC, GG and GC dinucleotides. A mutation hotspot at nucleotide 212 occurred at a higher frequency in MutaMouse and Big Blue Rat. In addition, CG dinucleotides were the most mutable in all spectra except two Big Blue Mouse spectra. Thus, spontaneous base-substitution spectra showed more variation in distribution, type and sequence context in Big Blue Mouse relative to spectra derived from MutaMouse and Big Blue Rat. The results of our analysis provide a baseline reference for mutation studies utilising the cII gene in transgenic rodent models. The potential differences in spontaneous base-substitution spectra should be considered when making comparisons between these test systems. The ease at which iMARS has allowed us to carry out an exhaustive investigation to assess mutation distribution, mutation type, strand bias, target sequences and motifs, as well as predict mutation hotspots provides us with a valuable tool in helping to distinguish true chemically induced hotspots from background mutations and gives a true reflection of mutation frequency.     

Using behavioural and state variables to identify proximate causes of population change in a seabird

Changes in animal population size are driven by the interactions between intrinsic processes and extrinsic forces, and identifying the proximate mechanisms behind population change remains a fundamental question in ecology. Here we report on how measuring behavioural and state proxies of food availability among populations experiencing different growth rates can be used to rapidly identify proximate drivers of population trends. In recent decades, the Cape gannet Morus capensis has shown a major distributional shift with historically large colonies in Namibia decreasing rapidly, whilst numbers at South African colonies have increased, suggesting contrasting environmental conditions in the two regions. We compared per capita growth rates of five of the six extant colonies with foraging range (using miniaturised Global Positioning System loggers), foraging work rate, food delivery rates and body condition of breeding adults. We found significant associations between the rate of population change, individual behaviour, energetic gain and body condition that indicate that recent population changes are associated with extrinsic effects. This study shows that behavioural and state data can be used to identify important drivers of population change, and their cost-effectiveness ensures that they are an appealing option for measuring the health of animal populations in numerous situations.     

Expression and characterization of a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein) in Arabidopsis

Arabidopsis (Arabidopsis thaliana) was transformed with a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein [roGFP]), with expression targeted to either the cytoplasm or to the mitochondria. Both the mitochondrial and cytosolic forms are oxidation-reduction sensitive, as indicated by a change in the ratio of 510 nm light (green light) emitted following alternating illumination with 410 and 474 nm light. The 410/474 fluorescence ratio is related to the redox potential (in millivolts) of the organelle, cell, or tissue. Both forms of roGFP can be reduced with dithiothreitol and oxidized with hydrogen peroxide. The average resting redox potentials for roots are -318 mV for the cytoplasm and -362 mV for the mitochondria. The elongation zone of the Arabidopsis root has a more oxidized redox status than either the root cap or meristem. Mitochondria are much better than the cytoplasm, as a whole, at buffering changes in redox. The data show that roGFP is redox sensitive in plant cells and that this sensor makes it possible to monitor, in real time, dynamic changes in redox in vivo.     

Patterns of sequence loss and cytosine methylation within a population of newly resynthesized Brassica napus allopolyploids

Allopolyploid formation requires the adaptation of two nuclear genomes within a single cytoplasm, which may involve programmed genetic and epigenetic changes during the initial generations following genome fusion. To study the dynamics of genome change, we synthesized 49 isogenic Brassica napus allopolyploids and surveyed them with 76 restriction fragment length polymorphism (RFLP) probes and 30 simple sequence repeat (SSR) primer pairs. Here, we report on the types and distribution of genetic and epigenetic changes within the S(1) genotypes. We found that insertion/deletion (indel) events were rare, but not random. Of the 57,710 (54,383 RFLP and 3,327 SSR) parental fragments expected among the amphidiploids, we observed 56,676 or 99.9%. Three loci derived from Brassica rapa had indels, and one indel occurred repeatedly across 29% (14/49) of the lines. Loss of one parental fragment was due to the 400-bp reduction of a guanine-adenine dinucleotide repeat-rich sequence. In contrast to the 4% (3/76) RFLP probes that detected indels, 48% (35/73) detected changes in the CpG methylation status between parental genomes and the S1 lines. Some loci were far more likely than others to undergo epigenetic change, but the number of methylation changes within each synthetic polyploid was remarkably similar to others. Clear de novo methylation occurred at a much higher frequency than de novo demethylation within allopolyploid sequences derived from B. rapa. Our results suggest that there is little genetic change in the S(0) generation of resynthesized B. napus polyploids. In contrast, DNA methylation was altered extensively in a pattern that indicates tight regulation of epigenetic changes.     

A spatially explicit model for an Allee effect: why wolves recolonize so slowly in Greater Yellowstone

A reduced probability of finding mates at low densities is a frequently hypothesized mechanism for a component Allee effect. At low densities dispersers are less likely to find mates and establish new breeding units. However, many mathematical models for an Allee effect do not make a distinction between breeding group establishment and subsequent population growth. Our objective is to derive a spatially explicit mathematical model, where dispersers have a reduced probability of finding mates at low densities, and parameterize the model for wolf recolonization in the Greater Yellowstone Ecosystem (GYE). In this model, only the probability of establishing new breeding units is influenced by the reduced probability of finding mates at low densities. We analytically and numerically solve the model to determine the effect of a decreased probability in finding mates at low densities on population spread rate and density. Our results suggest that a reduced probability of finding mates at low densities may slow recolonization rate.     

Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi

We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA+ and vapA-negative (including vapB+) plasmid genotypes. The vapA+ frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to approximately 10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA+) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA+ "horse-pathogenic" genotype determination.