Efficient targeted mutagenesis in Epichloë festucae using a split marker system.

A split-marker system for targeted gene deletion was developed for the model grass endophytic fungus Epichloë festucae. Compared to the conventional system that yields up to 25% homologous recombinants, the method resulted in 33-74% targeted deletions in E. festucae using as little as 1.5kb of targeting sequence.

Simultaneous recording of mouse retinal ganglion cells during epiretinal or subretinal stimulation.

We compared response patterns and electrical receptive fields (ERF) of retinal ganglion cells (RGCs) during epiretinal and subretinal electrical stimulation of isolated mouse retina. Retinas were stimulated with an array of 3200 independently controllable electrodes. Four response patterns were observed: a burst of activity immediately after stimulation (Type I cells, Vision Research (2008), 48, 1562-1568), delayed bursts beginning >25ms after stimulation (Type II), a combination of both (Type III), and inhibition of ongoing spike activity. Type I responses were produced more often by epiretinal than subretinal stimulation whereas delayed and inhibitory responses were evoked more frequently by subretinal stimulation. Response latencies were significantly shorter with epiretinal than subretinal stimulation. These data suggest that subretinal stimulation is more effective at activating intraretinal circuits than epiretinal stimulation. There was no significant difference in charge threshold between subretinal and epiretinal configurations. ERFs were defined by the stimulating array surface area that successfully stimulated spikes in an RGC. ERFs were complex in shape, similar to receptive fields mapped with light. ERF areas were significantly smaller with subretinal than epiretinal stimulation. This may reflect the greater distance between stimulating electrodes and RGCs in the subretinal configuration. ERFs for immediate and delayed responses mapped within the same Type III cells differed in shape and size, consistent with different sites and mechanisms for generating these two response types.

Blind deconvolution of time-of-flight mass spectra from atom probe tomography.

A major source of uncertainty in compositional measurements in atom probe tomography stems from the uncertainties of assigning peaks or parts of peaks in the mass spectrum to their correct identities. In particular, peak overlap is a limiting factor, whereas an ideal mass spectrum would have peaks at their correct positions with zero broadening. Here, we report a method to deconvolute the experimental mass spectrum into such an ideal spectrum and a system function describing the peak broadening introduced by the field evaporation and detection of each ion. By making the assumption of a linear and time-invariant behavior, a system of equations is derived that describes the peak shape and peak intensities. The model is fitted to the observed spectrum by minimizing the squared residuals, regularized by the maximum entropy method. For synthetic data perfectly obeying the assumptions, the method recovered peak intensities to within ±0.33 at%. The application of this model to experimental APT data is exemplified with Fe-Cr data. Knowledge of the peak shape opens up several new possibilities, not just for better overall compositional determination, but, e.g., for the estimation of errors of ranging due to peak overlap or peak separation constrained by isotope abundances.

Pseudogene rescue: an adaptive mechanism of codon reassignment.

Alterations to the genetic code--codon reassignments--have occurred many times in life's history, despite the fact that genomes are coadapted to their genetic codes and therefore alterations are likely to be maladaptive. A potential mechanism for adaptive codon reassignment, which could trigger either a temporary period of codon ambiguity or a permanent genetic code change, is the reactivation of a pseudogene by a nonsense suppressor mutant transfer RNA. I examine the population genetics of each stage of this process and find that pseudogene rescue is plausible and also readily explains some features of extant variability in genetic codes.

Epigenomic plasticity within populations: its evolutionary significance and potential.

Epigenetics has progressed rapidly from an obscure quirk of heredity into a data-heavy 'omic' science. Our understanding of the molecular mechanisms of epigenomic regulation, and the extent of its importance in nature, are far from complete, but in spite of such drawbacks, population-level studies are extremely valuable: epigenomic regulation is involved in several processes central to evolutionary biology including phenotypic plasticity, evolvability and the mediation of intragenomic conflicts. The first studies of epigenomic variation within populations suggest high levels of phenotypically relevant variation, with the patterns of epigenetic regulation varying between individuals and genome regions as well as with environment. Epigenetic mechanisms appear to function primarily as genome defences, but result in the maintenance of plasticity together with a degree of buffering of developmental programmes; periodic breakdown of epigenetic buffering could potentially cause variation in rates of phenotypic evolution.

A comprehensive index for assessing environmental stress in animals.

Numerous models and indices exist that attempt to characterize the effect of environmental factors on the comfort of animals and humans. Heat and cold indices have been utilized to adjust ambient temperature (Ta) for the effects of relative humidity (RH) or wind speed (WS) or both for the purposes of obtaining a "feels-like" or apparent temperature. However, no model has been found that incorporates adjustments for RH, WS, and radiation (RAD) over conditions that encompass hot and cold environmental conditions. The objective of this study was to develop a comprehensive climate index (CCI) that has application under a wide range of environmental conditions and provides an adjustment to Ta for RH, WS, and RAD. Environmental data were compiled from 9 separate summer periods in which heat stress events occurred and from 6 different winter periods to develop and validate the CCI. The RH adjustment is derived from an exponential relationship between Ta and RH with temperature being adjusted up or down from an RH value of 30%. At 45 degrees C, the temperature adjustment for increasing RH from 30 to 100% equals approximately 16 degrees C, whereas at -30 degrees C temperature adjustments due to increasing RH from 30 to 100% equal approximately -3.0 degrees C, with greater RH values contributing to a reduced apparent temperature under cold conditions. The relationship between WS and temperature adjustments was also determined to be exponential with a logarithmic adjustment to define appropriate declines in apparent temperature as WS increases. With this index, slower WS results in the greatest change in apparent temperature per unit of WS regardless of whether hot or cold conditions exist. As WS increases, the change in apparent temperature per unit of WS becomes less. Based on existing windchill and heat indices, the effect of WS on apparent temperature is sufficiently similar to allow one equation to be utilized under hot and cold conditions. The RAD component was separated into direct solar radiation and ground surface radiation. Both of these were found to have a linear relationship with Ta. This index will be useful for further development of biological response functions, which are associated with energy exchange, and improving decision-making processes, which are weather-dependent. In addition, the defined thresholds can serve as management and environmental mitigation guidelines to protect and ensure animal comfort.

Tympanic temperature in confined beef cattle exposed to excessive heat load.

Angus crossbred yearling steers (n = 168) were used to evaluate effects on performance and tympanic temperature (TT) of feeding additional potassium and sodium to steers exposed to excessive heat load (maximum daily ambient temperature exceeded 32°C for three consecutive days) during seasonal summer conditions. Steers were assigned one of four treatments: (1) control; (2) potassium supplemented (diet containing 2.10% KHCO3); (3) sodium supplemented (diet containing 1.10% NaCl); or (4) potassium and sodium supplemented (diet containing 2.10% KHCO3 and 1.10% NaCl). Overall, additional KHCO3 at the 2% level or NaCl at the 1% level did not improve performance or heat stress tolerance with these diet formulations. However, the addition of KHCO3 did enhance water intake. Independent of treatment effects, TT of cattle displaying high, moderate, or low levels of stress suggest that cattle that do not adequately cool down at night are prone to achieving greater body temperatures during a subsequent hot day. Cattle that are prone to get hot but can cool at night can keep average tympanic temperatures at or near those of cattle that tend to consistently maintain lower peak and mean body temperatures. In addition, during cooler and moderately hot periods, cattle change TT in a stair-step or incremental pattern, while under hot conditions, average TT of group-fed cattle moves in conjunction with ambient conditions, indicating that thermoregulatory mechanisms are at or near maximum physiological capacity.

Replication of KIAA0350, IL2RA, RPL5 and CD58 as multiple sclerosis susceptibility genes in Australians.

A recent genome-wide association study (GWAS) conducted by the International Multiple Sclerosis Genetics Consortium (IMSGC) identified a number of putative MS susceptibility genes. Here we have performed a replication study in 1134 Australian MS cases and 1265 controls for 17 risk-associated single nucleotide polymorphisms (SNPs) reported by the IMSGC. Of 16 SNPs that passed quality control filters, four, each corresponding to a different non-human leukocyte antigen (HLA) gene, were associated with disease susceptibility: KIAA0350 (rs6498169) P=0.001, IL2RA (rs2104286) P=0.033, RPL5 (rs6604026) P=0.041 and CD58 (rs12044852) P=0.042. There was no association (P=0.58) between rs6897932 in the IL7R gene and the risk of MS. No interactions were detected between the replicated IMSGC SNPs and HLA-DRB1*15, gender, disease course, disease progression or age-at-onset. We used a novel Bayesian approach to estimate the extent to which our data increased or decreased evidence for association with the six most-associated IMSGC loci. These analyses indicated that even modest P-values, such as those reported here, can contribute markedly to the posterior probability of 'true' association in replication studies. In conclusion, these data provide support for the involvement of four non-HLA genes in the pathogenesis of MS, and combined with previous data, increase to genome-wide significance (P=3 x 10(-8)) evidence of an association between KIAA0350 and risk of disease.

SNP mapping and candidate gene sequencing in the class I region of the HLA complex: searching for multiple sclerosis susceptibility genes in Tasmanians.

This study is an extension to previously published work that has linked variation in the human leukocyte antigen (HLA) class I region with susceptibility to multiple sclerosis (MS) in Australians from the Island State of Tasmania. Single nucleotide polymorphism (SNP) mapping was performed on an 865-kb candidate region (D6S1683-D6S265) in 166 Tasmanian MS families, and seven candidate genes [ubiquitin D (UBD), olfactory receptor 2H3 (OR2H3), gamma-aminobutyric acid B receptor 1 (GABBR1), myelin oligodendrocyte glycoprotein (MOG), HLA-F, HLA complex group 4 (HCG4) and HLA-G] were resequenced. SNPs tagging the extended MS susceptibility haplotype were genotyped in an independent sample of 356 Australian MS trios and SNPs in the MOG gene were significantly over-transmitted to MS cases. We identified significant effects on MS susceptibility of HLA-A*2 (OR: 0.51; P = 0.05) and A*3 (OR: 2.85; P = 0.005), and two coding polymorphisms in the MOG gene (V145I: P = 0.01, OR: 2.2; V142L: P = 0.04, OR: 0.45) after full conditioning on HLA-DRB1. We have therefore identified plausible candidates for the causal MS susceptibility allele, and although not conclusive at this stage, our data provide suggestive evidence for multiple class I MS susceptibility genes.

Spontaneous loss of a conditionally dispensable chromosome from the Alternaria alternata apple pathotype leads to loss of toxin production and pathogenicity.

The Alternaria alternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide, host-specific toxin, AM-toxin. We recently cloned a cyclic peptide synthetase gene, AMT, whose product catalyzes the production of AM-toxin and showed that it resides on chromosomes of 1.8 Mb or less, depending on the A. alternata apple pathotype strain. Reverse transcriptase (RT)-PCR, using primers specific to AMT, on laboratory sub-cultured strains previously shown to produce AM-toxin, identified one isolate that did not express the gene. A leaf necrosis bioassay confirmed an AM-toxin-minus phenotype. However, an original isolate of this strain which had not undergone sub-culture gave a positive result by both RTPCR and bioassay. Contour-clamped homogeneous electric field electrophoresis and Southern hybridization demonstrated the loss of a 1.1-Mb chromosome in the non-toxin-producing isolate. Since this chromosome can be entirely lost without affecting growth, but is necessary for pathogenicity, we propose it is a conditionally dispensable chromosome.