A Technique for Simultaneous Antikinetochore Immunofluorescence Staining and Q-Banding in Chromosomes from Human Lymphocytes

Antikinetochore immunofluorescence staining has been used in several studies to determine whether a second kinetochore is present, active, or both, in multicentric chromosomes. All of these studies have used tissue culture cells, and contended with the problem of obtaining well spread, banded metaphase chromosomes without affecting the kinetochore staining. We have adapted hypotonic, centrifugation and chromosome staining techniqnes to obtain simultaneous Q-banding and bright kinetochore staining of chromosomes from human lymphocytes.     

Directed evolution of a fungal peroxidase

The Coprinus cinereus (CiP) heme peroxidase was subjected to multiple rounds of directed evolution in an effort to produce a mutant suitable for use as a dye-transfer inhibitor in laundry detergent. The wild-type peroxidase is rapidly inactivated under laundry conditions due to the high pH (10.5), high temperature (50 degrees C), and high peroxide concentration (5-10 mM). Peroxidase mutants were initially generated using two parallel approaches: site-directed mutagenesis based on structure-function considerations, and error-prone PCR to create random mutations. Mutants were expressed in Saccharomyces cerevisiae and screened for improved stability by measuring residual activity after incubation under conditions mimicking those in a washing machine. Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of wild-type CiP. In the final two rounds, mutants were randomly recombined by using the efficient yeast homologous recombination system to shuffle point mutations among a large number of parents. This in vivo shuffling led to the most dramatic improvements in oxidative stability, yielding a mutant with 174 times the thermal stability and 100 times the oxidative stability of wild-type CiP.     

Novel protein-protein interactions of the Yersinia pestis type III secretion system elucidated with a matrix analysis by surface plasmon resonance and mass spectrometry

Binary complexes formed by components of the Yersinia pestis type III secretion system were investigated by surface plasmon resonance (SPR) and matrix-assisted laser desorption time-of-flight mass spectrometry. Pairwise interactions between 15 recombinant Yersinia outer proteins (Yops), regulators, and chaperones were first identified by SPR. Mass spectrometry confirmed over 80% of the protein-protein interactions suggested by SPR, and new binding partners were further characterized. The Yop secretion protein (Ysc) M2 of Yersinia enterocolitica and LcrQ of Y. pestis, formerly described as ligands only for the specific Yop chaperone (Syc) H, formed stable complexes with SycE. Additional previously unreported complexes of YscE with the translocation regulator protein TyeA and the thermal regulator protein YmoA and multiple potential protein contacts by YscE, YopK, YopH, and LcrH were also identified. Because only stably folded proteins were examined, the interactions we identified are likely to occur either before or after transfer through the injectosome to mammalian host cells and may have relevance to understanding disease processes initiated by the plague bacterium.     

Selection in a subdivided population with dominance or local frequency dependence

The interplay between population structure and natural selection is an area of great interest. It is known that certain types of population subdivision do not alter fixation probabilities of selected alleles under genic, frequency-independent selection. In the presence of dominance for fitness or frequency-dependent selection these same types of subdivision can have large effects on fixation probabilities. For example, the barrier to fixation of a fitter allele due to underdominance is reduced by subdivision. Analytic results presented here relate a subdivided population that conforms to a finite island model to an approximately equivalent panmictic population. The size of this equivalent population is different from (larger than) the actual size of the subdivided population. Selection parameters are also different in the hypothetical equivalent population. As expected, the degree of dominance is lower in the equivalent population. The results are not limited to dominance but cover any form of polynomial frequency dependence.     

Population genetics of the endangered Knysna seahorse,Hippocampus capensis

The evolutionary history of the endangered Knysna seahorse, Hippocampus capensis, and the extent of gene flow among its three known populations, were investigated using 138 mitochondrial DNA control region sequences. Similarly high levels of genetic diversity were found in two of the populations (Knysna and Keurbooms Estuaries), whereas diversity in the third population (Swartvlei Estuary) was lower. Although most haplotypes are shared between at least two populations, based on the haplotype frequency distributions the three assemblages constitute distinct management units. The extant population structure of H. capensis suggests that the Knysna seahorse originated in the large Knysna Estuary. The presence of seahorses in the two smaller estuaries is either the result of a vicariance event at the beginning of the present interglacial period, colonization of the estuaries via the sea, or a combination of the two.     

Statistical Analysis of Multiplex Brain Gene Expression Images

Analysis of variance (ANOVA) was employed to investigate 9,000 gene expression patterns from brains of both normal mice and mice with a pharmacological model of Parkinson's disease (PD). The data set was obtained using voxelation, a method that allows high-throughput acquisition of 3D gene expression patterns through analysis of spatially registered voxels (cubes). This method produces multiple volumetric maps of gene expression analogous to the images reconstructed in biomedical imaging systems. The ANOVA model was compared to the results from singular value decomposition (SVD) by using the first 42 singular vectors of the data matrix, a number equal to the rank of the ANOVA model. The ANOVA was also compared to the results from non-parametric statistics. Lastly, images were obtained for a subset of genes that emerged from the ANOVA as significant. The results suggest that ANOVA will be a valuable framework for insights into the large number of gene expression patterns obtained from voxelation.     

Microarray data quality analysis: lessons from the AFGC project. Arabidopsis Functional Genomics Consortium

Genome-wide expression profiling with DNA microarrays has and will provide a great deal of data to the plant scientific community. However, reliability concerns have required the development data quality tests for common systematic biases. Fortunately, most large-scale systematic biases are detectable and some are correctable by normalization. Technical replication experiments and statistical surveys indicate that these biases vary widely in severity and appearance. As a result, no single normalization or correction method currently available is able to address all the issues. However, careful sequence selection, array design, experimental design and experimental annotation can substantially improve the quality and biological of microarray data. In this review, we discuss these issues with reference to examples from the Arabidopsis Functional Genomics Consortium (AFGC) microarray project.     

Should we expect substitution rate to depend on population size?

The rate of nucleotide substitution is generally believed to be a decreasing function of effective population size, at least for nonsynonymous substitutions. This view was originally based on consideration of slightly deleterious mutations with a fixed distribution of selection coefficients. A realistic model must include the occurrence and fixation of some advantageous mutations that compensate for the loss of fitness due to deleterious substitutions. Some such models, such as so-called "fixed" models, also predict a population size effect on substitution rate. An alternative model, presented here, predicts the near absence of a population size effect on substitution rate. This model is based on concave log-fitness functions and a fixed distribution of mutational effects on the selectively important trait. Simulations of an instance of the model confirm the approximate insensitivity of the substitution rate to population size. Although much experimental evidence has been claimed to support the existence of a population size effect, the body of evidence as a whole is equivocal, and much of the evidence that is supposed to demonstrate such an effect would also suggest that it is very small. Perhaps the proposed model applies well to some genes and not so well to others, and genes therefore vary with regard to the population size effect.     

Convergent validity of six methods to assess physical activity in daily life.

The purpose was to examine the agreement (convergent validity) between six common measures of habitual physical activity to estimate durations of light, moderate, vigorous, and total activity in a range of free-living individuals. Over 7 consecutive days, 49 ethnic Chinese (30 men, 19 women), aged 15-55 yr, wore a Polar heart rate monitor, a uniaxial MTI, and triaxial Tritrac accelerometer, plus a Yamax pedometer for > or = 600 min/day. They also completed a daily physical activity log and on day 8 a Chinese version of the 7-day International Physical Activity Questionnaire. At each level of activity, there was good agreement between the two questionnaire-derived instruments and the two accelerometry-derived instruments, but wide variation across different instruments, with two- to fourfold differences in mean durations often seen. The heart rate monitor overestimated light activity and underestimated moderate activity compared with all other measures. Spearman correlation coefficients were low to moderate (0.2-0.5) across most measures of activity, with the pedometer showing correlations with total activity that were often superior to the other movement sensors. We conclude that, with the use of commonly accepted cut points for defining light, moderate, vigorous, and total activity, little convergent validity across the instruments was evident, suggesting these measures are sampling different levels of habitual physical activity and care is needed when comparing their results. To provide a more stable comparison of activity among different people, across studies, or against accepted physical activity promotion guidelines, further work is needed to fine tune the different cut points across a range of common activity monitors to provide more consistent results during free-living conditions.