<Emphasis Type="Italic">ScreenMill</Emphasis>: A freely available software suite for growth measurement,analysis and visualization of high-throughput screen data

Background  

Many high-throughput genomic experiments, such as Synthetic Genetic Array and yeast two-hybrid, use colony growth on solid media as a screen metric. These experiments routinely generate over 100,000 data points, making data analysis a time consuming and painstaking process. Here we describe ScreenMill, a new software suite that automates image analysis and simplifies data review and analysis for high-throughput biological experiments.     

Down-Regulation of Rad51 Activity during Meiosis in Yeast Prevents Competition with Dmc1 for Repair of Double-Strand Breaks

Interhomolog recombination plays a critical role in promoting proper meiotic chromosome segregation but a mechanistic understanding of this process is far from complete. In vegetative cells, Rad51 is a highly conserved recombinase that exhibits a preference for repairing double strand breaks (DSBs) using sister chromatids, in contrast to the conserved, meiosis-specific recombinase, Dmc1, which preferentially repairs programmed DSBs using homologs. Despite the different preferences for repair templates, both Rad51 and Dmc1 are required for interhomolog recombination during meiosis. This paradox has recently been explained by the finding that Rad51 protein, but not its strand exchange activity, promotes Dmc1 function in budding yeast. Rad51 activity is inhibited in dmc1Δ mutants, where the failure to repair meiotic DSBs triggers the meiotic recombination checkpoint, resulting in prophase arrest. The question remains whether inhibition of Rad51 activity is important during wild-type meiosis, or whether inactivation of Rad51 occurs only as a result of the absence of DMC1 or checkpoint activation. This work shows that strains in which mechanisms that down-regulate Rad51 activity are removed exhibit reduced numbers of interhomolog crossovers and noncrossovers. A hypomorphic mutant, dmc1-T159A, makes less stable presynaptic filaments but is still able to mediate strand exchange and interact with accessory factors. Combining dmc1-T159A with up-regulated Rad51 activity reduces interhomolog recombination and spore viability, while increasing intersister joint molecule formation. These results support the idea that down-regulation of Rad51 activity is important during meiosis to prevent Rad51 from competing with Dmc1 for repair of meiotic DSBs.     

Regulation of DNA Pairing in Homologous Recombination

Homologous recombination (HR) is a major mechanism for eliminating DNA double-strand breaks from chromosomes. In this process, the break termini are resected nucleolytically to form 3′ ssDNA (single-strand DNA) overhangs. A recombinase (i.e., a protein that catalyzes homologous DNA pairing and strand exchange) assembles onto the ssDNA and promotes pairing with a homologous duplex. DNA synthesis then initiates from the 3′ end of the invading strand, and the extended DNA joint is resolved via one of several pathways to restore the integrity of the injured chromosome. It is crucial that HR be carefully orchestrated because spurious events can create cytotoxic intermediates or cause genomic rearrangements and loss of gene heterozygosity, which can lead to cell death or contribute to the development of cancer. In this review, we will discuss how DNA motor proteins regulate HR via a dynamic balance of the recombination-promoting and -attenuating activities that they possess.     

Foraging ecology of the endangered wood stork recorded in the stable isotope signature of feathers

Down feathers and regurgitant were collected from nestling wood storks (Mycteria americana) from two inland and two coastal breeding colonies in Georgia. The stable isotopic ratios of carbon (¹³C/¹²C) and nitrogen (¹⁵N/¹⁴N) in these materials were analyzed to gain insights into the natal origins of juvenile storks and the foraging activities of adults. Down feathers differed in '¹³C between inland and coastal colonies, having average isotopic values that reflected the sources of carbon fixed in biomass at the base of the food web. Feathers from the inland colonies differed between colonies in '¹⁵N, while those from the coastal colonies did not. These patterns primarily reflected the foraging activities of parent storks, with individuals capturing differing percentages of prey of distinct trophic status at each colony. Collectively, the carbon and nitrogen isotopic signatures of feather keratin were used to distinguish nestlings from each colony, except for instances where storks from different colonies foraged in common wetlands. The stable isotopic composition of food items in regurgitant was used to reconstruct the trophic structure of the ecosystems in which wood storks foraged. Predicted foraging activities based on the isotopic composition of keratin were generally consistent with the percentage of prey types (freshwater vs. saltwater and lower trophic level vs. upper trophic level consumer) observed in regurgitant, except for the coastal colony at St. Simons Island, where the '¹³C of feathers strongly suggested that freshwater prey were a significant component of the diet. This inconsistency was resolved by aerial tracking of adults during foraging excursions using a fixed-wing aircraft. Observed foraging activities supported interpretations based on the stable isotope content of feathers, suggesting that the latter provided a better record of overall foraging activity than regurgitant analysis alone. Observed foraging patterns were compared to the predictions of a statistical model that determined habitat utilization based on habitat availability using a geographic information system (GIS) database. Observed foraging activities and those predicted from feathers both suggested that some adult storks preferred to feed their young freshwater prey, even when saltwater resources were more accessible in the local environment. This conclusion supports the contention that wood stork populations are sensitive to changes in the distribution of freshwater habitats along the southeastern coastal plain of the United States.     

Glyphosate sensitivity of 5-enol-pyruvylshikimate-3-phosphate synthase from Bacillus subtilis depends upon state of activation induced by monovalent cations

The 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase from Bacillus subtilis was activated by monovalent cations, catalytic activity being negligible in the absence of monovalent cations. The order of cation effectiveness (NH4+ greater than K+ greater than Rb+ greater than Na+ = Cs+ = Li+) indicated that the extent of activation was directly related to the unhydrated cation radius. Ammonium salts, at physiological concentrations, were dramatically more effective than other cations. Activation by ammonium was instantaneous, was not influenced by the counter ion, and gave a hyperbolic saturation curve. Hill plots did not show detectable cooperativity in the binding of ammonium. Double-reciprocal plots indicated that ammonium increases the maximal velocity and decreases the apparent Michaelis constants of EPSP synthase with respect to both phosphoenol pyruvate (PEP) and shikimate 3-phosphate (S3P). A direct relationship between sensitivity to inhibition by glyphosate and the activation state of EPSP synthase was demonstrated. Hill plots indicated a single value for glyphosate binding throughout the range of ammonium activation. Double-reciprocal plots of substrate saturation data obtained with ammonium-activated enzyme in the presence of glyphosate showed glyphosate to behave as a competitive inhibitor with respect to PEP and as a mixed-type inhibitor relative to S3P. The increased glyphosate sensitivity of ammonium-activated EPSP synthase is attributed to a lowering of the inhibitor constant of glyphosate with respect to PEP. Erroneous underestimates of sensitivities of some bacterial EPSP synthases to inhibition by glyphosate may result from failure to recognize cation requirements of EPSP synthases.     

Phylogeography of the California sheephead,Semicossyphus pulcher: the role of deep reefs as stepping stones and pathways to antitropicality

In the past decade, the study of dispersal of marine organisms has shifted from focusing predominantly on the larval stage to a recent interest in adult movement. Antitropical distributions provide a unique system to assess vagility and dispersal. In this study, we have focused on an antitropical wrasse genus, Semicossyphus, which includes the California sheephead, S. pulcher, and Darwin's sheephead, S. darwini. Using a phylogenetic approach based on mitochondrial and nuclear markers, and a population genetic approach based on mitochondrial control region sequences and 10 microsatellite loci, we compared the phylogenetic relationships of these two species, as well as the population genetic characteristics within S. pulcher. While S. pulcher and S. darwini are found in the temperate eastern Pacific regions of the northern and southern hemispheres, respectively, their genetic divergence was very small (estimated to have occurred between 200 and 600 kya). Within S. pulcher, genetic structuring was generally weak, especially along mainland California, but showed weak differentiation between Sea of Cortez and California, and between mainland California and Channel Islands. We highlight the congruence of weak genetic differentiation both within and between species and discuss possible causes for maintenance of high gene flow. In particular, we argue that deep and cooler water refugia are used as stepping stones to connect distant populations, resulting in low levels of genetic differentiation.     

Apparent ornithine decarboxylase activity, measured by 14CO2 trapping, after frozen storage of rat tissue and rat tissue supernatants

Ornithine decarboxylase (ODC) activity of rat tissues was measured by the standard 14CO2 trapping method after frozen storage (-60 or -70 degrees C) of the tissues or their 105,000g supernatants. True ODC activity was determined by two methods: (a) addition of the inhibitors alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, or aminooxyacetate (AOA), an inhibitor that blocks the decarboxylation of ornithine by mitochondrial enzymes; and (b) chromatographic analysis of the reaction products. In the frozen supernatants of liver and spleen, ODC activity changed only slightly after 1 day but increased 29 and 14%, respectively, by 30 days; activity in kidney supernatant decreased 17% after 1 day and remained near that level at 30 days. Kidney and spleen ODC activity was inhibited 90-100% by DFMO, but apparent liver ODC activity was inhibited only 60-75%. In the supernatant prepared from tissue stored frozen for 1 day, apparent ODC activity in liver increased 500% over that activity in the freshly prepared supernatant; at 23 days, apparent activity increased 755% for liver and 121% for kidney. After 23 days, DFMO did not inhibit apparent ODC activity in supernatants from frozen liver and inhibited ODC in frozen kidney by only 49%. With AOA, the ODC activities of the fresh and frozen supernatants were similar, indicating that the large increase in apparent ODC activity in frozen tissue was due to artifacts from the metabolism of ornithine via the mitochondrial pathway. HPLC analysis of the reaction products resulting from the incubation of uniformly labeled [14C]ornithine with the fresh and frozen preparations indicated no increase in putrescine with the frozen preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Demography of the western harvest mouse,Reithrodontomys megalotis,in eastern Kansas

Summary Reithrodontomys megalotis was live-trapped on three open field grids in eastern Kansas from August 1979 to August 1982. One grid was a control on which normal demography was monitored, and two were experimental grids where periodic removal of residents allowed the investigation of the demographic and fitness consequences of emigration. Popullations on the control grid showed an annual cycle in numbers, reaching peaks in density during the winter of each year, falling to low densities during the summer. Low summer densities were attributed, at least partially, to low trappability of R. megalotis during periods of high resource abundance. Reproduction was initiated in the spring of each year at approximately the same time as the emergence of new vegetative growth, and ceased in late fall of each year. The trappable population was composed almost entirely of adults, and the sex ratio was skewed significantly toward females. A statistically significant negative association between the number of M. ochrogaster residents and the reproductive activity of female R. megalotis residents was found. A canonical correlation analysis revealed a common seasonal component to the demography of the two species, and possible interspecific affects. Emigrating R. megalotis were a nonrandom sample of the population, with emigrants more likely to be subadult and juvenile males when compared to residents. No association was detected between the numbers of M. ochrogaster colonizing the removal grid and the numbers of R. megalotis colonizing the same removal grid, or between the number of M. ochrogaster residents on the control grid and the numbers of R. megalotis colonizing the removal grids. However, the number of R. megalotis residents on the control grid is positively correlated with the number of R. megalotis on the removal grids.     

An international collaborative study on the First International Standard of bovine luteinizing hormone for immunoassay

An ampouled freeze-dried preparation of bovine pituitary luteinizing hormone (bLH), coded EHC-bLH-1, has been evaluated in an international collaborative study and shown to be suitable and sufficiently stable to serve as a standard for bLH. Eight laboratories provided immunoassay data, one laboratory provided receptor assay data, and bioassay data were obtained from 4 laboratories. The geometric mean potency estimate obtained by immunoassays, expressed as milliunits of the USDA bLH-B-5 preparation per ampoule, was 25.6, which is consistent with the result obtained by in-vivo bioassays. The geometric mean estimate obtained by receptor assays or by in-vitro bioassays was lower, i.e. 13.2 milliunits per ampoule. The reason for this discrepancy is currently under investigation. With the authorization of the Expert Committee on Biological Standardization of the World Health Organization this preparation was established in 1985 as the International Standard for Luteinizing Hormone, Bovine, for Immunoassay with a unitage of 25 mi.u. per ampoule.