Estimating node degree in bait-prey graphs

MOTIVATION: Proteins work together to drive biological processes in cellular machines. Summarizing global and local properties of the set of protein interactions, the interactome, is necessary for describing cellular systems. We consider a relatively simple per-protein feature of the interactome: the number of interaction partners for a protein, which in graph terminology is the degree of the protein. RESULTS: Using data subject to both stochastic and systematic sources of false positive and false negative observations, we develop an explicit probability model and resultant likelihood method to estimate node degree on portions of the interactome assayed by bait-prey technologies. This approach yields substantial improvement in degree estimation over the current practice that naively sums observed edges. Accurate modeling of observed data in relation to true but unknown parameters of interest gives a formal point of reference from which to draw conclusions about the system under study. AVAILABILITY: All analyses discussed in this text can be performed using the ppiStats and ppiData packages available through the Bioconductor project (http://www.bioconductor.org).     

Estimating the number of coding mutations in genotypic- and phenotypic-driven N-ethyl-N-nitrosourea (ENU) screens

N-ethyl-N-nitrosourea (ENU) is a widely used mutagen in genotypic and phenotypic screens aimed at elucidating gene function. The high rate at which ENU induces point mutations raises the possibility that an observed phenotype may be to the result of another unidentified linked mutation. This article presents methods for estimating the probability of additional linked coding mutations (1) in a given region of DNA using both Poisson and Bayesian models and in (2) an F(1) animal exposed to ENU that has undergone b number of backcrosses. Applying these methods to the mouse data set of Quwailid et al., we estimate that the probability that a confounding mutation is linked to a cloned mutation when the candidate region is 5 Mb is very slim (p < 0.002). Where mutants are identified by genotypic methods, we show that backcrossing in the absence of marker-assisted selection is an inefficient means of eliminating linked confounding mutations.     

Glucose stimulates a decrease of the fatty acid saturation degree in Acinetobacter calcoaceticus

The fatty acid composition of Acinetobacter calcoaceticus 69-V was determined under various growth conditions. Saturated, unsaturated, and hydroxy fatty acids with chain lengths of 12–18 carbon atoms predominated in the fatty acid profile. With acetate or propanol as growth substrates, the ratio of saturated to unsaturated fatty acids varied with changes in the temperature. This was the only adaptive mechanism detected that compensated for the physical effects of temperature alterations on the cell membranes. The fatty acid composition of A. calcoaceticus grown at 40 °C had a saturation degree of approximately 50%; after growth at 20 °C it was approximately 35%. In the presence of a carbon and energy source, A. calcoaceticus was able to respond to temperature reductions under oxic conditions regardless of whether fatty acid biosynthesis was inhibited or not. This suggests an aerobic mechanism of fatty acid biosynthesis and the involvement of a fatty acid desaturase system. Addition of the non-growth substrate, glucose, helped the organism to adapt to lower temperature. The molecular mechanism of the aid is not really understood. The oxidation of glucose could provide the desaturase either with electrons directly via a pyrrolo-quinoline-quinone-linked glucose dehydrogenase or with NADH after fatty acid degradation has been initiated by ATP generated by the oxidation of glucose. Received: 19 June 1998 / Accepted: 28 December 1998     

Stringent mating-type-regulated auxotrophy increases the accuracy of systematic genetic interaction screens with Saccharomyces cerevisiae mutant arrays

A genomic collection of haploid Saccharomyces cerevisiae deletion strains provides a unique resource for systematic analysis of gene interactions. Double-mutant haploid strains can be constructed by the synthetic genetic array (SGA) method, wherein a query mutation is introduced by mating to mutant arrays, selection of diploid double mutants, induction of meiosis, and selection of recombinant haploid double-mutant progeny. The mechanism of haploid selection is mating-type-regulated auxotrophy (MRA), by which prototrophy is restricted to a particular haploid genotype generated only as a result of meiosis. MRA escape leads to false-negative genetic interaction results because postmeiotic haploids that are supposed to be under negative selection instead proliferate and mate, forming diploids that are heterozygous at interacting loci, masking phenotypes that would be observed in a pure haploid double-mutant culture. This work identified factors that reduce MRA escape, including insertion of terminator and repressor sequences upstream of the MRA cassette, deletion of silent mating-type loci, and utilization of α-type instead of a-type MRA. Modifications engineered to reduce haploid MRA escape reduced false negative results in SGA-type analysis, resulting in >95% sensitivity for detecting gene–gene interactions.     

Influence of the degree of saturation of iron stores on the gastrointestinal absorption of aluminum

The possible influence of iron metabolism in the regulation of aluminum gastrointestinal absorption in male Wistar rats has been studied. Three groups were considered: Fe overloaded Group 1; Fe normal Group 2; Fe depleted Group 3. All groups were exposed during 45 days to 40 mg of Al(OH)3 investigating the concentration of Al in serum and urine throughout the experiment and also the Al in brain at the end of that period. Results demonstrated that 24 h urinary Al excretion was significantly higher in Fe depleted rats than in Fe overloaded animals (p less than 0.01 and p less than 0.05 respectively). In addition, Al in brain showed the same pattern of deposition yielding in the Fe depleted rats nearly a three fold increase in the concentration of Al. By contrast, serum Al did not show any particular trend. These findings are in agreement with the fact that Fe metabolism may modulate Al gastrointestinal absorption suggesting that Al and Fe might share the same mechanism of gastrointestinal absorption.     

Super-sized deletions: Improved transposon excision screens using a mus309 mutant background

Over the past two decades, a large collection of transposable elements inserted at various locations in the Drosophila melanogaster genome has been assembled. These transposons are frequently utilized in imprecise excision screens to generate deletions in genes of interest. In general, these screens involve genetic manipulations to combine a non-autonomous transposon and the appropriate transposase in individual male or female flies. DNA double-strand breaks are created via transposase action in both somatic and germline cells of these individuals and inaccurate repair events are recovered in the progeny. Because deletion-prone repair of transposon-induced double-strand breaks is rare, these screens generally require a significant investment of time and resources. We recently reported that conducting imprecise excision screens in mus309 mutant flies, which lack the Drosophila ortholog of the Bloom Syndrome helicase, results in an increase in both the number and size of deletions recovered. Here, we provide additional information for Drosophila researchers wishing to utilize this technique. In addition, we discuss how the general principle behind this technique can be applied in other contexts where double-strand breaks are being generated for the purpose of genome modification.     

Estimating mutant microsatellite allele frequencies in somatic cells by small-pool PCR

Identifying microsatellite instability (MSI) by partitioning DNA into multiple small pools containing only single genome amounts of DNA results in trapping both progenitor and low-frequency mutant alleles into pools where they can be identified and counted following PCR. Statistical approaches determining both the frequencies and the significant differences between frequencies of these Poisson-distributed alleles are presented. Results indicate a level of sensitivity and quantification not possible by standard PCR methods. Using material from colon cancer patients with high levels of MSI in their tumors, we also present the molecular and robotic methods for carrying out such studies. Validation experiments indicated mutants detectable at frequencies >0.03 above background. Frequencies obtained in tumor tissue (>0.25) met the expectations of the approach. Significant levels of MSI were detected in the constitutive tissue of the patient carrying a germ-line mutation for mismatch repair, suggesting both mechanistic and clinical applications of the procedure.     

Estimating the degree of sperm precedence in laboratory mating experiments: a maximum likelihood method

Allozyme polymorphisms have been used frequently in laboratory mating experiments to study patterns of sperm utilization in multiply mated females. In some instances, due either to chance or to design, there is a diagnostic difference between male genotypes that allows unambiguous assignment of paternity. In other instances, there is some overlap in allelic composition of males, so that attribution of paternity is often uncertain. This paper presents a statistical method for analyzing data of the second sort obtained from twice-mated females, based on the principle of maximum likelihood. The method allows the estimation of a mating parameter, psi the frequency with which sperm from the first male fertilizes the female's eggs. Various hypotheses about the null value of psi may be tested by a likelihood ratio test statistic. Also presented is a method of testing for homogeneity in psi values across different broods produced by the same female.     

Estimating photosynthetic electron transport via chlorophyll fluorometry without Photosystem II light saturation

Estimates of thylakoid electron transport rates (J_e) from chlorophyll fluorometry are often used in combination with leaf gas exchange measurements to provide detailed information about photosynthetic activity of leaves in situ. Estimating J_e requires accurate determination of the quantum efficiency of Photosystem II (Φ_P), which in turn requires momentary light saturation of the Photosystem II light harvesting complex to induce the maximum fluorescence signal (F_M′). In practice, full saturation is often difficult to achieve, especially when incident photosynthetic photon flux density (Q) is high and energy is effectively dissipated by non-photochemical quenching. In the present work, a method for estimating the true F_M′ under high Q was developed, using multiple light pulses of varying intensity (Q′). The form of the expected relationship between the apparent F_M′ and Q′ was derived from theoretical considerations. This allowed the true F_M′ at infinite Q′ to be estimated from linear regression. Using a commercially available leaf gas exchange/ chlorophyll fluorescence measurement system, J_e was compared to gross photosynthetic CO₂ assimilation (A_G) under conditions where the relationship between J_e and A_G was expected to be linear. Both in C₄ leaves (Zea mays) in ambient air and also in C₃ leaves (Gossypium hirsutum) under non-photorespiratory conditions the apparent ratio between J_e and A_G declined at high Q when Φ_P was calculated from F_M′ measured simply using the highest available saturating pulse intensity. When F_M′ was determined using the multiple pulse / linear regression technique, the expected relationship between J_e and A_G at high Q was restored, indicating that the Φ_P estimate was improved. This method of determining F_M′ should prove useful for verifying when saturating pulse intensities are sufficient, and for accurately determining Φ_P when they are not. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of the degree of saturation of dietary fat on the hepatic production of lipoproteins in the African green monkey

The cholesteryl ester content of plasma low density lipoproteins (LDL) in monkeys has previously been shown to be related to the rate of hepatic cholesterol secretion and cholesteryl ester content of newly secreted lipoproteins in the isolated perfused liver. In the present studies, African green monkeys were fed diets containing cholesterol and 40% of calories as either butter or safflower oil in order to determine the effects of saturated versus polyunsaturated dietary fat on hepatic lipoprotein secretion. The rate of cholesterol accumulation in liver perfusates was correlated with the size of the donor's plasma LDL, but for any rate, a smaller plasma LDL was found in donor animals of the safflower oil group than in those of the butter group. Hepatic very low density lipoproteins (VLDL) were smaller in the safflower oil group but contained more cholesteryl ester and fewer triglyceride molecules per particle than those from the butter group. Livers from the safflower oil group contained more cholesteryl ester and less triglyceride than those from the butter group. The cholesteryl ester percentage composition of hepatic VLDL resembled that of the liver in each group. The data show that dietary polyunsaturated fat decreased plasma LDL size even though it increased the cholesteryl ester content of lipoproteins secreted by the liver. Therefore, intravascular formation of plasma LDL from hepatic precursor lipoproteins appears to include the removal of relatively greater amounts of cholesteryl esters from the precursor lipoproteins in polyunsaturated fat-fed animals.     

A green fluorescent protein-engineered haploid inducer line facilitates haploid mutant screens and doubled haploid breeding in maize

Northern corn leaf blight (NCLB) is a prevalent foliar disease in maize. Deployment of resistant cultivars is an effective way to control NCLB. In this study, 207 recombinant inbred lines derived from a K22 × By815 cross were planted in Yangling, China, in 2012 and 2013. NCLB score and lesion size were investigated after artificial inoculation. Significant phenotypic variation in NCLB resistance was observed in both years. Using a genetic map containing high-density single-nucleotide polymorphisms with average genetic distance of 0.74 cM, quantitative trait loci (QTL) for NCLB score and lesion size were analyzed. For NCLB score, four and three QTL were identified in 2012 and 2013, respectively. Two stable QTL were identified in both years. Of these, qNCLB5.04, located on chromosome 5 (bin 5.04), had the largest resistance effect, accounting for 19 and 20 % of the phenotypic variation in 2012 and 2013, respectively. For lesion size, six QTL were identified. Of these, one consensus QTL was associated with both lesion length and width, and the other five were associated only with lesion width. Among all QTL identified, only qNCLB5.04 was associated with both NCLB score and lesion size. Thus, our mapping results suggest that qNCLB5.04 could be a desirable target for marker-assisted selection for NCLB resistance in maize breeding programs.

     

Effect of aliphatic alcohols on growth and degree of saturation of membrane lipids in Acinetobacter calcoaceticus

The adaptive responses of the bacterium Acinetobacter calcoaceticus to different aliphatic alcohols on the level of the membrane fatty acids were studied in detail. The toxicity of the aliphatic alcohols increased with an increasing hydrophobicity. As alcohols are known to increase the fluidity of the membrane they consequently should cause the same adaptive effect on membrane level. Yet, cells of A. calcoaceticus react completely different to the alcohols: in the presence of long-chained alcohols they increase their degree of saturation, while in the presence of short-chained alcohols they decrease the degree of saturation. So, there are no observable differences in the adaptive responses of bacteria with the so-called anaerobic pathway, like Escherichia coli and Pseudomonas putida, and the bacterium carrying the so-called aerobic pathway like A. calcoaceticus. These results strongly indicate a physico-chemical difference in the membrane effect of both the partitioning and localisation of the different alcohols into the membrane and the membrane adaptive responses of the bacteria to these effects.     

Estimating regression parameters and degree of dependence for multivariate failure time data

Multivariate failure time data are frequently encountered in longitudinal studies when subjects may experience several events or when there is a grouping of individuals into a cluster. To take into account the dependence of the failure times within the unit (the individual or the cluster) as well as censoring, two multivariate generalizations of the Cox proportional hazards model are commonly used. The marginal hazard model is used when the purpose is to estimate mean regression parameters, while the frailty model is retained when the purpose is to assess the degree of dependence within the unit. We propose a new approach based on the combination of the two aforementioned models to estimate both these quantities. This two-step estimation procedure is quicker and more simple to implement than the EM algorithm used in frailty models estimation. Simulation results are provided to illustrate robustness, consistency, and large-sample properties of estimators. Finally, this method is exemplified on a diabetic retinopathy study in order to assess the effect of photocoagulation in delaying the onset of blindness as well as the dependence between the two eyes blindness times of a patient.     

The development of photosynthesis in a greening mutant of chlorella and an analysis of the light saturation curve

Photosynthetic oxygen evolution considerably precedes the rise in chlorophyll during the greening of a yellow mutant of Chlorella vulgaris. Dark-grown cells required 20 times more light to saturate photosynthesis than light-grown or normal cells. The chlorophyll appears to add first to active reaction centers, then to fill in a more general antenna. The carotenoid pigments seem to add more randomly to the reaction centers. The shape of the light saturation curves can be explained with the assumption that an excitation in the antenna can reach several reaction centers. The efficiency of the total unit is constant during the greening process.