The evolution of age-specific mortality rates in Drosophila melanogaster: genetic divergence among unselected lines.

Age-specific effects of spontaneous mutations on mortality rates in Drosophila are inferred from three large demographic experiments. Data were collected from inbred lines that were allowed to accumulate spontaneous mutations for 10, 19, and 47 generations. Estimates of age-specific mutational variance for mortality were based on data from all three experiments, totalling approximately 225,000 flies, using a model developed for genetic analysis of age-dependent traits (the character process model). Both within- and among-generation analyses suggest that the input of genetic variance is greater for early life mortality rates than for mortality at older ages. In females, age-specific mutational variances ranged over an order of magnitude from 5.96 x 10(-3) at 2 wk posteclosion to 0.02 x 10(-3) at 7 wk. The male data show a similar pattern. Age-specific genetic variances were substantially less at generation 47 than at generation 19-an unexplained observation that is likely due to block effects. Mutational correlations among mortality rates at different ages tend to increase with the accumulation of new mutations. Comparison of the mutation-accumulation lines at generations 19 and 47 with their respective control lines suggests little age-specific mutational bias.     

Argentine ant (Hymenoptera: Formicidae) trail pheromone enhances consumption of liquid sucrose solution

We investigated whether the Argentine ant, Linepithema humile (Mayr), trail pheromone, Z9-16:Ald, could enhance recruitment to and consumption of liquid sucrose solutions. All tests were done as paired comparisons with a 10% sucrose solution as food. In the laboratory, mixing 20 microl of a 10-microg/ml solution of the pheromone with 50 microl of the 10% sucrose solution increased the number of ants feeding by >150%. In a field test, we combined the trail pheromone with a 10% sucrose solution in 50-ml vials. These vials were covered with a plastic membrane that has 1.5-mm-diameter holes punched uniformly across its surface. Ants could drink from the holes after the vials were inverted. For half of the vials, 1 microg of the pheromone was put onto the plastic membrane before the vials were filled with a 10% sucrose solution. The remaining vials had no pheromone on the plastic membrane. After 4 h we measured the consumption in each vial. Bait consumption with the pheromone was enhanced by 29%. In a 2nd series of tests, vials were left outside for 24 h. The consumption rate was 33% higher with the pheromone compared with the controls that didn't have pheromone.     

Transformation of the gene for hypoxanthine phosphoribosyltransferase.

Purified DNA from wild-type Chinese ovary (CHO) cells has been used to transform three hypoxanthine phosphoribosyltransferase (HPRT) deficient murine cell mutants to the enzyme positive state. Transformants appeared at an overall frequency of 5 x 10(-8) colonies/treated cell and expressed CHO HPRT activity as determined by electrophoresis. One gene recipient, B21, was a newly isolated mutant of LMTK- deficient in both HPRT and thymidine kinase (TK) activities. Transformation of B21 to HPRT+ occurred at 1/5 the frequency of transformation to TK+; the latter was, in turn, an order of magnitude lower than that found in the parental LMTK- cells, 3 x 10(-6). Thus both clonal and marker-specific factors play a role in determining transformability. The specific activity of HPRT in transformant extracts ranged from 0.5 to 5 times the CHO level. The rate of loss of the transformant HPRT+ phenotype, as measured by fluctuation analysis, was 10(-4)/cell/generation. While this value indicates stability compared to many gene transferents, it is much greater than the spontaneous mutation rate at the indigenous locus. The ability to transfer the gene for HPRT into cultured mammalian cells may prove useful for mutational and genetic mapping studies in this well-studied system.     

Labeling of platelet surface proteins with 125I by the iodogen method

A procedure for the 125I-iodination of platelet suspensions is described. The procedure utilizes Iodogen, a solid-phase oxidizing agent similar to chloramine-T. Platelets were labeled under a variety of conditions, including in the presence of 0.1% albumin, and showed between 7 and 28% incorporation of 125I. Best labeling results were obtained at low platelet concentrations (3-5 x 10(8) platelets/ml), short reaction times (15 min), and with 2-ml glass vials coated with 100 micrograms of Iodogen. Analysis of the labeled platelet proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed that the same major protein bands were labeled by this procedure as were labeled by the lactoperoxidase procedure. At low platelet concentrations, the Iodogen procedure gives twice the amount of iodine incorporation.     

Analysis of mutational spectra by denaturing capillary electrophoresis

The point mutational spectrum over nearly any 75- to 250-bp DNA sequence isolated from cells, tissues or large populations may be discovered using denaturing capillary electrophoresis (DCE). A modification of the standard DCE method that uses cycling temperature (e.g., +/-5 degrees C), CyDCE, permits optimal resolution of mutant sequences using computer-defined target sequences without preliminary optimization experiments. The protocol consists of three steps: computer design of target sequence including polymerase chain reaction (PCR) primers, high-fidelity DNA amplification by PCR and mutant sequence separation by CyDCE and takes about 6 h. DCE and CyDCE have been used to define quantitative point mutational spectra relating to errors of DNA polymerases, human cells in development and carcinogenesis, common gene-disease associations and microbial populations. Detection limits are about 5 x 10(-3) (mutants copies/total copies) but can be as low as 10(-6) (mutants copies/total copies) when DCE is used in combination with fraction collection for mutant enrichment. No other technological approach for unknown mutant detection and enumeration offers the sensitivity, generality and efficiency of the approach described herein.     

Escherichia coli DNA polymerase IV contributes to spontaneous mutagenesis at coding sequences but not microsatellite alleles

Slipped strand mispairing during DNA synthesis is one proposed mechanism for microsatellite or short tandem repeat (STR) mutation. However, the DNA polymerase(s) responsible for STR mutagenesis have not been determined. In this study, we investigated the effect of the Escherichia colidinB gene product (Pol IV) on mononucleotide and dinucleotide repeat stability, using an HSV-tk gene episomal reporter system for microsatellite mutations. For the control vector (HSV-tk gene only) we observed a statistically significant 3.5-fold lower median mutation frequency in dinB(-) than dinB(+) cells (p<0.001, Wilcoxon Mann Whitney Test). For vectors containing an in-frame mononucleotide allele ([G/C](10)) or either of two dinucleotide alleles ([GT/CA](10) and [TC/AG](11)) we observed no statistically significant difference in the overall HSV-tk mutation frequency observed between dinB(+) and dinB(-) strains. To determine if a mutational bias exists for mutations made by Pol IV, mutational spectra were generated for each STR vector and strain. No statistically significant differences between strains were observed for either the proportion of mutational events at the STR or STR specificity among the three vectors. However, the specificity of mutational events at the STR alleles in each strain varied in a statistically significant manner as a consequence of microsatellite sequence. Our results indicate that while Pol IV contributes to spontaneous mutations within the HSV-tk coding sequence, Pol IV does not play a significant role in spontaneous mutagenesis at [G/C](10), [GT/CA](10), or [TC/AG](11) microsatellite alleles. Our data demonstrate that in a wild type genetic background, the major factor influencing microsatellite mutagenesis is the allelic sequence composition.     

A field-compatible method for measuring alternative respiratory pathway activities in vivo using stable O₂ isotopes

Plants can alter rates of electron transport through the alternative oxidase (AOX) pathway in response to environmental cues, thus modulating respiratory efficiency, but the (18)O discrimination method necessary for measuring electron partitioning in vivo has been restricted to laboratory settings. To overcome this limitation, we developed a field-compatible analytical method. Series of plant tissue subsamples were incubated in 12 mL septum-capped vials for 0.5-4 h before aliquots of incubation air were injected into 3.7 mL evacuated storage vials. Vials were stored for up to 10 months before analysis by mass spectrometry. Measurements were corrected for unavoidable contamination. Additional mathematical tools were developed for detecting and addressing non-linearity (whether intrinsic or due to contamination) in the data used to estimate discrimination values. Initial contamination in the storage vials was 0.03 ± 0.01 atm; storing the gas samples at -17 °C eliminated further contamination effects over 10 months. Discrimination values obtained using our offline incubation and computation method replicated previously reported results over a range of 10-31‰, with precision generally better than ±0.5‰. Our method enables large-scale investigations of plant alternative respiration along natural environmental gradients under field conditions.     

Prediction of growth of Bacillus megaterium spores as affected by gamma radiation dose and spore load

AIMS: To provide data on the interaction of radiation dose (x1) and microbial contamination load (x2), as predictor variables, on the percentage of vials exhibiting growth of Bacillus megaterium spores (y). METHODS AND RESULTS: The influence of a wide range of spore loads (1-50 000 spores of B. megaterium vial-1) and gamma radiation doses (0.2-10 kGy) on the contaminated samples was determined. Each contamination load was studied by adding the specified number of spores to 100 vials containing nutrient broth and exposing them to various doses of gamma radiation. Curves representing the number of contaminated vials against the dose of radiation were sigmoidal in shape and the data showed an indirect relationship. Data were analysed by regression analysis which revealed a significant correlation (R2=0.85). The relationship among the tested variables is exponential and can be described by the following equation: y = 1 - (1 - e(0.0173x(1)))(x(2)) It was also estimated that, for each increase of 1 in the number of spores per vial, there is an increase of 1 in the number of contaminated vials. CONCLUSION: The two variables (x1 and x2) have great influence on the radiation sterilization efficiency and the proposed mathematical model is valid for the prediction of this efficiency. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present investigation can be of useful industrial application and can help to set acceptance and rejection limits for the production of materials vulnerable to microbial contamination.     

Selection in Finite Populations with Multiple Alleles. III. Genetic Divergence with Centripetal Selection and Mutation

Natural selection for an intermediate level of gene or enzyme activity has been shown to lead to a high frequency of heterotic polymorphisms in populations subject to mutation and random genetic drift. The model assumes a symmetrical spectrum of mutational variation, with the majority of variants having only minor effects on the probability of survival. Each mutational event produces a variant which is novel to the population. Allelic effects are assumed to be additive on the scale of enzyme activity, heterosis arising whenever a heterozygote has a mean level of activity closer to optimal than that of other genotypes in the population.-A new measure of genetic divergence between populations is proposed, which is readily interpreted genetically, and increases approximately linearly with time under centripetal selection, drift and mutation. The parameter is closely related to the rate of accumulation of mutational changes in a cistron over an evolutionary time span.-A survey of published data concerning polymorphic loci in man and Drosophila suggests than an alternative model, based on the superiority of hybrid molecules, is not of general importance. Thirteen loci giving rise to hybrid zones on electrophoresis have a mean heterozygote frequency of 0.22 +/-.06, compared with a value of 0.23 +/-.04 for 16 loci classified as producing no hybrid enzyme.     

Diversity of phage types among archived cultures of the Demerec collection of Salmonella enterica serovar Typhimurium strains

The existence of several thousand Salmonella enterica serovar Typhimurium LT2 and LT7 cultures originally collected by M. Demerec and sealed in agar stab vials for 33 to 46 years is a resource for evolutionary and mutational studies. Cultures from 74 of these vials, descendants of cells sealed and stored in nutrient agar stabs several decades ago, were phage typed by the Callow and Felix, Lilleengen, and Anderson systems. Among 53 LT2 archived strains, 16 had the same phage type as the nonarchival sequenced LT2 strain. The other 37 archived cultures differed in phage typing pattern from the sequenced strain. These 37 strains were divided into 10 different phage types. Among the 19 LT7 strains, only one was similar to the parent by phage typing, while 18 were different. These 18 strains fell into eight different phage types. The typing systems were developed to track epidemics from source to consumer, as well as geographic spread. The value of phage typing is dependent upon the stability of the phage type of any given strain throughout the course of the investigation. Thus, the variation over time observed in these archived cultures is particularly surprising. Possible mechanisms for such striking diversity may include loss of prophages, prophage mosaics as a result of recombination events, changes in phage receptor sites on the bacterial cell surface, or mutations in restriction-modification systems.     

Diversity of Phage Types among Archived Cultures of the Demerec Collection of Salmonella enterica serovar Typhimurium Strains

The existence of several thousand Salmonella enterica serovar Typhimurium LT2 and LT7 cultures originally collected by M. Demerec and sealed in agar stab vials for 33 to 46 years is a resource for evolutionary and mutational studies. Cultures from 74 of these vials, descendants of cells sealed and stored in nutrient agar stabs several decades ago, were phage typed by the Callow and Felix, Lilleengen, and Anderson systems. Among 53 LT2 archived strains, 16 had the same phage type as the nonarchival sequenced LT2 strain. The other 37 archived cultures differed in phage typing pattern from the sequenced strain. These 37 strains were divided into 10 different phage types. Among the 19 LT7 strains, only one was similar to the parent by phage typing, while 18 were different. These 18 strains fell into eight different phage types. The typing systems were developed to track epidemics from source to consumer, as well as geographic spread. The value of phage typing is dependent upon the stability of the phage type of any given strain throughout the course of the investigation. Thus, the variation over time observed in these archived cultures is particularly surprising. Possible mechanisms for such striking diversity may include loss of prophages, prophage mosaics as a result of recombination events, changes in phage receptor sites on the bacterial cell surface, or mutations in restriction-modification systems.     

Polygenic Mutation in Drosophila Melanogaster: Estimates from Response to Selection of Inbred Strains

Replicated divergent artificial selection for abdominal and sternopleural bristle number from a highly inbred strain of Drosophila melanogaster resulted in an average divergence after 125 generations of selection of 12.0 abdominal and 8.2 sternopleural bristles from the accumulation of new mutations affecting bristle number. Responses to selection were highly asymmetrical, with greater responses for low abdominal and high sternopleural bristle numbers. Estimates of V(M), the mutational variance arising per generation, based on the infinitesimal model and averaged over the responses to the first 25 generations of selection, were 4.32 X 10(-3) V(E) for abdominal bristle number and 3.66 X 10(-3) V(E) for sternopleural bristle number, where V(E) is the environmental variance. Based on 10 generations of divergent selection within lines from generation 93, V(M) for abdominal bristle number was 6.75 X 10(-3) V(E) and for sternopleural bristle number was 5.31 X 10(-3) V(E). However, estimates of V(M) using the entire 125 generations of response to selection were lower and generally did not fit the infinitesimal model largely because the observed decelerating responses were not compatible with the predicted increasing genetic variance over time. These decelerating responses, periods of response in the opposite direction to artificial selection, and rapid responses to reverse selection all suggest new mutations affecting bristle number on average have deleterious effects on fitness. Commonly observed periods of accelerated responses followed by long periods of stasis suggest a leptokurtic distribution of mutational effects for bristles.     

Quantitative genetics of ovariole number in Drosophila melanogaster. II. Mutational variation and genotype-environment interaction.

The rare alleles model of mutation-selection balance (MSB) hypothesis for the maintenance of genetic variation was evaluated for two quantitative traits, ovariole number and body size. Mutational variances (VM) for these traits, estimated from mutation accumulation lines, were 4.75 and 1.97 x 10(-4) times the environmental variance (VE), respectively. The mutation accumulation lines were studied in three environments to test for genotype x environment interaction (GEI) of new mutations; significant mutational GEI was found for both traits. Mutations for ovariole number have a quadratic relationship with competitive fitness, suggesting stabilizing selection for the trait; there is no significant correlation between mutations for body size and competitive fitness. Under MSB, the ratio of segregating genetic variance, VG, to mutational variance, VM, estimates the inverse of the selection coefficient against a heterozygote for a new mutation. Estimates of VG/VM for ovariole number and body size were both approximately 1.1 x 10(4). Thus, MSB can explain the level of variation, if mutations affecting these traits are under very weak selection, which is inconsistent with the empirical observation of stabilizing selection, or if the estimate of VM is biased downward by two orders of magnitude. GEI is a possible alternative explanation.     

rpoS mutants in archival cultures of Salmonella enterica serovar typhimurium

Long-term survival under limited growth conditions presents bacterial populations with unique environmental challenges. The existence of Salmonella enterica serovar Typhimurium cultures undisturbed in sealed nutrient agar stab vials for 34 to 45 years offered a unique opportunity to examine genetic variability under natural conditions. We have initiated a study of genetic changes in these archival cultures. We chose to start with examination of the rpoS gene since, among gram-negative bacteria, many genes needed for survival are regulated by RpoS, the stationary-phase sigma factor. In each of 27 vials examined, cells had the rpoS start codon UUG instead of the expected AUG of Salmonella and Escherichia coli strains recorded in GenBank. Ten of the 27 had additional mutations in the rpoS gene compared with the X77752 wild-type strain currently recorded in GenBank. The rpoS mutations in the 10 strains included two deletions as well as point mutations that altered amino acid sequences substantially. Since these stored strains were derived from ancestral cells inoculated decades ago and remained undisturbed, it is assumed that the 10 rpoS mutations occurred during storage. Since the remaining 17 sequences were wild type (other than in the start codon), it is obvious that rpoS remained relatively stable during decades of sealed storage.     

利用反硝化细菌法测试水体硝酸盐氮氧同位素

反硝化细菌方法作为测试硝酸盐氮氧同位素组成的最新方法,具有可测试低浓度水样、对样品无需特殊处理、不会交叉污染和需样量少等诸多优点而得到迅速发展。本研究率先在国内实验室利用反硝化细菌法成功测试了硝酸盐氮氧同位素组成,将经过5～10d培养的反硝化细菌Pseudomonas aureofaciens离心,然后将菌液浓缩5倍,再向顶空进样瓶注入3mL菌液,密封后利用高纯氮气吹扫3h以上,注入50nmolNO3-水样经过夜培养灭活后,使用TraceGasPre-concentrator-Isoprime测试N2O同位素组成,结果表明,重现性和测试精度与国际上类似研究接近。该方法的建立对于国内开展河流及湖泊(水库)、降水等氮的生物地球化学循环将起到促进作用。     

Comparison of the Ames test and a newly developed assay for detection of mutagenic pollution of marine environments

Mismatch repair (MMR) genes, such as Msh2, are classified as "mutator" genes, responsible for the microsatellite instability identified in many tumors. In the current study, the mutation frequency and mutational spectrum in thymic lymphoma arising in Msh2 deficient mice are investigated. Thymic lymphoma developed in Msh2-/- background displayed an eight to nine-fold increase in mutation frequency compared to the normal thymi in Msh2 deficient animals. Sequencing demonstrated significantly different mutational spectra between normal thymus tissue and thymic lymphomas in Msh2-/- mice (P=0.02). The tumor mutational spectrum is characterized by an increase in base substitutions occurring at A:T sites, and multiple mutations, as well as a minor increase in -1 frameshifts. We analyzed mutations in different parts of the tumors, and different regional hotspots could be identified. Several hotspot mutations that are a rare event in normal tissues were identified in the tumor tissues. We conclude that thymic lymphomas arising in Msh2 deficient genetic background are hypermutable and the altered mutational spectrum might be an indication of infidelity of DNA replication during tumorigenesis.     

An economical system for liquid scintillation counting

Small glass shell vials (12 × 35 mm minivials), containing 2.0 ml of a dioxane-based scintillation solution plus a ¹⁴C-labeled sample, were placed in a conventional glass, 20-ml count vial and assayed in a scintillation spectrometer. Statistical comparison of counts recorded from ¹⁴C samples prepared both in the minivial system and conventional 20-ml count vials indicated that the two systems were equivalent with sample volumes of 10 and 100 μliters (1600-cpm solution) and 10 μliters (60-cpm solution). Conventional 20-ml glass or plastic count vials were both acceptable as containers for the minivials.There were no significant differences in the counts from samples in minivials placed on-center and off-center in the container vial. Cost per sample was reduced from 24.8 cents (conventional glass vials) to 4.7 cents (minivial system).     

Protection of histones isolated from elastase-treated mouse epidermis

Small glass shell vials (12 × 35 mm minivials), containing 2.0 ml of a dioxane-based scintillation solution plus a ¹⁴C-labeled sample, were placed in a conventional glass, 20-ml count vial and assayed in a scintillation spectrometer. Statistical comparison of counts recorded from ¹⁴C samples prepared both in the minivial system and conventional 20-ml count vials indicated that the two systems were equivalent with sample volumes of 10 and 100 μliters (1600-cpm solution) and 10 μliters (60-cpm solution). Conventional 20-ml glass or plastic count vials were both acceptable as containers for the minivials.There were no significant differences in the counts from samples in minivials placed on-center and off-center in the container vial. Cost per sample was reduced from 24.8 cents (conventional glass vials) to 4.7 cents (minivial system).     

High-efficiency DNA ligation for clamp attachment without polymerase chain reaction

We coupled ligation with mass action to achieve high-efficiency clamp attachment without polymerase chain reaction (PCR). Using a 10-fold molar excess of a GC-rich clamp of synthesized and hybridized oligonucleotides, we achieved the maximum clamp-ligation efficiency in which the clamp was ligated to >95% of 10(10)-10(12) restriction ends of a PCR-amplified fragment. The maximum efficiency was confirmed by ligating the clamp to 10(11)-10(12) restriction ends of human genomic DNA. Our approach can be added to a constant denaturant capillary electrophoresis (CDCE)-based method of analyzing rare point mutants at fractions as low as 10(-6); such mutants appear as small copy numbers in the initial samples. This CDCE-based method alone is applicable to only those DNA sequences juxtaposed with an internally occurring clamp of a higher melting temperature in genomic DNA. Since such sequences represent 9% of the human genome, the addition of clamp ligation significantly increases the scanning range for the human genome without reducing the initial mutant copy numbers. Furthermore, clamp ligation/attachment without PCR prevents PCR-created mutants from interfering with rare mutational analysis. In addition to those applications seeking high-efficiency DNA ligation, our approach can be generally applied to ligation of restriction ends.