A Novel Method for Generating Nested Deletions Using the in Vitro Bacteriophage T3 DNA Packaging System

To sequence a DNA segment inserted into a cosmid vector underthe directed sequencing strategy, we established a simple andrapid method for generating nested deletions which uses thein vitro packaging system of bacteriophage T3 DNA. The principleis based on the previous finding that this system can translocateany linear double-stranded DNA up to 40 kb into the phage capsidin a time-dependent manner and the encapsulated DNA becomesDNase-resistant. For this purpose, we constructed a cosmid vectorthat carries two different antibiotic selection markers at bothsides of the multiple cloning site, and after insertion of aDNA segment, the clone was linearized by -terminase at the cossite. After the packaging reaction in vitro followed by DNasetreatment, the encapsulated DNA was introduced into Escherichiacoli cells to give clones with unidirectional deletions by differentialantibiotic selection. Restriction and sequence analyses of deletionclones demonstrated that an ordered set of clones with nesteddeletions, ranging from less than 1 kb to 25 kb, was createdfrom either the end of the DNA segment. Thus, nested deletionclones that cover the entire region of a 40-kb cosmid insertcan be obtained by a single packaging reaction, and its restrictionmap can be simultaneously obtained.     

Specific and sensitive detection of Alcaligenes species from an agricultural environment

A quantitative real‐time PCR assay to specifically detect and quantify the genus Alcaligenes in samples from the agricultural environment, such as vegetables and farming soils, was developed. The minimum detection sensitivity was 106 fg of pure culture DNA, corresponding to DNA extracted from two cells of Alcaligenes faecalis. To evaluate the detection limit of A. faecalis, serially diluted genomic DNA from this organism was mixed with DNA extracted from soil and vegetables and then a standard curve was constructed. It was found that Alcaligenes species are present in the plant phytosphere at levels 10²–10⁴ times lower than those in soil. The approach presented here will be useful for tracking or quantifying species of the genus Alcaligenes in the agricultural environment.     

Amyloplast displacement is necessary for gravisensing in Arabidopsis shoots as revealed by a centrifuge microscope

The starch‐statolith hypothesis proposes that starch‐filled amyloplasts act as statoliths in plant gravisensing, moving in response to the gravity vector and signaling its direction. However, recent studies suggest that amyloplasts show continuous, complex movements in Arabidopsis shoots, contradicting the idea of a so‐called ‘static’ or ‘settled’ statolith. Here, we show that amyloplast movement underlies shoot gravisensing by using a custom‐designed centrifuge microscope in combination with analysis of gravitropic mutants. The centrifuge microscope revealed that sedimentary movements of amyloplasts under hypergravity conditions are linearly correlated with gravitropic curvature in wild‐type stems. We next analyzed the hypergravity response in the shoot gravitropism 2 (sgr2) mutant, which exhibits neither a shoot gravitropic response nor amyloplast sedimentation at 1  g . sgr2 mutants were able to sense and respond to gravity under 30  g conditions, during which the amyloplasts sedimented. These findings are consistent with amyloplast redistribution resulting from gravity‐driven movements triggering shoot gravisensing. To further support this idea, we examined two additional gravitropic mutants, phosphoglucomutase (pgm) and sgr9, which show abnormal amyloplast distribution and reduced gravitropism at 1  g . We found that the correlation between hypergravity‐induced amyloplast sedimentation and gravitropic curvature of these mutants was identical to that of wild‐type plants. These observations suggest that Arabidopsis shoots have a gravisensing mechanism that linearly converts the number of amyloplasts that settle to the ‘bottom’ of the cell into gravitropic signals. Further, the restoration of the gravitropic response by hypergravity in the gravitropic mutants that we tested indicates that these lines probably have a functional gravisensing mechanism that is not triggered at 1  g .     

The relationship between consumption of tyrosine and phenylalanine as precursors of catecholamine at breakfast and the circadian typology and mental health in Japanese infants aged 2 to 5 years

Background

This study aims to examine the relationship between tyrosine and phenylalanine intake at breakfast as precursors of dopamine, and scores on the Torsvall-Åkerstedt Diurnal Type Scale and of mental health in Japanese infants aged 2 to 5 years.

Results

An integrated questionnaire was administered to parents of 1,367 infants attending one of ten nursery schools governed by Kochi City or a kindergarten affiliated with the Faculty of Education at Kochi University (775 answers for analysis: 56.7%) in May and June 2008. Questionnaires included the Torsvall-Åkerstedt Diurnal Type Scale and questions on sleep habits (onset, offset, quality, quantity, and so on), meal habits (content and regularity of timing), and mental health (depressive states). Amount of tyrosine and phenylalanine intake was calculated based on a breakfast content questionnaire and data on the components of amino acids in foods. Infants who ingested more than 800 mg of tyrosine or phenylalanine at breakfast per meal were more morning-type than those who ingested less than 800 mg (ANOVA: P= 0.005). However, this relationship disappeared in the ANCOVA analysis (with the covariance of tryptophan intake, P= 0.894). Infants who ingested more than 800 mg of the two amino acids at breakfast showed significantly higher mental health scores (lower frequency of depressive states) than those who ingested less than 800 mg (ANOVA: P = 0.004). This relationship remained significant when ANCOVA analysis was performed with the covariance of tryptophan (ANCOVA: P= 0.017).

Conclusions

These results suggest that tyrosine and phenylalanine ingested at breakfast are not related with circadian phase, but are relate with mental health in infants.     

Selection responses of means and inbreeding depression for female fecundity in Drosophila melanogaster suggest contributions from intermediate-frequency alleles to quantitative trait variation

The extent to which quantitative trait variability is caused by rare alleles maintained by mutation, versus intermediate-frequency alleles maintained by balancing selection, is an unsolved problem of evolutionary genetics. We describe the results of an experiment to examine the effects of selection on the mean and extent of inbreeding depression for early female fecundity in Drosophila melanogaster. Theory predicts that rare, partially recessive deleterious alleles should cause a much larger change in the effect of inbreeding than in the mean of the outbred population, with the change in inbreeding effect having an opposite sign to the change in mean. The present experiment fails to support this prediction, suggesting that intermediate-frequency alleles contribute substantially to genetic variation in early fecundity.     

Irreversible cross-linking of heme to the distal tryptophan of stromal ascorbate peroxidase in response to rapid inactivation by H2O2

Ascorbate peroxidase (APX) isoforms localized in the stroma and thylakoid membrane of chloroplasts play a central role in scavenging reactive oxygen species generated by photosystems. These enzymes are inactivated within minutes by H2O2 when the reducing substrate, ascorbate, is depleted. We found that, when the enzyme is inactivated by H2O2, a heme at the catalytic site of a stromal APX isoform is irreversibly cross-linked to a tryptophan residue facing the distal cavity. Mutation of this tryptophan to phenylalanine abolished the cross-linking and increased the half-time for inactivation from <10 to 62 s. In contrast with H2O2-tolerant peroxidases, rapid formation of the cross-link in APXs suggests that a radical in the reaction intermediate tends to be located in the distal tryptophan so that heme is easily cross-linked to it. This is the first report of a mutation that improves the tolerance of chloroplast APXs to H2O2.     

Genetic variation and correlations among responses to five insecticides within natural populations of Drosophila melanogaster (Diptera: Drosophilidae)

To investigate the genetic basis of cross-resistance to insecticides, natural populations of Drosophila melanogaster (Meigen) were first collected from four different locations in Japan. After 10-80 isofemale lines of each population had been established in a laboratory, the susceptibility of each line to each of the insecticides permethrin, malathion, fenitrothion, prothiophos, and DDT was examined. Broad ranges of continuous variation in susceptibility to all the chemicals were observed within each natural population as a whole. In addition, highly significant correlations among responses to organophosphates were observed. However, based on the coefficients of determination, about less than half of variation in responses to one insecticide could be explained by variation in responses to another insecticide, suggesting that not only a common resistance factor but also other factors could be involved in a natural population. Genetic analyses by using resistant and susceptible inbred lines from the same natural population demonstrated that resistance to organophosphates in some resistant lines could be due to a single or tightly linked factors, and that resistance in the other line may be due to more than one major factor. These observations could suggest that several resistance factors may be involved within each natural population, and that some of major factors could contribute to correlations among responses to organophosphates. These major factors could then contribute to the broad ranges of continuous variation observed at the level of the populations.     

Gravity sensing and signaling

Gravitropism has attracted much attention from plant biologists. Recent studies have provided molecular evidence supporting two long-surviving hypotheses about the mechanism of gravitropism: the starch-statolith hypothesis and the Cholodney-Went hypothesis. Amyloplast movement along the gravity vector within gravity-sensing cells in the root and shoot is the most likely trigger of subsequent intracellular signaling. Several possible events leading from this signaling to differential auxin distribution within the sensing cells have been suggested recently.     

Age-specific mortality rates of reproducing and non-reproducing males of Drosophila melanogaster

The proximate and evolutionary causes of the levelling of mortality rates at late ages, observed in several species, remain obscure. To investigate the causes of mortality levelling late in life in Drosophila melanogaster, we examined the effect of reproduction on mortality patterns, by conducting population cage experiments with a total of more than 45,000 individuals. Several different genotypes of reproducing and non-reproducing males from F(1) crosses among isogenic lines were studied. Our results suggest that significant mortality levelling can occur even in non-reproducing males, but that reproduction also significantly affects mortality patterns. The results show that mortality levelling is strongly affected by the Gompertz initial mortality rate and exponential rate of increase parameters, probably through the effects of heterogeneity in mortality risks.     

Negative correlations between resistance to three organophosphate insecticides and productivity within a natural population of Drosophila melanogaster (Diptera: Drosophilidae)

To investigate the relationship between resistance to organophosphate insecticides and fitness components, we first measured resistance to three organophosphates, malathion, prothiophos, and fenitrothion, and productivity, a measure of fitness components, for each of the isofemale lines from the same natural population of Drosophila melanogaster (Meigen). Pearson correlation coefficients indicated that positive correlations among resistance to the organophosphates and negative correlations between resistance to each of the organophosphates and the productivity existed within the natural population. We further investigated the genetic basis of the correlations among resistance to the organophosphates and the productivity, by using chromosome-substituted lines between a resistant and a susceptible inbred line established from the same natural population. Chromosomal analyses indicated that the third chromosome from the resistant line exhibited not only significant, positive effects on resistance to all of the organophosphates tested but also a significant negative effect on the productivity, suggesting positive genetic correlations between resistance to each organophosphate and negative genetic correlations between resistance to each organophosphate and the productivity. In addition, a significant negative effect on the productivity was also detected from the second chromosome, which did not exhibit significant major effects on resistance to the organophosphates. This suggests that fitness components of resistant lines could be also affected by factors independent of insecticide resistance. The dynamics of genetic variation in resistance to the organophosphates within the natural population of D. melanogaster are discussed from the standpoint of negative genetic correlations between resistance to the organophosphates and the productivity.