Wheat plant DNA methylation pattern changes at chronic seed γ irradiation

Alterations of DNA methylation patterns of two wheat sorts Al’batros odesskii and Donetskaya-48 whose seeds were irradiated with a low dose rate (3 × 10^?7 Gy/s) for 4 months have been studied. Six restriction endonucleases were used in the experiments. Primary distinction in DNA methylation patterns of the studied sorts has been demonstrated. Chronic irradiation resulted in an increase of the methylation level on the recognion site for GluI and Sou3AI and in a decrease of this index in recognion sites for endonucleases GlaI and HpaII. The alterations of the methylation level in recognition sites for restrictases MboI and MspI were not found. The considerable increase of chromosome aberration level at the same dose of chronic irradiation has been shown. The role of DNA methylation pattern changes in development of irradiation damage and organism protective reactions is discussed.     

Survival of Drosophila melanogaster larvae on defined medium supplemented with naturally occurring nucleosides and nucleic acid bases.

Survival of Drosophila melanogaster larvae grown on defined medium supplemented with nucleic acid bases, ribonucleosides, and deoxyribonucleosides has been measured at doses from 10^?4M to 3.16 × 10^?2M. Purine-related compounds generally are more toxic than pyrimidine-related compounds.     

Dynamics of hybrid dysgenesis frequency in Drosophila melanogaster in controlled terms of protracted radiation exposure

The dynamics of hybrid dysgenesis frequency in F ₁ descendants of two lines of Drosophila melanogaster (lines Santon-s and radius incompletus (ri) brining P-mobile element), which was maintained for 20 generations were under the conditions of chronic radiation with three dose rate (1.2 × 10^?8; 0.3 × 10^?8; 0.12 × 10^?8 Gy/s), have been studied. Gradual changes have been shown in the dose dependences of the hybrid dysgenesis frequency for F ₁ descendants of every 20 generations from parents exposed to radiation. The complicated dynamics of the transitional process of the appearance of gonadal dysgenesis depending on the duration and dose rate of irradiation in ancestors, has been found. The cumulative effect of the prolonged irradiation appears in the form of adaptation at the lowest dose rate of irradiation and at exhaustion at the highest used dose rate. Problem of the transitional process peculiarities, including the hierarchy of protective and adaptive reactions under the conditions of the chronic irradiation has been discussed.     

Transposition of the hobo element in Drosophila melanogaster somatic cells

Somatic mutation and recombination test on wing cells of Drosophila melanogaster showed that the recombination frequency in the somatic tissues of strains studied correlated with the presence of a full-length copy of the hobo transposable element in the genome. Transposition of hobo in somatic tissue cells at a frequency 3.5 × 10^?2 per site per X chromosome was shown by fluorescence in situ hybridization with salivary gland polytene chromosomes of larvae of one of the D. melanogaster strains having a full-length hobo copy.     

Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. I. Dependence upon ecdysone concentration

The response of the three major classes of puff in salivary gland chromosomes of larval Drosophila melanogaster to varying β-ecdysone concentrations has been studied in in vitro cultured glands. Two (25AC and 68C) of the intermolt puffs regress at a rate dependent upon the hormone concentration. Three rapidly reacting puffs (23E, 74EF and 75B) respond in a graded way to β-ecdysone concentrations over a range of at least 600 ×. In contrast, five late-reacting puffs (62E, 78D, 22C, 63E, and 82F) do not respond below 5 × 10^?8M and at 2.5 × 10^?7M react maximally. The 50% response of the early puff sites 74EF and 75B and of the late puff sites occurs at 1 × 10^?7M. Two points are discussed in detail: whether ecdysone is necessary as a sustained stimulus or only as a trigger for the sequential puffing response and an evaluation of the absolute ecdysone concentration necessary for induction.     

Molecular evolution of thepaired gene inDrosophila: Cloning and characterization of the partialpaired gene fromDrosophila willistoni

A partialpaired gene ofDrosophila willistoni containing the paired box and extended homeo box was amplified by PCR and the nucleotide sequence of 1141 bp was determined. Comparison of thepaired genes inD. willistoni andD. melanogaster showed that the proportions of identical nucleotide sites in the coding region and identical amino acid sites were 73.8 and 86.5%, respectively. The amino acid sites in the N-terminal region, the paired box, and the extended homeo box were 88.5, 95.3, and 98.6% identical in the two species. The rates of amino acid substitution for these regions were estimated to be 1.73×10^?9, 0.67×10^?9, and 0.19×10^?9/site/year, respectively. In contrast, the connecting region between the two boxes has been highly diverged and evolved very rapidly, 18.3×10^?9/site/year, suggesting almost no functional constraint in the connecting region.     

Properties and inhibition of thymidylate synthetase in Drosophila melanogaster

A quantitative comparison of the incorporation of methyl-³H-thymidine and 6-³H-deoxyuridine into the DNA of Drosophila melanogaster in the presence and in the absence of 5-fluorouracil indicated that 5-fluorouracil inhibits the reaction converting dUMP to dTMP catalysed by thymidylate synthetase (methylenetetrahydrofolate:dUrd-5′-P C-methyltransferase, E.C. 2.1.1.b). The enzyme exhibits maximal activity at pH 7·5 to 8·0 and is protected from heat inactivation by deoxyuridine monophosphate. The addition of thiol compounds to the homogenization buffer results in the enhancement of synthetase activity. The K_m values for deoxyuridine monophosphate and 5,10-methylenetetrahydrofolate are 6·8 × 10^?6 M and 8·3 × 10^?5 M, respectively. Fluorodeoxyuridine monophosphate, trifluoromethyldeoxyuridine monophosphate, and methotrexate are inhibitors of the enzyme. 5-Bromodeoxyuridine and 5-iododeoxyuridine have no inhibitory effect. The results support the contention that, under conditions which induce morphological lesions in Drosophila, fluorinated pyrimidines and methotrexate inhibit the de novo synthesis of thymidylate whereas thymidine analogues function in some other manner.     

Germ line transposition of the copia retrotransposon in Drosophila melanogaster is restricted to males by tissue-specific control of copia RNA levels

Germ line transposition rates of the retrotransposon copia were directly measured in males and females of an inbred Drosophila melanogaster line, 2b3, which is highly polymorphic for copia insertion sites. The elevated germ line transposition rate of copia in this line (3–8?×?10^?3 per generation per element) is confined to males, with transposition in females being undetectable under the conditions of the experiment but at most 50-fold lower than the rate for males. To determine the molecular basis of this effect, copia RNA levels were measured in whole bodies and germ lines of male and female flies of both the unstable 2b3 line and a stable line, Oregon RC-iso, which shows normal rates of copia transposition. Both male and female 2b3 flies contain much more copia RNA than flies of the stable line. However, 2b3 male germinal tissues contain much higher levels of copia RNA than the equivalent female tissues. The highest copia expression is detected in maturing primary spermatocytes. Our data show that high rates of germ line copia transposition are restricted to males by tissue-specific control of RNA levels and suggest that transposition of copia only occurs in fly tissues containing more than a relatively high threshold level of copia RNA.     

Chromosomal aberrations induced by caffeine in somatic ganglia of Drosophila melanogaster

Nerve ganglia of third-instar larvae were treated with various doses of caffeine (5×10^?4, 10^?3, 5×10^?3, 10^?2 and 2×10^?2 M) for 2 h at 25±1°C. The ganglia were fixed at set time intervals after treatment so that the effect of caffeine in different stages of the cell cycle could be observed. Chromatid aberrations were induced only when the caffeine was administered in G₂ or approaching mitosis. No aberrations were observed after treatment in S or early G₂. In relation to the different doses administered, a threshold effect was evidenced, the number of aberrations increasing in a marked way at doses exceeding 5×10^?3 M. These data indicate, that the effect observed in Drosophila melanogaster is similar to that described by Kihlman in animals and plants treated with caffeine at temperatures below 30°C.Results obtained in non-cytological tests (non-disjunction, chromosome loss, lethal recessives, dominant lethals) have so far given incomplete indications as to the mutagenicity of caffeine in Drosophila. The results we have obtained with the cytological test seem to contribute to a better definition of the mutagenecity.     

Suggestion of GLYAT gene underlying variation of bone size and body lean mass as revealed by a bivariate genome-wide association study

Bone and muscle, two major tissue types of musculoskeletal system, have strong genetic determination. Abnormality in bone and/or muscle may cause musculoskeletal diseases such as osteoporosis and sarcopenia. Bone size phenotypes (BSPs), such as hip bone size (HBS), appendicular bone size (ABS), are genetically correlated with body lean mass (mainly muscle mass). However, the specific genes shared by these phenotypes are largely unknown. In this study, we aimed to identify the specific genes with pleiotropic effects on BSPs and appendicular lean mass (ALM). We performed a bivariate genome-wide association study (GWAS) by analyzing ~690,000 SNPs in 1,627 unrelated Han Chinese adults (802 males and 825 females) followed by a replication study in 2,286 unrelated US Caucasians (558 males and 1,728 females). We identified 14 interesting single nucleotide polymorphisms (SNPs) that may contribute to variation of both BSPs and ALM, with p values <10^?6 in discovery stage. Among them, the association of three SNPs (rs2507838, rs7116722, and rs11826261) in/near GLYAT (glycine-N-acyltransferase) gene was replicated in US Caucasians, with p values ranging from 1.89 × 10^?3 to 3.71 × 10^?4 for ALM–ABS, from 5.14 × 10^?3 to 1.11 × 10^?2 for ALM–HBS, respectively. Meta-analyses yielded stronger association signals for rs2507838, rs7116722, and rs11826261, with pooled p values of 1.68 × 10^?8, 7.94 × 10^?8, 6.80 × 10^?8 for ALB–ABS and 1.22 × 10^?4, 9.85 × 10^?5, 3.96 × 10^?4 for ALM–HBS, respectively. Haplotype allele ATA based on these three SNPs was also associated with ALM–HBS and ALM–ABS in both discovery and replication samples. Interestingly, GLYAT was previously found to be essential to glucose metabolism and energy metabolism, suggesting the gene’s dual role in both bone development and muscle growth. Our findings, together with the prior biological evidence, suggest the importance of GLYAT gene in co-regulation of bone phenotypes and body lean mass.     

Fetal lung and placental methylation is associated with in utero nicotine exposure

In utero smoke exposure has been shown to have detrimental effects on lung function and to be associated with persistent wheezing and asthma in children. One potential mechanism of IUS effects could be alterations in DNA methylation, which may have life-long implications. The goal of this study was to examine the association between DNA methylation and nicotine exposure in fetal lung and placental tissue in early development; nicotine exposure in this analysis represents a likely surrogate for in-utero smoke. We performed an epigenome-wide analysis of DNA methylation in fetal lung tissue (n = 85, 41 smoke exposed (48%), 44 controls) and the corresponding placental tissue samples (n = 80, 39 smoke exposed (49%), 41 controls) using the Illumina HumanMethylation450 BeadChip array. Differential methylation analyses were conducted to evaluate the variation associated with nicotine exposure. The most significant CpG sites in the fetal lung analysis mapped to the PKP3 (P = 2.94 × 10^?03), ANKRD33B (P = 3.12 × 10^?03), CNTD2 (P = 4.9 × 10^?03) and DPP10 (P = 5.43 × 10^?03) genes. In the placental methylome, the most significant CpG sites mapped to the GTF2H2C and GTF2H2D genes (P = 2.87 × 10^?06 ? 3.48 × 10^?05). One hundred and one unique CpG sites with P-values < 0.05 were concordant between lung and placental tissue analyses. Gene Set Enrichment Analysis demonstrated enrichment of specific disorders, such as asthma and immune disorders. Our findings demonstrate an association between in utero nicotine exposure and variable DNA methylation in fetal lung and placental tissues, suggesting a role for DNA methylation variation in the fetal origins of chronic diseases.     

Characterization of a hormone receptor defect in the androgen-insensitivity mutant

Mouse kidney cytosol contains specific receptors that reversibly bind dihydrotestosterone at a concentration of 43 f moles/mg protein. [Nonstandard abbreviation: DHT, dihydrotestosterone, 17 β-hydroxy-5 α-androstan-3-one.] The equilibrium dissociation constant of the receptor-dihydrotestosterone complex is 1.3 × 10^?9M for females and 1.7 × 10^?9M for castrated males. The complex sediments at 8–9S in glycerol gradients. In males bearing the androgen-insensitivity mutation (analogous to human testicular feminization), the specific dihydrotestosterone receptor activity is decreased about 8 fold. The residual binding activity has wild type affinity (K_D = 1.5 × 10^?9M) for dihydrotestosterone and also sediments at 8–9S. Kidney cytosol from castrated mutant mice displays a new binding component with low affinity and high capacity for dihydrotestosterone.     

Allelic asymmetry of the Lethal hybrid rescue (Lhr) gene expression in the hybrid between Drosophila melanogaster and D. simulans: confirmation by using genetic variations of D. melanogaster

In the cross between Drosophila melanogaster females and D. simulans males, hybrid males die at the late larval stage, and the sibling females also die at later stages at high temperatures. Removing the D. simulans allele of the Lethal hybrid rescue gene (Lhr ^sim ) improves the hybrid incompatibility phenotypes. However, the loss-of-function mutation of Lhr ^sim (Lhr ^sim0 ) does not rescue the hybrid males in crosses with several D. melanogaster strains. We first describe the genetic factor possessed by the D. melanogaster strains. It has been suggested that removing the D. melanogaster allele of Lhr (Lhr ^mel ), that is Lhr ^mel0 , does not have the hybrid male rescue effect, contrasting to Lhr ^sim0 . Because the expression level of the Lhr gene is known to be Lhr ^sim  > Lhr ^mel in the hybrid, Lhr ^mel0 may not lead to enough of a reduction in total Lhr expression. Then, there is a possibility that the D. melanogaster factor changes the expression level to Lhr ^sim  < Lhr ^mel . But in fact, the expression level was Lhr ^sim  > Lhr ^mel in the hybrid irrespectively of the presence of the factor. At last, we showed that Lhr ^mel0 slightly improves the viability of hybrid females, which was not realized previously. All of the present results are consistent with the allelic asymmetry model of the Lhr gene expression in the hybrid.     

Methylation of SOCS3 is inversely associated with metabolic syndrome in an epigenome-wide association study of obesity

Epigenetic mechanisms, including DNA methylation, mediate the interaction between gene and environment and may play an important role in the obesity epidemic. We assessed the relationship between DNA methylation and obesity in peripheral blood mononuclear cells (PBMCs) at 485,000 CpG sites across the genome in family members (8-90 y of age) using a discovery cohort (192 individuals) and a validation cohort (1,052 individuals) of Northern European ancestry. After Bonferroni-correction (P_α=0.05 = 1.31 × 10^?7) for genome-wide significance, we identified 3 loci, cg18181703 (SOCS3), cg04502490 (ZNF771), and cg02988947 (LIMD2), where methylation status was associated with body mass index percentile (BMI%), a clinical index for obesity in children, adolescents, and adults. These sites were also associated with multiple metabolic syndrome (MetS) traits, including central obesity, fat depots, insulin responsiveness, and plasma lipids. The SOCS3 methylation locus was also associated with the clinical definition of MetS. In the validation cohort, SOCS3 methylation status was found to be inversely associated with BMI% (P = 1.75 × 10^?6), waist to height ratio (P = 4.18 × 10^?7), triglycerides (P = 4.01 × 10^?4), and MetS (P = 4.01 × 10^?7), and positively correlated with HDL-c (P = 4.57 × 10^?8). Functional analysis in a sub cohort (333 individuals) demonstrated SOCS3 methylation and gene expression in PBMCs were inversely correlated (P = 2.93 × 10^?4) and expression of SOCS3 was positively correlated with status of MetS (P = 0.012). We conclude that epigenetic modulation of SOCS3, a gene involved in leptin and insulin signaling, may play an important role in obesity and MetS.     

The genetic basis for mating-induced sex differences in starvation resistance in Drosophila melanogaster

Multiple genetic and environmental factors interact to influence starvation resistance, which is an important determinant of fitness in many organisms, including Drosophila melanogaster. Recent studies have revealed that mating can alter starvation resistance in female D. melanogaster, but little is known about the behavioral and physiological mechanisms underlying such mating-mediated changes in starvation resistance. In the present study, we first investigated whether the effect of mating on starvation resistance is sex-specific in D. melanogaster. As indicated by a significant sex × mating status interaction, mating increased starvation resistance in females but not in males. In female D. melanogaster, post-mating increase in starvation resistance was mainly attributed to increases in food intake and in the level of lipid storage relative to lean body weight. We then performed quantitative genetic analysis to estimate the proportion of the total phenotypic variance attributable to genetic differences (i.e., heritability) for starvation resistance in mated male and female D. melanogaster. The narrow-sense heritability (h²) of starvation resistance was 0.235 and 0.155 for males and females, respectively. Mated females were more resistant to starvation than males in all genotypes, but the degree of such sexual dimorphism varied substantially among genotypes, as indicated by a significant sex × genotype interaction for starvation resistance. Cross-sex genetic correlation was greater than 0 but less than l for starvation resistance, implying that the genetic architecture of this trait was partially shared between the two sexes. For both sexes, starvation resistance was positively correlated with longevity and lipid storage at genetic level. The present study suggests that sex differences in starvation resistance depend on mating status and have a genetic basis in D. melanogaster.     

Uptake and binding of β-ecdysone in imaginal discs of Drosophila melanogaster

The kinetics of uptake and retention of β-ecdysone by imaginal discs from late third instar larvae of Drosophila melanogaster correspond well with those of the first synthetic response of discs to hormone, an increase in RNA synthesis.Competition studies indicate the presence of two types of hormone binding sites, specific and non-specific. The specific sites are saturated at hormone concentrations which fully induce morphogenesis. Results are consistent with the hypothesis that analogs which induce morphogenesis at differing concentrations bind to the same sites. Experiments with the inhibitors N-ethylmaleimide, actinomycin d, and cycloheximide suggest that the binding sites are pre-existing in the cell and require functional sulfhydryl groups for binding.Specific binding, binding that is competed by excess unlabeled β-ecdysone, is saturable (70–80 nM). Kinetic rate constants for this specific binding were estimated to be k_a = 1.5 × 10⁵M^?1 min^?1, k_d = 3 × 10^?2 min^?1. The equilibrium dissociation constant calculated from the kinetic rate constants was K_eq = 2 × 10^?7M compared to 1.7 × 10^?7M β-ecdysone required to induce morphogenesis in vitro and 2.5 × 10^?7M determined to be the in vivo concentration at the time of induction of morphogenesis.     

Nutritional control of xanthine dehydrogenase. II. Effects on xanthine dehydrogenase and aldehyde oxidase of culturing wild-type and mutant Drosophila on different levels of molybdenum

Two new mutants, deficient in aldehyde oxidase and xanthine dehydrogenase, have been isolated from a wild-type stock of Drosophila melanogaster and have been provisionally termed lxd ^c and lxd ^d, respectively, as both mutants appear to be allelic with lxd (low xanthine dehydrogenase). An analysis has been made of the effects of dietary molybdenum on lxd, lxd ^c, lxd^d, lao (low aldehyde oxidase), mal (maroon-like eye color), and pac (Pacific) wild-type flies. On the lower dietary levels of 10 ^?3 M and 10 ^?2 M molybdenum, increases in specific activity of both enzymes were observed only in lxd. Furthermore, two- to three-fold increases in specific activity of both enzymes occurred in all strains, except mal, when cultured on 5×10 ^?2 M molybdenum. The lxd and lxd ^c strains failed to survive on this high concentration of the ion. Similar concentrations of molybdenum had no effect in vitro. An extra electrophoretic band of xanthine dehydrogenase was observed on polyacrylamide gel from extracts of wild-type flies cultured on certain levels of molybdenum, but its appearance was not always correlated with the increases in specific activity.