Insects provide an accessible system to study the contribution of DNA methylation to complex epigenetic phenotypes created to regulate gene expression, chromatin states, imprinting and dosage compensation. The members of genus Drosophila have been used as a model system to study aspects of biology like development, behaviour and genetics. Despite the popularity of Drosophila melanogaster as a genetic and epigenetic model organism, DNA methylation studies are limited due to low levels of genomic 5-methylcytosine. Our study employs a sensitive liquid chromatography-mass spectrometry (LCMS) based method to quantify the levels of 5-methylcytosine from the genomic DNA in different members of the genus Drosophila. Our results reveal that, despite being phylogenetically related, there is a marked variation in the levels of 5-methylcytosine between the genomes of the members of genus Drosophila. Also, there is a change in the genomic levels of 5-methylcytosine through each life cycle stage of holometabolous development in D. melanogaster.

Drosophila melanogaster lacks DNMT1/DNMT3 based methylation machinery. Despite recent reports confirming the presence of low DNA methylation in Drosophila; little is known about the methyltransferase. Therefore, in this study, we have aimed to investigate the possible functioning of DNA methyltransferase in Drosophila. The 14 K oligo microarray slide was incubated with native cell extract from adult Drosophila to check the presence of the methyltransferase activity. After incubation under appropriate conditions, the methylated oligo sequences were identified by the binding of anti 5-methylcytosine monoclonal antibody. The antibody bound to the methylated oligos was detected using Cy3 labeled secondary antibody. Methylation sensitive restriction enzyme mediated PCR was used to assess the methylation at a few selected loci identified on the array. It could be seen that a few of the total oligos got methylated under the assay conditions. Analysis of methylated oligo sequences provides evidence for the presence of de novo methyltransferase activity and allows identification of its sequence specificity in adult Drosophila. With the help of methylation sensitive enzymes we could detect presence of CpC methylation in the selected genomic regions. This study reports presence of an active DNA methyltransferase in adult Drosophila, which exhibits sequence specificity confirmed by presence of asymmetric methylation at corresponding sites in the genomic DNA. It also provides an innovative approach to investigate methylation specificity of a native methyltransferase.

Heterogeneity among isogenic cells/individuals has been known for at least 150 years. Even Mendel, working on pea plants, realized that not all tall plants were identical. However, Mendel was more interested in the discontinuous variation between genetically distinct individuals. The concept of environment dictating distinct phenotypes among isogenic individuals has since been shown to impact the evolution of populations in numerous examples at different scales of life. In this review, we discuss how phenotypic heterogeneity and its evolutionary implications exist at all levels of life, from viruses to mammals. In particular, we discuss how a particular disease condition (cancer) is impacted by heterogeneity among isogenic cells, and propose a potential role that phenotypic heterogeneity might play toward the onset of the disease.

Although genomic DNA of Drosophila melanogaster has been shown to contain little cytosine methylation, the distribution of this genome-wide methylation patterns in different life stages remains to be elucidated. We have developed an immunochemical method using cDNA microarray to assess methylation. In the present work, this methylation microarray method was employed to identify DNA methylation in and around the genes in different life stages of D. melanogaster. This led to the identification of methylated genes in three stages of D. melanogaster, viz. embryo, pupa and adult. It is noteworthy that there was differential methylation in genes in different life cycle stages. Remarkably, a few functional annotation clusters showed negative correlation between transcription of a particular gene and its methylation status. In this analysis, some of the genes attributed to characteristic biological processes of particular life stage in D. melanogaster were found to be methylated in other life stages. Our analysis while providing a methylation map also suggests that gene-specific DNA methylation is altered during the life cycle stages of D. melanogaster. Keywords:Developmental regulation, DNA methylation, Drosophila development, epigenetics, gene-specific methylation, 5 methyl cytosine.DNA methylation is involved in the regulation of several molecular processes in an eukaryotic system. Methylation plays an important role in gene silencing 1,2 , chromatin remodelling 3 and repression of transposon activity 4 . In vertebrates, X chromosome inactivation and genomic imprinting are influenced by DNA methylation, wherein these phenomena are essential for the normal development of an organism. Invertebrates such as insects show significantly distinct life stages and developmental stagespecific regulation of gene expression which plays a crucial role in the transitions between differentiated lifehistory stages. Many research groups have emphasized the presence of DNA methylation in invertebrates and have advocated that DNA methylation might be the mechanism for developmental gene regulation involved in life cycle transition [5][6][7][8][9][10] . The genome-wide DNA methylation maps of insects like Drosophila melanogaster 9 , Bombyx mori 11 , Solenopsis invicta 12 and Apis mellifera 13,14 have been generated using whole genome bisulphite sequencing. Therefore it is important to study whether these methylation signatures play an important role in life cycle processes in invertebrates.Though Drosophila lacks the canonical DNMT1, DNMT3A and DNMT3B, the presence of very low levels of DNA methylation has been reported in D. melanogaster [15][16][17][18][19][20][21][22] . Presence of methylated DNA has been a controversial issue since few reports suggest that D. melanogaster lacks DNA methylation and DNMT2 is indeed an RNA methyltransferase that uses a DNA methyltransferase type of mechanism [23][24][25] . Capuano et al. 19 employed a sensitive LC-MS/MS method to report the presence of 0.034% cytosine DNA methylation in D. melanogaster...

28Drosophila melanogaster undergoes holometabolous development, has very low levels of 29 DNA methylation, and is known to possess a single known methyltransferase, dDNMT2. 30 This study compares the DNA methylation patterns between the two life cycle stages of D. 31 melanogaster using a combination of DNA immunoprecipitation and high throughput 32 sequencing techniques. 33 Our results indicate, a change in the chromosomal distribution of the sparse DNA 34 methylation concerning genes and natural transposable elements between in the embryo and 35 the adult stages of D. melanogaster. The differentially methylated regions localised on genes 36 involved in the regulation of cell cycle processes of mitotic cell divisions and chromosomal 37 segregation. dDNMT2 knockout flies exhibited altered patterns of DNA methylation. The 38 observed differences in DNA methylation were in genes involved in cellular communication 39 and cytoskeletal functions. The variation in DNA methylation between the two life cycle 40 stages is indicative that it could have a role in regulatory processes during development and, 41 dDNMT2 may have a role as a co-factor for the hitherto undiscovered DNA 42 methyltransferase in D. melanogaster. 43 44 Keywords: differential DNA methylation, 5-methylcytosine, DNMT2, asymmetric DNA 45 methylation, non-CpG methylation 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60DNA methylation in Drosophila melanogaster was first reported in the embryos by a 61 combination of photoacoustic spectroscopy and polyclonal antibodies to be 0.008 mol percent 62 of 5-methylcytosine (5mC) 1,2 . These results were later corroborated by enzymatic digestion 63 of DNA with McrBC, high-performance liquid chromatography (HPLC) and 2D-Thin layer 64 chromatography approximately to report the presence of one 5mC molecule in 1,000-2,000 65 cytosines molecules in adult fly 3 . There was no sequence-specific data in Drosophila 66 genome until the analysis of the early embryos followed by sequencing of select bisulfite 67 modified regions which revealed methylation to be predominant in non-CpG dinucleotides 4 . 68Subsequently, high-throughput sequencing of sodium bisulfite-treated 69 immunoprecipitated fragments bearing methylation from stage 5 embryos with 10,000X 70 coverage reported less than 1% of genome-wide DNA methylation in the stage-5 embryos 5 . 71DNA samples of D. melanogaster in a mixed population of adult males and females (w 1118 72 strain) was subjected to liquid chromatography selective reaction monitoring (LC-SRM) 73 method to report 0.034% of methylated cytosines in the genome which is 10−100 fold below 74 the suggested detection limit of bisulphite sequencing 6 whereas the female adult (Oregon-R 75 strain) was reported to harbour 0.002% methylation of cytosines 7 . The whole genome 76 bisulfite-sequencing also correlated with the 55% increment in DNA methylation of the 77 infected testes; however, this did not show any correlation with cytoplasmic incompatibility 8 . 78The changes in dietary specification also...

No abstract

Adaptive divergence leading to speciation is the major evolutionary process generating diversity in life forms. The most commonly observed form of speciation is allopatric speciation which requires that gene flow be prevented between populations by physical or temporal barriers, as they adapt to their respective environments. Eventually, these adaptive responses drive the populations far apart in the genotypic space such that individuals from the two populations become reproductively isolated. A widely accepted theory is that speciation simply occurs as a by-product of adaptive response of the populations 1,2. Several ecological and laboratory examples of allopatric speciation exist 3-6. However, we know little about the nature (pre- or post-zygotic) of barriers that arise first in this process. Understanding the first barriers that arise between populations is key, as populations diverge towards becoming distinct species. In recent years, fungi been used as model organisms to answer questions related to evolution of reproductive isolation 3,7-9. Here we show rapid evolution of pre-zygotic barriers between allopatric yeast populations. We further demonstrate that these pre-zygotic barriers arise due to altered mating kinetics of the evolved population. Moreover, our non-adaptive evolution experiments with yeast under limited selection pressure also show rapid emergence of reproductive isolation. Overall, our results show that evolution of pre-zygotic reproductive barriers can occur as result of natural selection or drift. These barriers result because of altered mating kinetics or mate preference.