Mining histone methyltransferases and demethylases from whole genome sequence

Epigenetic regulation through post-translational modification of histones, especially methylation, is wellconserved in evolution. Although there are several insect genomes sequenced, an analysis with a focus on theirepigenetic repertoire is limited. We have utilized a novel work-flow to identify one or more domains as highpriority domain (HPD), if present in at least 50% of the genes of a given functional class in the referencegenome, namely, that of Drosophila melanogaster. Based on this approach, we have mined histone methyltransferases and demethylases from the whole genome sequence of Aedes aegypti (Diptera), the pea aphidAcyrthosiphon pisum, the triatomid bug Rhodnius prolixus (Hemiptera), the honeybee Apis mellifera (Hymenoptera), the silkworm Bombyx mori (Lepidoptera) and the red flour beetle Tribolium castaneum(Coleoptera). We identified 38 clusters consisting of arginine methyltransferases, lysine methyltransferases anddemethylases using OrthoFinder, and the presence of HPD was queried in these sequences using InterProScan.This approach led us to identify putative novel members and currently inaccurate ones. Other than the highpriority domains, these proteins contain shared and unique domains that can mediate protein–protein interaction. Phylogenetic analysis indicates that there is different extent of protein sequence similarity; averagesimilarity between histone lysine methyltransferases varies from 41% (for active mark) to 48% (for repressivemark), arginine methyltransferases is 51%, and demethylases is 52%. The method utilized here facilitatesreliable identification of desired functional class in newly sequenced genomes

Application of SNaPshot for analysis of thiopurine methyltransferase gene polymorphism.

Background & objectives: Mercaptopurine, azathioprine, and thioguanine, used as antineoplastic agents and immunosuppressants are catabolized by thiopurine methyltransferase (TPMT) enzyme, which exhibits genetic polymorphism. Genotyping patients and the population to which the patients belong, is important for effective treatment and reducing toxicity. There is a need for faster methods for genotyping. Hence the present study was planned to test the application of SNaPshot technique for analysis of the three common TPMT alleles: TPMT*2, TPMT*3A, and TPMT*3C in DNA from healthy Indian volunteers as well as to apply the method on cDNA samples obtained from children with acute lymphoblastic leukaemia (ALL). Method: A total of 120 healthy volunteers and 25 patients were analysed by multiplexed SNaPshot reaction. Genomic DNA was the template for most of the analyses, but additionally the cDNA synthesized for translocation detection was used as the template in case of patients with ALL. The results of SNaPshot reaction were confi rmed by direct sequencing. Results: The TPMT genotype could be reliably identifi ed by SNaPshot analysis in multiplex reactions both in genomic DNA samples and cDNA. The overall frequency of the three common polymorphisms was observed to be 4.9 per cent, arising from heterozygosity for TPMT*3C (4.1%) and TPMT*3A (0.8%). Interpretation & conclusion: SNaPshot method for TPMT polymorphism analysis works faster with the potential for high throughput. By simultaneously interrogating the genotype at multiple sites, the method can provide future opportunity to multiplex, though multiplexing has not been done in the present analysis. Heterozygosity for TPMT*3C (719 A>G) was detected in 4.1 per cent of the study population and no homozygosity was observed. Our results indicated that TPMT*3C was the most common polymorphism in Indian population, while TPMT3*A, associated with the absence of catalytic activity of TPMT, was very rare.

Spontaneous Expression Of Fragile Site at Xq27.3 In a Fragile X patient.

Fragile site at Xq27.3 is classified as a rare fragile site which is observed only under conditions of folate depletion Here we report a case where fragile site at Xq27.3 was detected in normal RPMI 1640 medium without induction, in the lymphocytes of a patient clinically diagnosed as fragile X patient. At the molecular level both an expansion and methylation of (CGG)n repeat at FMRI was detected.

Modified bases in transfer RNA.

Transfer RNA is uniquely enriched with modified bases. A large body of information has accumulated about the occurrence, nature and distribution of modified bases in tRNA. But similar investigations on the enzymes involved in this post-transcriptional modification have been hampered by the instability of the enzymes and lack of suitable substrates. The present review summarises briefly, the occurrence and methods of detection of modified bases, the enzymes involved in their formation and also certain suggestive evidence for the role of modification in cellular metabolism.