Genome and transcriptome analysis of the mealybug Maconellicoccus hirsutus: Correlation with its unique phenotypes.

Mealybugs are aggressive pests with world-wide distribution and are suitable for the study of different phenomena like genomic imprinting and epigenetics. Genomic approaches facilitate these studies in absence of robust genetics in this system. We sequenced, de novo assembled, annotated Maconellicoccus hirsutus genome. We carried out comparative genomics it with four mealybug and eight other insect species, to identify expanded, specific and contracted gene classes that relate to pesticide and desiccation resistance. We identified horizontally transferred genes adding to the mutualism between the mealybug and its endosymbionts. Male and female transcriptome analysis indicates differential expression of metabolic pathway genes correlating with their physiology and the genes for sexual dimorphism. The significantly lower expression of endosymbiont genes in males relates to the depletion of endosymbionts in males during development.

Correlation of drug resistance with single nucleotide variations through genome analysis and experimental validation in a multi-drug resistant clinical isolate of M. tuberculosis.

BACKGROUND: Genome sequencing and genetic polymorphism analysis of clinical isolates of M. tuberculosis is carried out to gain further insight into molecular pathogenesis and host-pathogen interaction. Therefore the functional evaluation of the effect of single nucleotide variation (SNV) is essential. At the same time, the identification of invariant sequences unique to M. tuberculosis contributes to infection detection by sensitive methods. In the present study, genome analysis is accompanied by evaluation of the functional implication of the SNVs in a MDR clinical isolate VPCI591. RESULT: By sequencing and comparative analysis of VPCI591 genome with 1553 global clinical isolates of M. tuberculosis (GMTV and tbVar databases), we identified 141 unique strain specific SNVs. A novel intergenic variation in VPCI591 in the putative promoter/regulatory region mapping between embC (Rv3793) and embA (Rv3794) genes was found to enhance the expression of embAB, which correlates with the high resistance of the VPCI591 to ethambutol. Similarly, the unique combination of three genic SNVs in RNA polymerase ß gene (rpoB) in VPCI591 was evaluated for its effect on rifampicin resistance through molecular docking analysis. The comparative genomics also showed that along with variations, there are genes that remain invariant. 173 such genes were identified in our analysis. CONCLUSION: The genetic variation in M. tuberculosis clinical isolate VPCI591 is found in almost all functional classes of genes. We have shown that SNV in rpoB gene mapping outside the drug binding site along with two SNVs in the binding site can contribute to quantitative change in MIC for rifampicin. Our results show the collective effect of SNVs on the structure of the protein, impacting the interaction between the target protein and the drug molecule in rpoB as an example. The study shows that intergenic variations bring about quantitative changes in transcription in embAB and in turn can lead to drug resistance.

Distinguishing between biochemical and cellular function: Are there peptide signatures for cellular function of proteins?

The genome annotation and identification of gene function depends on conserved biochemical activity. However, in the cell, proteins with the same biochemical function can participate in different cellular pathways and cannot complement one another. Similarly, two proteins of very different biochemical functions are put in the same class of cellular function; for example, the classification of a gene as an oncogene or a tumour suppressor gene is not related to its biochemical function, but is related to its cellular function. We have taken an approach to identify peptide signatures for cellular function in proteins with known biochemical function. ATPases as a test case, we classified ATPases (2360 proteins) and kinases (517 proteins) from the human genome into different cellular function categories such as transcriptional, replicative, and chromatin remodelling proteins. Using publicly available tool, MEME, we identify peptide signatures shared among the members of a given category but not between cellular functional categories; for example, no motif sharing is seen between chromatin remodelling and transporter ATPases, similarly between receptor Serine/Threonine Kinase and Receptor Tyrosine Kinase. There are motifs shared within each category with significant E value and high occurrence. This concept of signature for cellular function was applied to developmental regulators, the polycomb and trithorax proteins which led to the prediction of the role of INO80, a chromatin remodelling protein, in development. This has been experimentally validated earlier for its role in homeotic gene regulation and its interaction with regulatory complexes like the Polycomb and Trithorax complex. Proteins 2017; 85:682-693. © 2016 Wiley Periodicals, Inc.

Fluoride exposure abates pro-inflammatory response and induces in vivo apoptosis rendering zebrafish (Danio rerio) susceptible to bacterial infections.

The present study describes the immunotoxic effect of chronic fluoride exposure on adult zebrafish (Danio rerio). Zebrafish were exposed to fluoride (71.12 mg/L; 1/10 LC50) for 30 d and the expression of selected genes studied. We observed significant elevation in the detoxification pathway gene cyp1a suggesting chronic exposure to non-lethal concentration of fluoride is indeed toxic to fish. Fluoride mediated pro-oxidative stress is implicated with the downregulation in superoxide dismutase 1 and 2 (sod1/2) genes. Fluoride affected DNA repair machinery by abrogating the expression of the DNA repair gene rad51 and growth arrest and DNA damage inducible beta a gene gadd45ba. The upregulated expression of casp3a coupled with altered Bcl-2 associated X protein/B-cell lymphoma 2 ratio (baxa/bcl2a) clearly suggested chronic fluoride exposure induced the apoptotic cascade in zebrafish. Fluoride-exposed zebrafish when challenged with non-lethal dose of fish pathogen A. hydrophila revealed gross histopathology in spleen, bacterial persistence and significant mortality. We report that fluoride interferes with system-level output of pro-inflammatory cytokines tumour necrosis factor-α, interleukin-1ß and interferon-γ, as a consequence, bacteria replicate efficiently causing significant fish mortality. We conclude, chronic fluoride exposure impairs the redox balance, affects DNA repair machinery with pro-apoptotic implications and suppresses pro-inflammatory cytokines expression abrogating host immunity to bacterial infections.

Proximity of H2A.Z containing nucleosome to the transcription start site influences gene expression levels in the mammalian liver and brain.

Nucleosome positioning maps of several organisms have shown that Transcription Start Sites (TSSs) are marked by nucleosome depleted regions flanked by strongly positioned nucleosomes. Using genome-wide nucleosome maps and histone variant occupancy in the mouse liver, we show that the majority of genes were associated with a single prominent H2A.Z containing nucleosome in their promoter region. We classified genes into clusters depending on the proximity of H2A.Z to the TSS. The genes with no detectable H2A.Z showed lowest expression level, whereas H2A.Z was positioned closer to the TSS of genes with higher expression levels. We confirmed this relation between the proximity of H2A.Z and expression level in the brain. The proximity of histone variant H2A.Z, but not H3.3 to the TSS, over seven consecutive nucleosomes, was correlated with expression. Further, a nucleosome was positioned over the TSS of silenced genes while it was displaced to expose the TSS in highly expressed genes. Our results suggest that gene expression levels in vivo are determined by accessibility of the TSS and proximity of H2A.Z.

Modeling SNP mediated differential targeting of homologous 3'UTR by microRNA.

We had previously proposed that the post-transcriptional regulation through microRNA as a mechanism for incomplete penetrance and variable expressivity, leads to lack of correlation between genotype and phenotype. Here we report the validation of miRNA-target interactions we predicted earlier and demonstrate the regulation of endogenous JAG1 by hsa-miR-214 and hsa-miR-124, and TGFBR2 by hsa-miR-34b*, through luciferase activity of reporter constructs and also the expression levels of the endogenous genes. Using these targets, we have modeled the diploid state for miRNA target site with heterozygosity for the SNP and demonstrate the differential targeting of an otherwise identical 3'UTR. We show that SNP rs8708 (A > G) at the target site of hsa-miR-214 can relieve the repression while an SNP rs11466532 (C > T) enhances the repression of reporter expression by hsa-miR-34b*. We discuss the results in the light of its implications in the context of penetrance of dominant mutations in miRNA targeted genes, using JAG1 as an example. These observations imply that disease causing mutations in JAG1 linked to the SNP rs8708G will be poorly targeted by hsa-miR-214 when present against a normal allele of JAG1 with rs8708A and will show penetrance of JAG1 mutations as Alagille syndrome, while mutant JAG1 linked to rs8708A against rs8708G on the normal allele will show either no disease or much attenuated symptoms and hence exhibit incomplete penetrance.

Nucleosomal occupancy and CGG repeat expansion: a comparative analysis of triplet repeat region from mouse and human fragile X mental retardation gene 1.

The expansion of CGG repeats in the 5'-untranslated region (5'UTR) of FMR1 gene is the molecular basis of fragile X syndrome in most of the patients. The nature of the flanking sequences in addition to the length and interruption pattern of repeats is predicted to influence CGG repeat instability in the FMR1 gene. We investigated nucleosome occupancy as a contributor to CGG repeat instability in a transgenic mouse model containing unstable (CGG)(26,) from human FMR1 cloned downstream of nucleosome-excluding sequence. We observe that the transgene has an open chromatin structure compared to the stable endogenous mouse Fmr1 within the same nucleus. CGG repeats in mouse Fmr1 are flanked by nucleosomes unlike the repeats in the transgene in all the tissues examined. Further in vitro chromatin reconstitution experiments show that DNA fragment without the SV40ori/EPR (nucleosome-excluding sequence) forms more stable chromatin than the one containing it, despite having the same number of CGG repeats. The correlation between nucleosomal organisation of the FMR1 gene and CGG repeat instability was supported by significantly lower frequency of repeat expansion in mice containing an identical transgene without the SV40ori/EPR. Our studies demonstrate that flanking DNA sequences can influence repeat instability through modulation of nucleosome occupancy in the region.

Identification of a polycomb responsive region in human HoxA cluster and its long-range interaction with polycomb enriched genomic regions.

Polycomb and Trithorax group proteins (PcG, TrxG) epigenetically regulate developmental genes. These proteins bind with specific DNA elements, the Polycomb Response Element (PRE). Apart from mutations in polycomb/ trithorax proteins, altered cis-elements like PRE underlie the modified function and thus disease etiology. PREs are well studied in Drosophila, while only a few human PREs have been reported. We have identified a polycomb responsive DNA element, hPRE-HoxA3, in the intron of the HoxA3 gene. The hPRE-HoxA3 represses luciferase reporter activity in a PcG-dependent manner. The endogenous hPRE-HoxA3 element recruits PcG proteins and is enriched with repressive H3K27me3 marks, demonstrating that hPRE-HoxA3 is a part of the PcG-dependent gene regulatory network. Furthermore, it interacts with D11-12, the well-known PRE in the human Hox cluster. hPRE-Hox3 is a part of the 3-dimensional chromosomal domain organization as it is involved in the long-range interaction with other PcG enriched regions of Hox A, B, C, and D clusters.

SIN-3 functions through multi-protein interaction to regulate apoptosis, autophagy, and longevity in Caenorhabditis elegans.

SIN3/HDAC is a multi-protein complex that acts as a regulatory unit and functions as a co-repressor/co-activator and a general transcription factor. SIN3 acts as a scaffold in the complex, binding directly to HDAC1/2 and other proteins and plays crucial roles in regulating apoptosis, differentiation, cell proliferation, development, and cell cycle. However, its exact mechanism of action remains elusive. Using the Caenorhabditis elegans (C. elegans) model, we can surpass the challenges posed by the functional redundancy of SIN3 isoforms. In this regard, we have previously demonstrated the role of SIN-3 in uncoupling autophagy and longevity in C. elegans. In order to understand the mechanism of action of SIN3 in these processes, we carried out a comparative analysis of the SIN3 protein interactome from model organisms of different phyla. We identified conserved, expanded, and contracted gene classes. The C. elegans SIN-3 interactome -revealed the presence of  well-known proteins, such as DAF-16, SIR-2.1, SGK-1, and AKT-1/2, involved in autophagy, apoptosis, and longevity. Overall, our analyses propose  potential mechanisms by which SIN3 participates in multiple biological processes and their conservation across species and identifies candidate genes for further experimental analysis.

Chromatin remodeling protein INO80 has a role in regulation of homeotic gene expression in Drosophila.

The homologues of yeast INO80 are identified across phyla from Caenorhabditis elegans to human. In Drosophila it has been shown that dINO80 forms a complex with Pleiohomeotic but does not interact with Hox PRE (polycomb responsive element). As some proteins of the INO80 complex are implicated in homeotic gene regulation, we examined if dINO80 is involved in regulation of homeotic genes. We find that dINO80 null mutants generated by imprecise excision of P-element are late embryonic lethals and show homeotic transformation. We detect misexpression of homeotic genes like Sex-comb reduced, Antennapedia, Ultrabithorax and Abdominal-B in dIno80 mutant embryos by immunostaining which is further substantiated by quantitative PCR. Polycomb phenotype in dIno80-Pc is enhanced in double mutants. Concurrently, the localization of dINO80 to sequences upstream of misexpressed genes in vivo shows that dINO80 is involved in homeotic gene regulation and probably through its interactions with PcG-trxG complexes.

Single nucleotide polymorphism in the genes of mce1 and mce4 operons of Mycobacterium tuberculosis: analysis of clinical isolates and standard reference strains.

BACKGROUND: The presence of four mammalian cell entry (mce) operons in Mycobacterium tuberculosis suggests the essentiality of the functions of the genes in these operons. The differential expression of the four mce operons in different phases of in vitro growth and in infected animals reported earlier from our laboratory further justifies the apparent redundancy for these genes in the genome.Here we investigate the extent of polymorphism in eight genes in the mce1 and mce4 operons of M. tuberculosis from four standard reference strains (H37Rv, H37Ra, LVS (Low Virulent Strain) and BCG) and 112 clinical isolates varying in their drug susceptibility profile, analysed by direct sequencing and Sequenom MassARRAY platform. RESULTS: We discovered 20 single nucleotide polymorphisms (SNPs) in the two operons. The comparative analysis of the genes of mce1 and mce4 operons revealed that yrbE1A [Rv0167] was most polymorphic in mce1 operon while yrbE4A [Rv3501c] and lprN [Rv3495c] had the highest number of SNPs in the mce4 operon. Of 20 SNPs, 12 were found to be nonsynonymous and were further analysed for their pathological relevance to M. tuberculosis using web servers PolyPhen and PMut, which predicted five deleterious nonsynonymous SNPs. A mutation from proline to serine at position 359 of the native Mce1A protein was most deleterious as predicted by both PolyPhen and PMut servers. Energy minimization of the structure of native Mce1A protein and mutated protein was performed using InsightII. The mutated Mce1A protein showed structural changes that could account for the effects of this mutation. CONCLUSIONS: Our results show that SNPs in the coding sequences of mce1 and mce4 operons in clinical isolates can be significantly high. Moreover, mce4 operon is significantly more polymorphic than mce1 operon (p < 0.001). However, the frequency of nonsynonymous substitutions is higher in mce1 operon and synonymous substitutions are more in mce4 operon. In silico modeling predict that nonsynonymous SNP at mce1A [Rv0169], a virulence gene could play a pivotal role in causing functional changes in M. tuberculosis that may reflect upon the biology of the bacteria.

Incomplete penetrance and variable expressivity: is there a microRNA connection?

Incomplete penetrance and variable expressivity are non-Mendelian phenomena resulting in the lack of correlation between genotype and phenotype. Not withstanding the diversity in mechanisms, differential expression of homologous alleles within cells manifests as variations in penetrance and expressivity of mutations between individuals of the same genotype. These phenomena are seen most often in dominantly inherited diseases, implying that they are sensitive to concentration of the gene product. In this framework and the advances in understanding the role of microRNA (miRNA) in fine-tuning gene expression at translational level, we propose miRNA-mediated regulation as a mechanism for incomplete penetrance and variable expressivity. The presence of miRNA binding sites at 3' UTR, co-expression of target gene-miRNA pairs for genes showing incomplete penetrance and variable expressivity derived from available data lend support to our hypothesis. Single nucleotide polymorphisms in the miRNA target site facilitate the implied differential targeting of the transcripts from homologous alleles.

Functional analysis of an intergenic non-coding sequence within mce1 operon of M.tuberculosis.

BACKGROUND: The mce operons play an important role in the entry of M. tuberculosis into macrophages and non-phagocytic cells. Their non-redundant function as well as complex regulation is implied by the phenotype of mce mutants. Recently, mce1 operon was found to extend over 13 genes, fadD5 (Rv0166) being the first gene of the operon. The presence of a non-coding sequence of 200 base pairs between Rv0166 and Rv0167 is peculiar to mce1 among the four mce operons of M.tuberculosis. We have examined the function of this region. RESULTS: We predicted putative promoter activity of the 200 base pairs of non-coding, intergenic region between Rv0166 and Rv0167 in silico using MEME software and designate it as intergenic promoter, IGPr. We demonstrate both promoter activity and a putative negative regulatory function of this fragment by reporter assays carried out in the surrogate host M.smegmatis. We find that the repressive elements not only control the native promoter but also repress a heterologous promoter of M.smegmatis. The higher activity of the intergenic promoter in a clinical isolate in comparison with the wild type sequence from M.tuberculosis H37Rv could be correlated with a point mutation within the negative element. We have mapped two transcription start sites for mce1 operon both of which are utilized in M.tuberculosis H37Rv as well as the clinical isolate VPCI591. Our studies show that the promoter activity in the non-coding region is relevant not only in reporter gene expression but also in the expression of mce1 operon in M. tuberculosis cells grown in synthetic medium. CONCLUSION: The mce operon of M.tuberculosis H37Rv potentially can be transcribed from two promoters P1 and P2, former mapping upstream of Rv0166 and the latter in the non-coding intergenic region between Rv0166 and Rv0167. The transcription initiation from P1 results in a transcript with Rv0166 while that from P2 will be without it. The sequences between the translation start site of Rv0167 and the promoter P2 have a negative regulatory role, as point mutation within the sequence leads to enhanced activity of P2 as well as a heterologous promoter from M.smegmatis. The mutation detected in the clinical isolate VPCI591 therefore behaves like a gain-of-function mutation.

Mining histone methyltransferases and demethylases from whole genome sequence.

Epigenetic regulation through post-translational modification of histones, especially methylation, is well conserved in evolution. Although there are several insect genomes sequenced, an analysis with a focus on their epigenetic 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 reference genome, 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 aphid Acyrthosiphon 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 and demethylases 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; average similarity between histone lysine methyltransferases varies from 41% (for active mark) to 48% (for repressive mark), arginine methyltransferases is 51%, and demethylases is 52%. The method utilized here facilitates reliable identification of desired functional class in newly sequenced genomes.

The regulatory function of dIno80 correlates with its DNA binding activity.

The INO80 complex, including the Ino80 protein, forms a highly conserved canonical complex that remodels chromatin in the context of multiple cellular functions. The Drosophila homologue, dIno80, is involved in homeotic gene regulation during development as a canonical Pho-dIno80 complex. Previously, we found that dIno80 regulates homeotic genes by interacting with epigenetic regulators, such as polycomb and trithorax, suggesting the occurrence of non-canonical Ino80 complexes. Here using spectroscopic methods and gel retardation assays, we identified a set of consensus DNA sequences that DNA binding domain of dIno80 (DBINO) interacts with having differential affinity and high specificity. Testing these sequences in reporter assays, showed that this interaction can positively regulate transcription. These results suggest that, dIno80 has a sequence preference for interaction with DNA leading to transcriptional changes.

Epigenetic regulation and transcriptional memory in development; selection facilitating prudence.

The epigenetic mechanisms regulating developmental gene expression are examples of a strategy to generate unique expression profiles with global regulators controlling several genes. In a simplified view, a common set of tools, that include DNA motif recognizing proteins (recruiters), binding/interacting surfaces (ARPs- actin related proteins), epigenetic writers (histone methyltransferases, acetylases), readers (chromatin remodeling proteins, PRC1 members) and erasers (demethylases, deacetylases) form complexes which not only regulate transcription, but also retain the transcriptional memory through mitosis. There are two arms of epigenetic regulation: covalent modification of DNA and the post-translational modification of histones. In this review, we discuss both of these aspects briefly to illustrate functional diversity. We discuss our efforts at utilization of the genome sequence data for de novo identification of new players and their functional validation in this remarkable process.

Epigenetic regulation of cytomegalovirus major immediate-early promoter activity in transgenic mice.

The expression of genes in transgenic mice is known to be influenced by the site of integration even when they carry their own promoter elements and transcription factor binding sites. The cytomegalovirus (CMV) promoter, a strong promoter often used for transgene expression in mammalian cells in culture, is known to be silenced by DNA methylation and histone deacetylation but there is no report on the role of histone methylations in its regulation. We generated two transgenic lines carrying green fluorescence protein coding gene as reporter driven by cytomegalovirus major immediate-early promoter/enhancer. We observe that silencing of CMV promoter is dependent on the site of transgene integration, except in testis, and the nature of DNA and histone methylations strongly correlate with the expression status of the reporter. We find that silenced CMV promoter interacts in vivo, with Methyl CpG binding protein 2 (MeCP2), a recruiter of histone deacetylases (HDACs) and histone (H3K9) methyl transferase. Histone H3K4methylation, the active chromatin mark, is also associated with silenced promoter, suggesting bivalent marking of the promoter and its susceptibility to reactivation on induction.

Genomic analysis of local isolate of Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease (JD or paratuberculosis) in animals and has also been implicated in Crohn's disease of humans. It has been shown that MAP is endemic in animal population of India. Understanding of heterogeneity among MAP strains is important both for diagnosis and design control measures. Genotyping and epidemiological investigations revealed that MAP 'Bison type' was the predominant strain infecting domestic ruminant population in India. MAP 'Bison type' has also been reported from USA. A number of comparative genomics studies have been conducted to understand 'Cattle type' and 'Sheep type' strains. However, present study was the first attempt to characterize MAP 'Bison type' S5 using different markers including IS900, ISMAP02, IS1311, LSPs and SSRs. Study showed that MAP S5 is similar to MAP K10 in terms of number of IS900, IS1311 and ISMAP02 elements. There was high sequence similarity for IS900 and ISMAP02 between MAP K10 and MAP S5. However, this study also reported genetic differences between two strains. In some IS1311 loci, TG gap at 64th and 65th position was observed in MAP S5. Further sequencing of few more MAP isolates confirmed that this gap was specific to indigenous MAP 'Bison type' and can be further used as molecular signature. ISMAP02 locus 1 was observed at polymorphic position in MAP S5 compared to MAP K10. MAP 'Bison type' S5 also showed polymorphic profile for LSP(P)4. Polymorphism was also observed in SSRs. This pilot study may form the basis for future epidemiological investigations.

Application of SNaPshot for analysis of thiopurine methyltransferase gene polymorphism.

BACKGROUND & OBJECTIVE: 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). METHODS: 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 confirmed by direct sequencing. RESULTS: The TPMT genotype could be reliably identified 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.

Characterization of Mce4A protein of Mycobacterium tuberculosis: role in invasion and survival.

BACKGROUND: The mce4 operon is one of the four homologues of mammalian cell entry (mce) operons of Mycobacterium tuberculosis. The mce4A (Rv3499c) gene within this operon is homologous to mce1A (Rv0169), that has a role in host cell invasion by M. tuberculosis. Our earlier reports show that mce4 operon is expressed during the stationary phase of growth of the bacillus in culture and during the course of infection in mammalian hosts. M. tuberculosis carrying mutation in mce4 operon shows growth defect and reduced survival in infected mice. However, the intracellular localization of Mce4A protein and its direct role in cell entry or survival of the bacillus has not been demonstrated so far. RESULTS: By transmission electron microscopy we have demonstrated that recombinant Mce4A protein facilitates the invasion of non-pathogenic strain of E. coli into non-phagocytic HeLa cells. We observe that mce4A gene has a role comparable to mce1A in the survival of recombinant E. coli in human macrophages. Using antibodies raised against Mce4A protein, we show that the protein is localized in the cell wall fraction of M. tuberculosis H37Rv stationary phase culture only. CONCLUSION: Mce4A protein is expressed during the stationary phase of broth culture and localizes in the cell wall fraction of M. tuberculosis. Mce4A protein expressed in non-pathogenic E. coli enables it to enter and survive within HeLa cells and the macrophages. As Mce4A protein is expressed during later phase of mycobacterial growth, our results raise the possibility of it playing a role in maintenance of persistent tubercular infection.