Professor & Head Department of Molecular Biology And Genetic Engineering, College of Basic Sciences and Humanities, G. B. Pant University of Agriculture & Technology, PANTNAGAR 263 145 Uttarakhand India
ak_gupta2k@rediffmail.com
Dr Anil Kumar is presently working as Professor & Head in the Department of Molecular Biology & Genetic Engineering and actively involved in teaching and research since last 25 years. Based on his meritorious services for the cause of teaching and research he has been given the responsibility of Coordinator, Human Resource Development in Agricultural Biotechnology, Coordinator, Sub-DIC in Bioinformatics and Co-coordinator in Programme Mode Support in Agricultural Biotechnology funded by Department of Biotechnology, Govt. of India., New Delhi. Beside it he successfully run several competitive projects with the financial assistance of ICAR, DRDE, UPDASP, DBT, DST and others. He published more than 80 research papers in reputed national and international journals, around 50 popular articles and book chapters and seven books in his credit. Dr Anil Kumar did his Masters in Biochemistry with specialization in Microbiology with special emphasis to understand the molecular mechanisms of Bacterial sporulation from G.B. Pant University of Agriculture & Technology, Pantnagar in 1984. The preliminary study later led to the identification of multiple molecular forms of GS. He did his Ph.D. in 1990 from University of Rajasthan, Jaipur working in the field of Cancer Biology and applications of Hybridoma Technology to characterize differentiation and tumor associated antigen(s). The study concluded that dbc-AMP induced differentiation of cultured human neuroblastoma call line SK-N-SH is associated with the expression and suppression of antigenic components present in the cellular organization of neuroblastoma cells. Subsequently, he was involved in teaching as demonstrator, JRO and Lecturer in Medical Institutions till 1993 where he also carried out fragmentary work on Biochemical applications in the area of Pregnancy and infertility related disorders as well as in other healthy and diseased conditions and derived useful information based on biochemical and Immunological testing Then he was awarded the several Regional Service awards and others to participate in several International conferences, symposium and meetings held at several countries including Hongkong, Taiwan, USA, Japan, Australia and Neatherland. Further, He joined back to his almameter in Plant Biotechnology research group at Pantnagar and was involved in signal transduction research in relation to Host-pathogen interaction taking Karnal bunt-wheat and Alternaria blight-Brassica as model systems. He is engaged on three aspects of Molecular Plant Pathology i.e. Plant disease surveillance through Molecular Diagnostics, Pathogen Indexing Programme through Molecular Pathotyping and Characterization of disease resistance and Pathogenesis through Molecular signaling investigating the role of MAP kinases and Cystatin gene families as candidate genes. Dr Anil Kumar is Professor & Head of the department since 2006 and was Incharge since 2004. He visited the University of Jerusalem, Rehovot as FAO Fellow during 2000-2001. Dr Kumar also visited USA under World Bank-NATP programme during 2004-05 and worked on molecular characterization and cloning of Dof transcription factors for investigating its role in carbon and nitrogen metabolism. Based on his long experience, Dr. Anil is developing animal cell culture facilities for production of hybridoma and cell culture based assays which can be used for screening pharmaceutically important molecules and a proposal for establishment of ‘Centre of Bioprospecting’ is under consideration by the Uttarakhand Government for re-inventing the old principles of Auyrvedic biology into newer perspectives. Dr Kumar has also been awarded the ‘Shiksha Rattan’ and ‘Scientist of Excellence Award’ for his meritorious services to the cause of Biotechology education and research. Dr. Anil Kumar after assuming the charge of Prof & Head, Department of Molecular Biology & Genetic Engineering of G.B. Pant University of Agriculture & Technology, Pantnagar in 2006, is instrumental in establishing new wing of Biotechnology. He has created well defined 10 function based laboratories including one UG and PG teaching labs having state-of art facilities for carrying out research and teaching. These independent laboratories are providing facilities for carrying out research in several areas including Bioseperation Animal Cell Culture, Molecular Diagnostics, Molecular Cytogenetics, Plant Tissue Culture, Plant Stress, Biosafety is well as frontier research in area of ‘Science of Omics’: Genomics, Proteomics, Metabolomics and Bioinformatics. Dr. Kumar has tried newer innovations in enhancing the quality of teaching standards by Organizing Student’s Colloquia on various themes, providing conceptual basis in logical manner, scintillating the mind of young students by giving problem solving approaches. He has designed the B. Tech (Biotch), M.Sc./M.V.Sc. and Ph.D (Molecular Biology & Biotechnology) based on pyramidal approach having combination of basic, fundamental, specialized training with emphasis to develop quest for science the students. Beside it, he has given in the master’s curricula in Nanoscience and Nanotechnology among by the university to acts chairman for designing responsibility. Dr. Anil Kumar always insisted on sequential flow of information with emphasis on hands-on training. Accordingly, he has designed several teaching aids in the form of techniques manuals (6 No.): Cell Biology, Molecular Biology, Immunology & Serology, Animal Cell Culture, Genetic Engineering and Bioinformatics. Beside it he developed 3 books on biosafety related issues in the form of frequently asked questions. His one book on “Biotechnology” in Medicine and Agriculture: Principle and Practices” covering application in medicine, agriculture, Veterinary and environment is under publication by IK International Publishing House and soon appear in 2011 for the post-graduate students. Dr. Anil Kumar has good command on the Molecular Cell Biology and Cellular and Molecular Immunology. Due to this rare combination of knowledge, he can easily go back and forth to work in both animal and plant systems to explore newer cellular and molecular basis of signal transduction having application both in medical and agricultural sciences.
Abstract: We have studied the functions of the Trichoderma virens TmkB, a homologue of the yeast cell-wall integrity MAP kinase Slt2, using gene knockout. The functions of TmkB were compared to those of the pathogenicity MAP kinase homologue (TmkA). Like the tmkA loss-of-function mutants, tmkB mutants exhibited reduced radial growth and constitutive conidiation in dark as well as in liquid shake cultures. The tmkB mutants, in contrast to tmkA mutants, had cell-wall integrity defects, as shown by autolysis of the mycelia and increased sensitivity to cell-wall degrading enzymes. Interestingly, the tmkB mutants were not autolytic on the synthetic Vogels minimal medium. The tmkB mutants had attenuated ability to overgrow the plant pathogen Sclerotium rolfsii, while retaining the ability to overgrow Rhizoctonia solani and Pythium spp., a phenotype also exhibited by the tmkA mutants. This first functional analysis of a cell-wall integrity MAPK in Trichoderma spp., a group of economically important fungi, shows the importance of this signaling pathway in biocontrol. Common phenotypes of the TmkA and TmkB pathways suggest that the two MAPKs may share some substrates, perhaps subunits of key transcription factors, thus dependent on two phosphorylation events for their activity.
Abstract: Phytocystatins constitute a multigene family that regulates the activity of endogenous and/or exogenous cysteine proteinases. Cereal crops like wheat are continuously threatened by a multitude of pathogens, therefore cystatins offer to play a pivotal role in deciding the plant response. In order to study the need of having diverse specificities and activities of various cystatins, we conducted comparative analysis of six wheat cystatins (WCs) with twelve rice, seven barley, one sorghum and ten corn cystatin sequences employing different bioinformatics tools. The obtained results identified highly conserved signature sequences in all the cystatins considered. Several other motifs were also identified, based on which the sequences could be categorized into groups in congruence with the phylogenetic clustering. Homology modeling of WCs revealed 3D structural topology so well shared by other cystatins. Protein-protein interaction of WCs with papain supported the notion that functional diversity is a con-sequence of existing differences in amino acid residues in highly conserved as well as relatively less conserved motifs. Thus there is a significant conservation at the sequential and structural levels; however, concomitant variations maintain the functional diversity in this protein family, which constantly modulates itself to reciprocate the diversity while counteracting the cysteine proteinases.
Abstract: Two genotypes showing differential immunity against Karnal bunt (Tilletia indica) were used to investigate the role of three members of cystatin gene family in growth stage dependent immunity in wheat (Triticum aestivum L.). Three members of cystatin gene family (WC1, WC2, and WC4) were cloned and sequenced. Analysis of sequenced data showed that there was 76-99% nucleotide and protein sequence identity between different genes of the wheat cystatin. In silico amino acid sequence analysis revealed the presence of a conserved signature pattern of residues and also the functional domains were presumed to be actively involved in imparting cysteine protease inhibition capability. The semi-quantitative and quantitative levels of these members were measured by means of RT-PCR, northern blotting, western blotting, and by ELISA techniques. The members of cystatin gene family were expressed in both resistant (HD 29) and susceptible genotypes (WH 542); however, the expression level was significantly (P < 0.001) higher in resistant compared to susceptible genotype at all the stages of wheat spikes. The patterns of expression of WC2, WC4 were similar except in the levels in S(1) and S(2) stages as it remained constant (P > 0.05) in contrary to WC1 family whose expression gradually increased from S(v) to S(2) stage. According to the intensity of the detected band in RT PCR, northern blot and western blot, WC1 family seems to be expressed more than the other gene families. The immunoassay results further showed that WC1 protein was abundantly expressed in resistant genotype and high expression was observed at the S2 stage as compared to susceptible genotype (P < 0.001) suggesting that low level of expression of WC1 in S2 stage is responsible for KB infection. The results of the present study clearly indicate the role of cystatin gene family in differential and stage dependent immunity against KB.
Abstract: Seed development is a complex process controlled by temporal and spatial expression of many transcription factors (TF) inside the developing seed. In the present study, transcript profiles of all the 30 members of rice DofTFs from flowering to seed development stages were analyzed. It was found that 16 Dof genes besides a previously characterized Dof gene 'RPBF' are differentially expressed during the seed development and unlike RPBF are not seed specific. Based on the expression patterns, these rice DofTFs were categorized into four groups-6 genes were constitutive while 4 genes were up-regulated and 3 genes were down regulated and four genes were maximally expressed at specific stages of seed development viz. one gene at flowering, two genes at watery ripe and one gene at milky stage. The involvement of more than one gene at different stages of seed development is suggestive of combinatorial regulation of their downstream genes involved in seed development. In silico expression analysis of wheat and Arabidopsis Dof Tfs also revealed that more than 50% of the Dof genes are expressed during the seed development process. Further in silico study of regulatory elements present in the promoters of these genes revealed the presence of some unique and common motifs in the promoters of rice and wheat Dof genes which indicate that Dof genes are possibly involved in ethylene and jasmonate signaling pathways affecting grain filling and grain quality. These Dof genes containing ethylene responsive motifs in their promoter region could possibly be the targets of recently identified Sub1 gene which codes for a ethylene responsive factor.
Abstract: Finger millet (Eleusine coracana L.) is an important crop used for food, forage, and industrial products. Three DNA marker techniques, random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and cytochrome P(450) gene based markers were used for the detection of genetic polymorphism in 83 accessions of finger millet collected from various geographical regions of India and Africa. A total of 18 RAPD, 10 SSR and 10 pairs of cytochrome P(450) gene based markers were generated 56.17, 70.19 and 54.29% polymorphism, respectively. Mean polymorphism information content (PIC) for each of these marker systems (0.280 for RAPD, 0.89 for SSR and 0.327 for cytochrome P(450) gene based markers) suggested that SSR marker were highly effective in determining polymorphism. The phenograms based on the three markers data indicate that genotypes from different geographical regions are clearly distinguishable as separate clusters. Mantel test employed for detection of goodness of fit established cophenetic correlation values above 0.90 for all the three marker systems. The dendrograms and PCA plots derived from the binary data matrices of the three marker systems are highly concordant. High bootstrap values were obtained at major nodes of phenograms through WINBOOT software. Based on the results of present study, SSR and cytochrome P(450) gene based markers appear to be particularly useful for the estimation of genetic diversity. This study reveals the potential of RAPD, SSR and gene based markers for characterizing germplasm of Eleusine coracana and narrow down the vast germplasm into distinct core groups.
Abstract: Magnaporthe grisea, the blast fungus is one of the main pathological threats to finger millet crop worldwide. A systematic search for the blast resistance gene analogs was carried out, using functional molecular markers. Three-fourths of the recognition-dependent disease resistance genes (R-genes) identified in plants encodes nucleotide binding site (NBS) leucine-rich repeat (LRR) proteins. NBS-LRR homologs have only been isolated on a limited scale from Eleusine coracana. Genomic DNA sequences sharing homology with NBS region of resistance gene analogs were isolated and characterized from resistant genotypes of finger millet using PCR based approach with primers designed from conserved regions of NBS domain. Attempts were made to identify molecular markers linked to the resistance gene and to differentiate the resistant bulk from the susceptible bulk. A total of 9 NBS-LRR and 11 EST-SSR markers generated 75.6 and 73.5% polymorphism respectively amongst 73 finger millet genotypes. NBS-5, NBS-9, NBS-3 and EST-SSR-04 markers showed a clear polymorphism which differentiated resistant genotypes from susceptible genotypes. By comparing the banding pattern of different resistant and susceptible genotypes, five DNA amplifications of NBS and EST-SSR primers (NBS-05(504,) NBS-09(711), NBS-07(688), NBS-03(509) and EST-SSR-04(241)) were identified as markers for the blast resistance in resistant genotypes. Principal coordinate plot and UPGMA analysis formed similar groups of the genotypes and placed most of the resistant genotypes together showing a high level of genetic relatedness and the susceptible genotypes were placed in different groups on the basis of differential disease score. Our results provided a clue for the cloning of finger millet blast resistance gene analogs which not only facilitate the process of plant breeding but also molecular characterization of blast resistance gene analogs from Eleusine coracana.
Abstract: The genome of rabies virus encodes five proteins; the nucleoprotein, the phosphoprotein, the matrix protein, the glycoprotein, and the RNA-dependent RNA polymerase. Among these, the glycoprotein is the most important as it is the major contributor to pathogenicity and virus neutralizing antibody response. Keeping in mind that glycoprotein is the only protein exposed on the surface of virus and is thought to be responsible for the interaction with the cell membrane, it was attempted to target glycoprotein by a ligand polyethylene glycol 4000, which blocks its active site, as seen by molecular operating environment software, so that it may be possible to prevent the spread of virus into the host. The ligand polyethylene glycol 4000 was retrieved from Research Collaboratory for Structural Bioinformatics protein data bank by providing the glycoprotein sequence to the databank. In this study it was observed that the ligand was successfully docked on a major portion of antigenic site II of glycoprotein by mimicking the virus neutralizing antibodies. This knowledge may be important for the development of novel therapies for the treatment of rabies and other viral diseases in the future.
Abstract: Immune responses against Bm95 recombinant cattle tick antigen and its protective efficacy for control of Rhipicephalus (Boophilus) microplus ticks were determined in experimental crossbred cow calves. Anti-Bm95 antibody titers, as assessed by indirect ELISA, in immunized calves ranged from 196.1+/-13.7 on day 0 to 7979.9+/-312.5 on day 110 post-primary immunization. The rise in antibody titer was statistically significant (p<0.01) throughout the study period. Besides this, constantly higher lymphoproliferative response (LPR), as assessed by lymphocyte stimulation test, was observed from 10 days post-immunization, but a positive LPR of antigen stimulated cells in immunized animals was recorded only on day 50 and day 70 post-immunization. Following challenge of immunized calves with larvae of R. microplus, significant increase (p<0.01) in rejection percentage, mean number of damaged ticks, mean percentage of dead ticks, and decrease in engorgement weight were recorded in immunized animals. Also, there were significant differences (p<0.01) in preoviposition period, oviposition period, egg mass weight and percent hatchability between the immunized and control calves. The percent reduction in number of adult females in vaccinated calves, reduction in mean weight of egg masses, percent reduction in mean weight and reduction in fertility of engorged females collected from vaccinated calves were determined and the efficacy of Bm95 recombinant cattle tick antigen was 81.27%.
Abstract: Nitrogen is a crucial macronutrient needed in the greatest amount of all mineral elements required by plants. Development of crop varieties with high nitrogen use efficiency (NUE) is imperative for sustainable agriculture. Understanding how plant genes respond to different nitrogen conditions is essential for formulating approaches, for manipulating genes, for improving NUE. In the present study we analyzed the activity of three different enzymes involved in nitrogen assimilation viz., GS, GOGAT and GDH along with physiological parameters like chlorophyll variable yield (Fv/Fmax), photosynthesis rate and total chlorophyll content at four different growth stages of wheat plant development under different nitrogen treatments. For this study two different wheat varieties UP-2644 and Raj-4097 having high and low protein content, respectively in the grains were chosen. Gene expression profile of a Dof transcription factor (TaDof1 of wheat) was also included in the study to assess its role in nitrogen metabolism. Densitometry analysis at S(2) and S(3) stage of wheat spikes of both the wheat varieties grown at different nitrogen treatments showed that TaDof1 expression was up-regulated in low nitrogen treatment. In S(3) stage, in high protein content wheat variety UP-2644, TaDof1 expression was elevated in low and normal nitrogen treatment as compared to high nitrogen treatment. The gene expression profile of Dof 1 was found to coincide with the enzyme activities of GS, GOGAT at the S(3) stage. The activities of these enzymes were prolonged in the high protein content variety. Since, Dof transcription factor(s) have been previously reported to control the expression of genes involved nitrogen assimilation i.e., GS and GOGAT and may be the elevated expression of Dof 1 at the grain filling stage over expresses the GS and GOGAT genes thereby prolonging their activities.
Abstract: We describe the development of a user friendly tool that would assist in the retrieval of information relating to Cry genes in transgenic crops. The tool also helps in detection of transformed Cry genes from Bacillus thuringiensis present in transgenic plants by providing suitable designed primers for PCR identification of these genes. The tool designed based on relational database model enables easy retrieval of information from the database with simple user queries. The tool also enables users to access related information about Cry genes present in various databases by interacting with different sources (nucleotide sequences, protein sequence, sequence comparison tools, published literature, conserved domains, evolutionary and structural data). AVAILABILITY: http://insilicogenomics.in/Cry-btIdentifier/welcome.html.
Abstract: Possible involvement of apoptosis was investigated in pathotoxin-treated and nutritionally-depleted in vitro cultured calli by comparing levels of p53-like protein. Antibodies raised against human p53 were used to detect and quantify p53 in B. campestris. Expression of p53-like protein increased from proliferating to static growth stage and reached to constant level at decaying stage. Both ELISA and dot immuno-binding assay showed that p53-like protein was over expressed in toxin treated and nutritionally depleted calli. Almost similar changes were seen in senescent damage in Brassica species indicating involvement of p53 dependent pathways.
Abstract: Polymerase chain reaction (PCR) based RAPD profiles, in conjunction with six primers, of Karnal bunt of wheat and rice bunt exhibiting distinct polymorphic DNA. A total of 84 RAPD loci were observed on polyacrylamide gel for both Tilletia sps. Out of 84, 16 loci were found monomorphic, while other 68 loci were unique. Usefulness of random primers was also checked with other seed borne fungal pathogens of wheat and rice. None of primers gave amplification with Magnaporthe grisea, a causative agent of rice blast. However, distinct RAPD profiles were obtained with Alternaria triticina, Fusarium monaliforme, Helminthosporium sativum and Rhizoctonia solani. These six arbitrary primers could distinguish T. indica, a quarantine fungal pathogen from a non-quarantine fungal pathogen, T. barclayana. The two Tilletia sps. could be discriminated not only on the basis of distinct RAPD profiles, but also by presence of few unique gene fragments amplified using all six primers.
Abstract: Polyclonal antibodies raised against intact teliospores of T. indica in New Zealand albino rabbits were used for the development of indirect immunofluorescence tests. Specificity of anti-teliospore antibodies was evaluated by cross reactivity studies on other bunt, smut and related pathogens. The characteristic reactivity pattern indicated that the antibodies reacted with Tilletia species only. Chemical modifications, heat and enzyme treatments followed by indirect immunofluorescence tests were employed to delineate the molecular nature of the surface antigens. There was partial or no loss in immunoreactivity by methanol, periodate, heat or trypsin treatments. Extensive periodate treatment altered the fluorescence pattern due to changes in configuration of carbohydrate antigen present in episporium. Sequential treatment of periodate and trypsin showed diminished fluorescence due to access of proteolytic enzyme into inner site of episporium thereby cleaving peptide epitope(s) after reorientation of carbohydrate moietiesby periodate treatment. It indicated glycoprotein nature or peptide nature of epitopes on the teliospore surface.
Abstract: Karnal bunt of wheat, incited by a phytopathogen Tilletia indica (Syn. Neovossia indica) is a floret infecting disease. In the floral tissues fungus proliferates and produces massive amount of black spores. In smut fungi, belonging to order Ustilaginales, communication between cells is necessary to regulate growth, differentiation and monokaryotic to dikaryotic transition during pathogenic and sexual development. Neighbouring cells are able to communicate with each other by direct cell to cell contact through plasma membrane bound signaling molecules or through formation of gap junctions and alternatively through secretion of chemical signals if cells are some distance away. Current research efforts toward understanding of pathogenic and sexual development in phytopathogenic fungi, offer a number of opportunities. These include the analysis of molecular signal(s) for direct contribution of sexual interactions to ability of smut and bunt pathogens to cause disease. These efforts will provide not only to explore the mechanisms of pathogenesis, but also to enhance knowledge of basic cellular biology of an economically important group of fungi.
Abstract: Indirect enzyme linked immunosorbent assays (ELISA) were developed using polyclonal antibodies against soluble cytoplasmic (SCA) and insoluble cell wall antigens (ICWA) for monitoring modulation of mycelial antigens during growth cycle of T. indica. With SCA, continuous decrease in ELISA reactivity was observed in maturing fungus cultures, suggesting that SCA were expressed predominantly during early vegetative phase and their decreasing role was apparent as the fungus matures possibly towards sporogenous mycelium. In case of ICWA, the reaction profile showed an increase up to exponential phase of growth probably due to increase in the cell division and branching of mycelium. But later, ICWA antibody reactivity was decreased which may be due to conversion of mycelial phase to sporogenous phase, a quiescent stage of growth. Characterization of changes in antigenic configuration during developmental cycle of Tilletia indica by these antibodies could prove to be useful in identification of developmentally related and virulence marker(s).
