Rational design of DNA sequence-specific zinc fingers

We developed a rational scheme for designing DNA binding proteins. The scheme was applied for a zinc finger protein and the designed sequences were experimentally characterized with high DNA sequence specificity. Starting with the backbone of a known finger structure, we initially calculated amino acid sequences compatible with the expected structure and the secondary structures of the designed fingers were then experimentally confirmed. The DNA-binding function was added to the designed finger by reconsidering a section of the amino acid sequence and computationally selecting amino acids to have the lowest protein-DNA interaction energy for the target DNA sequences. Among the designed proteins, one had a gap between the lowest and second lowest protein-DNA interaction energies that was sufficient to give DNA sequence-specificity.     

In vitro activation of the IkappaB kinase complex by human T-cell leukemia virus type-1 Tax

Human T-cell leukemia virus type-I expresses Tax, a 40-kDa oncoprotein that activates IkappaB kinase (IKK), resulting in constitutive activation of NFkappaB. Herein, we have developed an in vitro signaling assay to analyze IKK complex activation by recombinant Tax. Using this assay in combination with reporter assays, we demonstrate that Tax-mediated activation of IKK is independent of phosphatases. We show that sustained activation of the Tax-mediated activation of the NFkappaB pathway is dependent on an intact Hsp90-IKK complex. By acetylating and thereby preventing activation of the IKK complex by the Yersinia effector YopJ, we demonstrate that Tax-mediated activation of the IKK complex requires a phosphorylation step. Our characterization of an in vitro signaling assay system for the mechanism of Tax-mediated activation of the IKK complex with a variety of mutants and inhibitors results in a working model for the biochemical mechanism of Tax-induced activation.     

<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Seed traits,fatty acid profile and genetic diversity assessment in <Emphasis Type="Italic">Pongamia pinnata</Emphasis> (L.) Pierre germplasm

Phenotypic variation of important seed traits like seed length, seed breadth, seed thickness, 100 seed weight and seed oil content were recorded in a total of 157 collected accessions of Pongamia. Out of these, fatty acid profiles of 38 accessions selected based on their high and low oil content was analyzed. Fatty acid profile revealed high variability in stearic, oleic and linoleic acid which varied from 0.42 to 10.61 %, 34.34 to 74.58 %, and 7.00 to 31.28 % respectively. Variations in palmitic and linolenic acid were small. Iodine value, saponification number and cetane number (CN) of fatty acid methyl esters (FAME) of seed oil ranges from 186.99 to 201.25, 81.13 to 108.19 and 46.16 to 56.47 respectively. Fatty acid compositions, degree of unsaturation and CN are the important parameters, which are used to determine quality of FAME were used as biodiesel. Some of the Pongamia accessions identified were higher in oil content while some accessions showed higher degree of unsaturation and a few of them had CN values higher than 55. Genetic diversity analysis with six TE-AFLP primers generated a total of 334 bands out of which 174 (52.10 %) were polymorphic. The genetic similarity ranged from 0.11 to 0.47. These findings clearly showed high level of genetic diversity and all economically desirable traits were not present in a single genotype of Pongamia. All these traits could be selected from these CPTs and transfer to a single elite variety through selection and breeding programme and could be utilized for large scale multiplication and plantation to produce high quantity and quality biodiesel in future.     

Analysis of genetic diversity and population structure in <Emphasis Type="Italic">Capsicum</Emphasis> landraces from North Eastern India using TE-AFLP markers

North eastern (NE) India harbours a precious germplasm repository of Capsicum in the form of various landraces. The present study was undertaken to characterise the extent of genetic variation present in different Capsicum landraces from north eastern India. A set of 171 Capsicum accessions were characterised using three-endonuclease amplified fragment length polymorphism (AFLP) markers. Out of 416 bands obtained from six primer combinations, 254 (61 %) were polymorphic. The pairwise genetic dissimilarity among accessions ranged from 0.03 to 0.97. Cluster analysis based on neighbour joining showed two major clusters. Cluster I contained most of the bhut jolokia accessions whereas cluster II contained all of the Capsicum annuum genotypes. Similar grouping was observed with population STRUCTURE analysis as well as principle coordinate analysis. Analysis of molecular variance (AMOVA) revealed 45 and 54 % variation among and within populations, respectively. This information on population structure analysis and molecular characterisation will be helpful for effective utilisation of this germplasm in Capsicum improvement programs.     

Co-localization of glyceraldehyde-3-phosphate dehydrogenase with ferredoxin-NADP reductase in pea leaf chloroplasts

In immunogold double-labeling of pea leaf thin sections with antibodies raised against ferredoxin-NADP reductase (EC 1.18.1.2, FNR) and antibodies directed against the A or B subunits of the NADP-linked glyceraldehyde-3-P dehydrogenase (GAPD) (EC 1.2.1.13), many small and large gold particles were found together over the chloroplasts. Nearest neighbor analysis of the distribution of the gold particles indicates that FNR and the NADP-linked GAPD are co-localized, in situ. This suggests that FNR might carry FADH2 or NADPH from the thylakoid membrane to GAPD, or that ferredoxin might carry electrons to FNR co-localized with GAPD in the stroma. Crystal structures of the spinach enzymes are available. When they are docked computationally, the proteins appear, as modeled, to be able to form at least two different complexes. One involves a single GAPD monomer and an FNR monomer (or dimer). The amino acid residues located at the putative interface are highly conserved on the chloroplastic forms of both enzymes. The other potential complex involves the GAPD A2B2 tetramer and an FNR monomer (or dimer). The interface residues are conserved in this model as well. Ferredoxin is able to interact with FNR in either complex.     

Comparative in-vitro biodegradation studies of epoxy and its silicone blend by selected microbial consortia

A total of six bacterial isolates were developed into two consortia and tested for utilization of epoxy silicone blends (ESBs; % w/w: 3.0) and epoxy as the sole carbon source. In-vitro biodegradation studies in minimal broth revealed that higher biomass and more sustained growth of consortia were obtained in the presence of epoxy and/or ESBs when these were incubated under aerobic conditions for 15 days. Treated samples were analyzed by Fourier transform infrared spectroscopy (FTIR) and simultaneous thermogravimetric–differential thermogravimetry–differential thermal analysis (TG–DTG–DTA), which indicated the breakage and formation of bonds in the polymer backbone. Moreover, a weight loss of 34.17 and 36.9% was found in epoxy and ESBs, respectively after 15 days of treatment with consortium-1. Further, in-vitro growth statistics study revealed more CFU count at mid-logarithmic phase in the presence of epoxy/ESBs unlikely to the absence of the polymers. However, the generation time was not affected. In the present study, consortium-1, comprising of Microbacterium sp., Pseudomonas putida and Bacterium Te 68R showed better biodegradation in comparison to consortium-2, wherein, P. putida and Pseudomonas aeruginosa were present. Overall, these results suggest that epoxy/ESBs polymers could be degraded by a biologically mediated process if a suitable consortium is used.     

Optimization of extraction and purification of glucoamylase produced by Aspergillus awamori in solid-state fermentation

Various parameters such as solvent selection, concentration, soaking time, and temperature were tested in a single bioreactor in order to determine optimum extraction conditions of glucoamylase, when produced simultaneously with protease by Aspergillus awamari nakazawa MTCC 6652. Optimum conditions were achieved in a 10% glycerol solution soaked for 2 h at 40°C, followed by concentration of extracted glucoamylase (9,157 U/gds) by acetone precipitation (1:2, v/v), which yielded 51.9% recovery. Ion exchange chromatography and gel filtration showed specific activities of 270.5 and 337.5 U/mg, respectively, while SDS-PAGE and zymogram analysis of glucoamylase indicated the presence of three starch-hydrolyzing isoforms with molecular weights of approximately 109.6, 87.1, and 59.4 kDa, respectively