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M. Sc. Dissertations

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2012 - 20137
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Subject: Bioinformatics × Type: Thesis × Thesis Type: M.Sc ×
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Now showing 1 - 7 of 7
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    ThesisItemOpen Access
    Subunit-subunit interactions in the heterotetrameric structure of rice (Oryza sativa) ADP-Glucose Pyrophosphorylase
    (CCSHAU, 2012) Dawar, Chhavi; Jain, Sunita
    ADP-glucose pyrophosphorylase, a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of large (LS) and small subunits (SS). Current evidence indicates that the two subunits play distinct roles in enzyme function. The LS is involved in mainly allosteric regulation through its interaction with the catalytic SS. Recently the crystal structure of the SS homotetramer from potato tuber has been solved, but no crystal structure of the native heterotetrameric enzyme is currently available for any species. In this study, homology modeling of the three-dimensional structure of the LS and SS from rice (Oryza sativa) was done using Swiss Model Server to construct the heterotetrameric assembly of the enzyme. The SS from the crystal structure of potato tuber was taken as the template. The models were evaluated using PROCHECK, VERIFY3D and ERRAT from SAVES. Further the two subunits were docked using GRAMM-X rigid docking server first to obtain the stable heterodimer orientation (LS as receptor and SS as ligand) and then the heterotetrameric orientation. The initial heterotetrameric orientation was further refined using RosettaDock Server and idealization of bond geometry and removal of unfavorable non-bonded contacts was performed by energy minimization with force field GROMOS96 from Swiss- Pdb Viewer. A minimized energy state of -69853 KJ/mol for the heterotetramer was obtained. MD simulation of the representative heterodimer and heterotetramer was performed using NAMD on an 8-node Linux cluster operating at 0.1 TFLOPS. The studies indicated that the tail-to-tail interaction of the LS and SS was more stable with -437086 kcal/mol energy as compared to the head-to-head orientation (-396097 kcal/mol) also the heterotetramer energy was minimized to -767011 kcal/mol. Following this the subunit-subunit interaction studies were carried out on the heterotetramer using NACCESS and Dimplot programs. NACCESS indicated interface residues; 57 residues for SS out of which 30 were hydrophobic and 27 were hydrophilic and 63 residues (31 hydrophobic and 32 hydrophilic residues) for LS. Dimplot plotted the hydrophobic interactions and hydrogen bonds between different chains of the heterotetramer. Lastly superimposition of the deduced heterotetramer on the template homotetramer (1YP2) was done and found to be quite similar as evident by 0.822Å RMSD.
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    ThesisItemOpen Access
    Multiple Sequence Alignment: Comparison and Evaluation
    (2012) Ritu; Jain, Sunita
    The present investigation was undertaken to compare and evaluate eleven tools of Multiple Sequence Alignment, namely Bali-phy, ClustalW, DCA, Dialign, Kalign, MAFFT, Multalin, MUSCLE, PRALINE, PROBCONS and T-COFFEE and to present a choice to the users of MSA regarding the optimal program. Nineteen sequences, each of cytochrome C and hexokinase were aligned by these tools using their default parameters. On comparing the various features of MSA tools, it was found that linux was the most favoured operating system and Fasta was the most favoured input as well as output format. PRALINE was the only web-based and Bali-phy was the only stand-alone tool, while the rest of the tools were both stand-alone as well as web-based. Various MSA programs were based on different alignment algorithms and scoring matrices. Alignments produced were used to generate phylogenetic trees. Comparison of these phylogenetic trees with the reference phylogenetic tree i.e. NCBI Taxonomy Common Tree, showed that while the tree obtained from the T-COFFEE alignment tool was nearest to the NCBI Taxonomy Common Tree, phylogenetic trees produced by none of the MSA tools were consistent. Multalin tool was found to give the highest diversity of position scores on Scorecons server for both cytochrome C and hexokinase sequences; while TCOFFEE, MUSCLE, Kalign and MAFFT programs gave low scores. The benchmark database, BAliBASE was used in the current study for cytochrome C sequences. It was found that MSA programs Praline, MAFFT, MUSCLE and PROBCONS gave high SP scores for both divergent sequences and sub-families, while T-COFFEE gave low score. Inconsistent phylogenetic trees and discrepancy between Scorecons and BAliBASE scores from various MSA tools may be attributed to the use of highly divergent sequences from bacteria to human in the current study.
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    ThesisItemOpen Access
    Prediction of codon bias in rice (Oryza sativa) genes
    (CCSHAU, 2012) Savita; Jain, Sunita
    Patterns and degrees of codon usage bias vary not only among different organisms, but also among genes in the same organism. Codon usage bias (CUB) conveys useful information about the selection on synonymous codons induced by gene expression, translation efficiency and about the distinctive evolutionary properties. In the present study, by using 28502 complete coding sequences (CDS) of rice (Oryza sativa) with full length cDNA support, a detailed analysis of the codon bias was performed by employing Automated Codon Usage Analysis (ACUA) tool. 27897 genes demonstrated codon bias as their ENc (effective number of codons) values varied from 20 to less than 61. On basis of GC3 content values, 267 biased genes were taken as high GC 3(GC3>=0.8) whereas 27630 biased genes were termed as low GC3(GC3 <0.8) genes. Quantification of genomic compositional asymmetry and dinucleotide frequency distribution analysis were further performed on selected biased genes to provide possible reasons of differences in nucleotide composition and compositional gradients along the coding regions of all the genes. GC3 gradient analysis results revealed that from the 5' end to 3' end of the open reading frame, high GC3 biased genes had a very slight positive gradient and low GC 3biased genes had strong negative gradient. CG3 skewness analysis showed that C3 preference even though higher for high GC 3genes than low GC 3genes, varied differentially as a function of distance from ATG codon. The analysis for CG3- and GC3- skewness as a function of distance from the startcodon in rice genes clearly depicted the prevailing tendency for GC 3 to decrease toward the 3' end. By dinucleotide frequencies ratio distribution analysis, 45/267 high GC 3 biased genes were detected at CG/GC dinucleotide frequencies ratio peak centered at 1.1, whereas 1729/27630 and 1762/27630 low GC3 genes with CG/GC dinucleotide frequencies ratio peaks centered at 0.5 and 1.1, respectively were observed that clearly depicted that high GC 3 genes had a significantly higher frequency of CG dinucleotides than low GC 3genes. The differential pattern of GC 3bias in rice genes shown in our study corroborate with the previous studies that plant codon usage might be affected by translational selection.
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    ThesisItemOpen Access
    Identification of microRNA in rice (Oryza sativa)
    (CCSHAU, 2012) Mamta Rani; Sudhir Kumar
    Th e pr e s e n t i n ve s t i g a t i on h a s be e n un d er t a k en t o i d e n t i f y m i c r oRNA i n Or y z a s a t i v a u s i n g c om p u t a t i on a l a p pr oa c h . Th e m i RNA p l a y s i g n i f i c a n t r ol e i n pl a n t bi ol og y a s n e g a t i ve r e g u l a t or of g e n e e x p r e s s i on a n d h a ve b e e n s h own t o r e g u l a t e d i ve r s e fu n c t i on s i n c l u di n g t r a n s c r i pt i on , c a t a l ys i s , t r a n s p or t er a c t i vi t y, s t r e s s r e s p on s e , p r ot e i n d e g r a d a t i on , n ut r i e n t m e t a bol i sm , r oot g r owt h , or g a n d e ve l op m e n t s u c h a s l e a f m or ph og e n e s i s . In p l a n t s , m os t o f t h e mi RNA bi n d t o c od i n g s e q u e n c e or op e n r e a di n g f r a m e of t h e i r m RNA t a r g e t s wi t h p er fe c t or n e a r l y p e r fe c t c om p l em e n t a r i t i e s a n d i n d u c e s t a r g e t m RNA c l e a va g e . I n t h e p r e s e n t s t u d y, 1 2 9 m i RNAs we r e i d e n t i f i e d i n r i c e g e n om e b y i d e n t i f yi n g p a l i n dr om e s e q u e n c e s a n d pr e d i c t i on of s e c on d a r y s t r u c t ur e b y R N A f o l d , t h en f i l t e r ou t t h e s e q u e n c e s wh os e MF E s e c on d a r y s t r u c t ur e wa s l e s s t h a n - 4 0 k c a l a n d f i n a l l y a l l r e p or t e d m i RNAs we r e ve r i f i e d a g a i n s t kn own s e q u e n c e s o f m i RNAs i n m i RBa s e d a t a ba s e . T wo n ew m i RN A ( Os a - MI R8 1 2 m , Os a - MI R8 1 2 o) we r e r e p or t e d on t h i r d a n d s e v e n t h c h r om os om e . Nu m b e r s o f m i RNA r e p or t e d we r e d i f f e r e n t on d i f fe r e n t ch r om o s om e s ; h i gh er m i RNA ( 1 7 ) we r e r e p or t e d on s i xt h c h r om os om e wh i l e on l y t h r e e m i RNA we r e i d e n t i f i e d on 1 2 t h c h r om os om e . Th e m os t a bu n d a n t l y e x p r e s s e d m i RNA fa m i l i e s i n r i c e s e e d l i n g s a r e MI R1 6 9 a n d MI R1 5 6 on c h r om os om e 2 n d a n d 4 t h . S om e m i RNA ( Os a - MI R4 2 6 , Os a - MI R4 1 7 , Os a MI R4 2 0 Os a - MI R4 1 6 ) we r e f ou n d t o be c on s e r v e d be t we e n r i c e a n d A r a bi d o p s i s wh i l e Os a MI R4 4 4 e , Os a - MI R1 4 2 7 , Os a - MI R1 4 3 0 , Os a - MI R1 4 3 7 , Os a - MI R1 6 7 , Os a - MI R1 4 4 0 we r e f ou n d t o be a bs e n t i n A r a b i d o p s i s bu t pr e s e n t i n c l os e l y r e l a t e d m on oc ot s . T h e mi RNA Os a MI R3 9 9 d , Os a - MI R2 1 2 3 a we r e r e p or t e d on f i r s t a n d t h i r d c h r om os om e a n d i n vol v e d i n s t r e s s r e s p on s e s . Ot h e r m i RNA l i k e Os a - MI R8 1 2 e , Os a - MI R2 8 7 3 , Os a - MI R5 4 9 1 , Os a MI R5 5 3 4 b, Os a - MI R8 1 2 g , Os a - MI R8 1 9 k a n d Os a - MI R2 1 0 2 i d e n t i f i e d on f i r s t , t h i r d a n d n i n t h ch r om os om e a n d h a ve r e g u l a t or y r ol e i n p l a n t pos t em br yog e n i c d e ve l op m e n t , p ol l en a n d s e e d d e ve l op m e n t , d i f f e r e n t r ol e s i n s u p er i or a n d i n fe r i or s p i k e l e t s of r i c e . C on s e r ve d m i RNAs - t a r g e t pa i r s we r e f ou n d t o be a s s oc i a t e d wi t h d e ve l op m e n t a l pr oc e s s e s a n d t r a n s c r i p t i on a l r e g u l a t i on , wh i l e n on - c on s e r ve d p a i r s we r e i n vol ve d i n s i gn a l t r a n s d u c t i on a n d r e s p on s e t o s t r e s s pr oc e s s e s . A n on - s i gn i f i c a n t c or r e l a t i on ( 0. 2 7 ) h a s be e n fou n d be t we e n g e n e s a n d m i RNAs p r e d i c t e d i n r i c e g e n om e .
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    ThesisItemOpen Access
    Virtual high-throughput screening of peroxisome proliferator-activated receptor Inhibitors
    (CCSHAU, 2012) Sharma, Drishta; Sudhir Kumar
    Peroxisome proliferator-activated receptors are ligand activated transcription factors that belong to the nuclear receptor super family and are involved in control of energy, lipid and glucose homeostasis. There are three different isotypes of the receptor namely, PPARα, PPARβ/δ and PPARγ. The moderate (or partial) levels of PPAR-γ activation coordinate an evolutionary beneficial and adaptive response. Current evidence shows that there are natural PPAR activators, which brings this tightly regulated system out of balance, and contributes to the pathogenesis of life-style associated diseases, such as obesity, type2 diabetes, and atherosclerosis. This also indicates that inhibiting PPAR activity, rather than activating, might be the preferred therapeutic strategy to treat the above disorders. In this study, virtual high throughput screening of PPAR inhibitors was performed using UCSF DOCK 6.0. The three sets of databases were prepared namely, NCI database, ZIN C (target-wise) database and ZINC (lead-like) database. PPARγ was selected as the receptor as the three isotypes were 80% similar. The ouput of the first run (NCI database) contained 1516 compounds. The output of second run (ZINC target-wise) contained 20 compounds. The output of third run (ZINC lead-like) contained 103 compounds. Scoring was done for each compound by summing the energies calculated by DOCK. The cluster analysis was done using Ches-Mapper. In the first run, NCI dataset was used, in which 1516 compounds were taken and resulted in the identification of 8 ligands. Out of 20 compounds obtained from virtual screening of ZINC (target-wise), two of them were identified as the ligands. In the third run, ZINC (lead-like) database was used in which 7 ligands were obtained. Lastly, the obtained hits were compared with the known inhibitors and it was found that the groups present in the inhibitors are similar to the groups present in the hits.
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    ThesisItemOpen Access
    Modelling and comparison of in silico mutagenesis generated ADP-glucose pyrophosphorylase large subunits in rice (Oryza sativa L.) with Rev6 mutant of maize (Zea mays L.)
    (CCSHAU, 2013) Vicky; Jain, Sunita
    ADP – Glucose Phosphorylase (AGPase) is a key enzyme for starch synthesis in plant kingdom. It is a heterotetrameric enzyme consisting of two large subunits (LS) encoded by Shrunken-2 (Sh2) and two small subunit by Brittle-2 (Bt2). Manipulation of AGPase is a prime target to increase starch production and in turns crop yield as observed in maize where Rev6 mutated structure of AGPase resulted in increase in starch content & kernal weight. In spite of 93% identity in amino acid sequences of AGPase LS in maize and rice, the crop yield increase was not observed in rice with same insertion as in Rev6 mutant of maize. In this study, in silico mutagenesis in normal LS sequence of AGPase in rice was carried out using Mutation Pressure Simulator. The mutation in Sh2 gene (LS) involved substitution mutations in sequence SDHPEE and insertion of 6 nucleotides in Sh2 gene, thereby insertion of two amino acid residues Ser and Tyr at specific position. Then, the homology modeling of the normal and 9 mutated LS from rice was done using MODELLER9.11. The models were refined for energy minimization using GROMOS force field and verified with various structure refinement programs at WHATIF Server. The models were further evaluated using PROCHECK, VERIFY3D and ERRAT from SAVES. All the models were visualized in UCSF Chimera and in normal LS of rice AGPase, secondary structure accounted by six amino acids (SDHPEE) sequence just before the site of insertion of additional amino acid (S,Y) was observed. This secondary structure was also present in S490T, D491N and D491E mutants but it got disappeared similar to Rev6 mutant of maize in S490A, P493A, E494Q, E495D and E495Q mutants. These mutated models of rice were compared with normal LS of rice and Rev6 mutant of maize using Superpose Version1.0. The RMSD was found to be < 2 Å for all mutants with respect to normal LS whereas only P493A had RMSD 1.76Å (below 2Å) with respect to Rev6 mutant. The surface topography analysis using CASTp Server showed that the number of binding pockets increased (81 -90) in all mutants as compared to normal LS (69) except P493A in which it reduced to 65. Lastly, the surface topology of the rice mutants was compared with Rev6 mutant to find the most similar mutant. The only rice mutant that displayed almost similar changes in surface topology like Rev6 mutant of maize was observed to be P493A. As P493A mutant of rice was found to be the most similar to Rev6 mutant as evident by 1.76Å RMSD and quite similar structural and topology differences so it may be expected to behave in rice similar to the Rev6 mutant of maize.
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    ThesisItemOpen Access
    Modeling and structural comparison of betaine aldehyde dehydrogenase 2 of fragrant and non-fragrant soybean (Glycine max) and rice (Oryza sativa L.)
    (CCSHAU, 2013) Shilpi; Sudhir Kumar
    Betaine aldehyde dehydrogenase2 (BADH2) is an important protein involved in presence of the fragrance in soybean and rice. It was predicted that loss of function of BADH2 leads to the fragrance. In rice 8bp deletion leads to the premature stop codon and hence non-functioning truncated BADH2 protein but in soybean a change in amino acid glycine 334 with aspartate334 leads to the fragrance without any shortening of protein. In this study, homology modeling of the BADH2 protein of fragrant and non-fragrant soybean (Glycine max) and rice (Oryza sativa) was done using Swiss Model Server and Modeller. The crystal structure of BADH2 of Pisum sativum and of Zea mays were taken as the template for soybean and rice, respectively. On verification by Anolea, QMEAN and GROMOS, the models generated by Swiss Model Server were selected. The selected models were evaluated using PROCHECK, VERIFY3D and ERRAT from SAVES. On comparison of fragrant and non-fragrant soybean BADH2, no structural differences were observed (RMSD 0.016). However, nonfragrant soybean BADH2 differed from non-fragrant rice only at three places viz. E260, G472 and E473 (RMSD 0.588). The BADH2 variant of fragrant rice superimposed on non-fragrant rice BADH2from 1-266 position only (RMSD 0.318). The surface topologies of the proteins were analyzed using CASTp server. A total of 65 pockets were observed in soybean and 66 and 35 in non-fragrant and fragrant riceBADH2 protein, respectively. Two binding pockets (7 and 32) were in contact with each other in non-fragrant soybean BADH2 while in fragrant soybean a single pocket was present at the corresponding position with no matching increase in area and volume.P335, however was a part of non-fragrant soybean (pocket 7) but not of fragrant soybean BADH2.Further, the refinement of results was done by active site prediction tool of SCFBio and interestingly K319 near the conserved region EGCRLD/GPIVS showed conformation change in fragrant soybean. Apart from this S255, L256, L258, S262of another highly conserved region also showed conformation change in two soybean BADH2 variants. Position variation of R296, K460 and R461 in a nearby side chains were also observed.