Pavan Umate

Genome-wide comprehensive analysis of human helicases

Helicases are motor proteins which catalyze the unwinding of duplex nucleic acids in an ATP-dependent manner. They are involved in almost all the nucleic acid transactions. In the present study, we report a comprehensive analysis of helicase gene family in human and its comparison with homologs in model organisms. Human genome encodes for 95 non-redundant helicase proteins of which 64 are RNA helicases and 31 are DNA helicases. 57 RNA helicases are validated based on annotations and occurrence of conserved helicase signature motifs. These include 14 DExH and 37 DExD subfamily members, six other members such as, U5.snRNP, ATR-X, Suv3, FANCJ, and two of superkiller viralicidic activity 2-like helicases. 31 DNA helicases are also identified, which include RecQ, MCM, and RuvB-like helicases. Finding set of helicases in human and almost similar sequences in model organisms suggests that the “core” members of helicase gene family are highly conserved throughout evolution. The present study gives an overview of members of RNA and DNA helicases encoded by the human genome along with their conserved motifs, phylogeny, and homologs in model organisms. The study on comparing these homologs will spread light on the organization and complexity of helicase gene family in model organisms. The comprehensive analysis of human helicases presented in this study will further provide an invaluable resource for elaborate biological research on these helicases. Dedicated to the memory of Professor Arturo Falaschi

Genome-wide analysis of lipoxygenase gene family in Arabidopsis and rice.

The enzymes called lipoxygenases (LOXs) can dioxygenate unsaturated fatty acids, which leads to lipoperoxidation of biological membranes. This process causes synthesis of signaling molecules and also leads to changes in cellular metabolism. LOXs are known to be involved in apoptotic (programmed cell death) pathway, and biotic and abiotic stress responses in plants. Here, the members of LOX gene family in Arabidopsis and rice are identified. The Arabidopsis and rice genomes encode 6 and 14 LOX proteins, respectively, and interestingly, with more LOX genes in rice. The rice LOXs are validated based on protein alignment studies. This is the first report wherein LOXs are identified in rice which may allow better understanding the initiation, progression and effects of apoptosis, and responses to bitoic and abiotic stresses and signaling cascades in plants

MICROPROPAGATION OF TERMINALIA BELLIRICA ROXB. – A SERICULTURE AND MEDICINAL PLANT

SummaryA protocol for micropropagation of plants via axillary bud proliferation from nodal explants of Terminalia bellirica Roxb. seedlings has been established. Explants were cultured on Murashige and Skoog medium with different concentrations of 6-benzyladenine (BA; 4.4, 8.9, 13.3, 17.8, or 22.2 μM) or kinetin (Kn; 4.6, 9.3. 14.0, 18.6, or 23.2 μM). Within the range evaluated, the medium containing 13.3 μM BA showed the highest shoot length (1.9=0.2 cm) in the primary culture. When separated and transferred to fresh subculture medium with lower levels of BA (2.2. 4.4, 6.6, or 8.9 μM) or Kn (2.3, 4.6, 6.9, or 9.3 μM), the nodal segments from individual regenerants (obtained initially from seedling nodes) showed efficient shoot induction at 4.4 μM BA. Rooting of the shoots was achieved under in vitro conditions on two media tested, i.e., modified Gamborgs (B5) medium or Woody Plant Medium, both supplemented with 4.9 μM indole-3-butyric acid. Regenerated plants were established in the greenhouse.

Comparative Genomics of the Lipid-body-membrane Proteins Oleosin, Caleosin and Steroleosin in Magnoliophyte, Lycophyte and Bryophyte

Lipid bodies store oils in the form of triacylglycerols. Oleosin, caleosin and steroleosin are unique proteins localized on the surface of lipid bodies in seed plants. This study has identified genes encoding lipid body proteins oleosin, caleosin and steroleosin in the genomes of five plants: Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Selaginella moellendorffii and Physcomitrella patens. The protein sequence alignment indicated that each oleosin protein contains a highly-conserved proline knot motif, and proline knob motif is well conserved in steroleosin proteins, while caleosin proteins possess the Dx[D/N]xDG-containing calcium-binding motifs. The identification of motifs (proline knot and knob) and conserved amino acids at active site was further supported by the sequence logos. The phylogenetic analysis revealed the presence of magnoliophyte- and bryophyte-specific subgroups. We analyzed the public microarray data for expression of oleosin, caleosin and steroleosin in Arabidopsis and rice during the vegetative and reproductive stages, or under abiotic stresses. Our results indicated that genes encoding oleosin, caleosin and steroleosin proteins were expressed predominantly in plant seeds. This work may facilitate better understanding of the members of lipid-body-membrane proteins in diverse organisms and their gene expression in model plants Arabidopsis and rice.

Genome-wide analysis of thioredoxin fold superfamily peroxiredoxins in Arabidopsis and rice

A broad range of peroxides generated in subcellular compartments, including chloroplasts, are detoxified with peroxidases which are called as peroxiredoxins (Prx). The Prx are ubiquitously distributed in all organisms including bacteria, fungi, animals, and also in cyanobacteria and plants. Recently, the Prx have emerged as new molecules in antioxidant defense in plants. Here, the members which belong to Prx gene family in Arabidopsis and rice are been identified. Overall, the Prx members constitute a small family with 10 and 11 genes in Arabidopsis and rice respectively. The prx genes of rice are assigned to their functional groups based on homology search against Arabidopsis protein database. Deciphering the Prx functions in rice will add novel information to the mechanism of antioxidant defense in plants. Further, the Prx also forms the part of redox signaling cascade. Here, the Prx family has been described for rice.

Efficient In-Vitro Regeneration from Mature Leaf Explants of Scoparia dulcis L., an Ethnomedicinal Plant

ABSTRACT A plant regeneration protocol via multiple-shoot induction using leaf explants from field-grown mature plants of Scoparia dulcis L. (Scrophulariaceae), an ethnomedicinal plant, was established. Explants were cultured on Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP, 13.2, 17.6 and 22.2 μM) in combination with indole-3-acetic acid (IAA, 0.5 and 2.8 μM) or napthalene-3-acetic acid (NAA, 0.5 and 2.6 μM). The maximum number of shoots (14.0 ± 1.14) with longest shoot length (2.97 ± 0.18) were obtained directly (without an intervening callus phase) from the leaf explants using a combination of BAP (22.2 μM) and IAA (0.5 μM). BAP used in combination with NAA produced fewer shoots per explant as compared with the use of IAA and the regeneration was mediated by callus formation. For root induction, the elongated shoots were separated and transferred onto MS medium supplemented with indole-3-butyric acid (IBA, 4.9 μM). Rooted plantlets (90%) were successfully transferred to soil in the greenhouse with a 75% survival rate.

microRNA access to the target helicases from rice.

Major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs are single-stranded RNA molecules of around 22 nucleotides in length. Most miRNAs show imperfect homology with their targets. The biogenesis mechanisms of miRNAs are different for plant and animals. Silencing of genes by miRNAs may serve as an appropriate tool to speed-up analyses of gene functions in a post-genome era. We have identified in silico a set of miRNAs that control helicase gene expression by regulating its mRNA stability and translation in rice. Our analysis revealed that several rice helicases have distinct miRNA specificities. Such analyses will be a prerequisite to refining our understanding of target selection and regulation of helicase gene expression by miRNAs in rice. Further, we discuss recent findings on miRNA gene family and its gene structure, criteria for miRNA annotation and on miRNA biogenesis that involve transcription, processing and maturation of miRNAs.

Architectures of the unique domains associated with the DEAD-box helicase motif

Helicases are motor proteins of biological system, which catalyze the opening of energetically stable duplex nucleic acids in an ATP-dependent manner and thereby are involved in almost all aspects of nucleic acid metabolism including cell cycle progression. They contain several conserved domains including the DEAD-box and also several unique domains associated with these. The Pfam database (http://pfam.janelia.org/) is a large collection of protein families, each represented by multiple sequence alignments and hidden Markov models (HMMs). A diverse range of proteins are found in nature, and the functional specificity to each protein, to a greater extent, is imparted by its domain architecture. To this extent, a DEAD-box ATP-dependent RNA helicase (LOC_Os01g36890; Genomic sequence length: 6284 nucleotides; CDS length: 1299 nucleotides; Protein length: 432 amino acids) was studied. The protein sequence was imported for domain search on Pfam. This particular Pfam entry after covering a large proportion of the sequences in the underlying database has generated a more comprehensive coverage across a wide range of phyla of the known domains that are associated with the typical DEAD-box helicase motif. A total of 362 domain architectures were recollected from the Pfam database for the Family: DEAD (PF00270). We have therefore systematically analyzed the domains closely associated with DEAD-motif, which occur in a variety of proteins and can provide insights into their function.

Subcellular localization of proteins of Oryza sativa L. in the model tobacco and tomato plants

The cellular localization and molecular interactions are indicative of functions of a protein. The development of a simple and efficient method for subcellular localization of a protein is indispensable to elucidate gene function in plants. In this study, we assessed the feasibility of Agrobacterium-mediated transformation (agroinfiltration) of tobacco and tomato leaf tissue to follow intracellular targeting of proteins from rice fused to green fluorescent protein (GFP). For this, a simple in planta assay for subcellular localization of rice proteins in the heterologous host systems of tobacco and tomato leaf via transient transformation was developed. We have tested the applicability of this method by expressing GFP fusions of the putative antiphagocytic protein 1 (APP1) (OsAPP, LOC_Os03g56930) and ZOS3-18 – C2H2 zinc-finger protein (OsZF1, LOC_Os03g55540) from Oryza sativa L. subsp. japonica in tobacco and tomato leaf tissues. Our results demonstrate the suitability of GFP as a reporter in gene expression studies in tomato cv MicroTom. The use of GFP-fused proteins from rice for subcellular targeting in the heterologous hosts of tobacco and tomato plant systems has been confirmed.

In Vitro HIV Type-1 Reverse Transcriptase Inhibitory Activity from Leaf Extracts of Scoparia dulcis L.

Aqueous and methanolic crude extracts of Scoparia dulcis L. leaves were screened for activity against human immunodeficiency virus (HIV) type-1 reverse transcriptase (RT) activity. The results were expressed as IC50. Zidovudine, which was used as a standard drug, showed an IC50 of 38.0 μg mL−1, whereas the aqueous and methanolic extracts from leaves of S. dulcis showed an IC50 of 60.0 and 47.0 μg mL−1, respectively. In this study, methanolic extract of S. dulcis leaves showed remarkable HIV type-1 reverse transcriptase inhibitory activity comparable to that of a standard such as zidovudine. Observed results using standardized parameters indicated that S. dulcis has promising HIV type-1 reverse transcriptase inhibitory activity.