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S K Sopory

Publications and source records attributed to S K Sopory.

At least 37 records · Page 2Linked to original sources
PubMed2002

Directional genome walking using PCR.

We describe here a PCR-based "directional genome walking" protocol. The basic procedure for the amplification consists of two rounds of PCR. A primary PCR was performed, on the genomic DNA using a biotinylated primer specific to a known sequence in the genome along with four universal walker primers that were designed with partial degeneracy. The biotinylated primary PCR products were immobilized on streptavidin-linked paramagnetic beads. This step removed all nonspecific amplification products, and the purified template was used for the second PCR using a nested primer and the walker primer-2 to increase specificity. This technique is potentially useful for cloning promoter regions and has been successfully used to isolate 5'-flanking genomic regions of many cDNA clones previously isolated by us.

Base Sequence↗
PubMed2001

Cloning and expression of a nuclear encoded plastid specific 33 kDa ribonucleoprotein gene (33RNP) from pea that is light stimulated.

We report the cloning and sequencing of both cDNA and genomic DNA of a 33 kDa chloroplast ribonucleoprotein (33RNP) from pea. The analysis of the predicted amino acid sequence of the cDNA clone revealed that the encoded protein contains two RNA binding domains, including the conserved consensus ribonucleoprotein sequences CS-RNP1 and CS-RNP2, on the C-terminus half and the presence of a putative transit peptide sequence in the N-terminus region. The phylogenetic and multiple sequence alignment analysis of pea chloroplast RNP along with RNPs reported from the other plant sources revealed that the pea 33RNP is very closely related to Nicotiana sylvestris 31RNP and 28RNP and also to 31RNP and 28RNP of Arabidopsis and spinach, respectively. The pea 33RNP was expressed in Escherichia coli and purified to homogeneity. The in vitro import of precursor protein into chloroplasts confirmed that the N-terminus putative transit peptide is a bona fide transit peptide and 33RNP is localized in the chloroplast. The nucleic acid-binding properties of the recombinant protein, as revealed by South-Western analysis, showed that 33RNP has higher binding affinity for poly (U) and oligo dT than for ssDNA and dsDNA. The steady state transcript level was higher in leaves than in roots and the expression of this gene is light stimulated. Sequence analysis of the genomic clone revealed that the gene contains four exons and three introns. We have also isolated and analyzed the 5' flanking region of the pea 33RNP gene.

Amino Acid Sequence↗
PubMed2001

Cytoplasmic kinase and phosphatase activities can induce PsaF gene expression in the absence of functional plastids: evidence that phosphorylation/dephosphorylation events are involved in interorganellar crosstalk.

PsaF is a nuclear gene for subunit III of the reaction center of photosystem I, and its expression is stimulated by cytokinins and light, when monitored at the mRNA level or at the level of GUS activity directed by chimeric promoter::uidA gene fusions in transgenic tobacco. These inductive effects can be mimicked by pertussis toxin, serotonin, phorbol acetate myristate or Ca2+, suggesting the involvement of heterotrimeric G proteins, phospholipids and Ca2+-dependent processes. Both breakdown products of the phosphatidylinositol cycle, inositol triphosphate (IP3) and diacylglycerol (or its homolog phorbol myristate acetate, PMA) appear to be involved. The IP3-dependent pathway requires kinase activity, and the signal operates via a 42-bp Ca2+-responsive element located between positions -220 and -178, while the PMA-dependent pathway requires phosphatase activity and a binding element that lies further upstream in the promoter. The effects of G proteins, phospholipids and Ca2+ on GUS gene expression are restricted to tissues with functional plastids, while modulation of phosphatase and kinase activities activates the responsive PsaF promoter regions even in photobleached material. Thus, activation of kinases and phosphatases can bypass the plastid-mediated inhibition of PsaF gene expression in tobacco seedlings. One cytoplasmic target which reflects the functional state of the plastids is protein kinase C. The enzyme can be efficiently phosphorylated in protein extracts from seedlings in which plastid function is impaired, but not in extracts from green tissue.

Calcium↗
PubMed2001

Zea mays CCaMK: autophosphorylation-dependent substrate phosphorylation and down-regulation by red light.

The role of protein kinases has been extensively studied in various signal transduction pathways and they are one of the most important components that link the signal perception to the final response. However, not many studies have been reported, especially from the plant systems, that show the regulation pattern of the kinase itself under different conditions. A calcium/calmodulin-dependent protein kinase has already been purified and characterized from etiolated maize coleoptiles. In this paper a detailed study of how the kinase itself is regulated at the autophosphorylation level is provided. Evidence is also given that the autophosphorylation of kinase effects its activity towards substrate phosphorylation. It is further shown that the kinase is an important component of the light signalling pathway as the level of the kinase itself decreases by red light irradiation.

Amino Acids↗
PubMed2000

A novel protein kinase from Brassica juncea stimulated by a protozoan calcium binding protein. Purification and partial characterization.

A novel protein kinase (BjCCaBPk) from etiolated Brassica juncea seedlings has been purified and partially characterized. The purified enzyme migrated on SDS/PAGE as a single band with an apparent molecular mass of 43 kDa. The optimum pH for the kinase activity was 8.0. It was stimulated more than sixfold by the protozoa Entamoeba histolytica calcium binding protein EhCaBP (10.5 nM) but not by calmodulin (CaM) when used at equimolar concentration. Moreover the kinase also did not bind CaM-Sepharose. There was neither inhibition of the kinase activity in the presence of W-7 (a CaM antagonist), KN-62 (a specific calcium/CaM kinase inhibitor) and anti-CaM Ig, nor any effect on BjCCaBPk activity of staurosporine (a protein kinase C inhibitor). Furthermore a CaM-kinase specific substrate, syntide-2, proved to be a poor substrate for the BjCCaBPk compared with histone III-S. The phosphorylation of histone III-S involved serine residues. Southern and Northern blot analysis showed the presence of EhCaBP homologues in Brassica. The data suggest that BjCCaBPk may be a novel protein kinase with an affinity towards a calcium binding protein like EhCaBP.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1999

Glyoxalase I from Brassica juncea is a calmodulin stimulated protein.

Brassica juncea glyoxalase I (S-lactoylglutathione-lyase, EC 4.4.1. 5) is a 56 kDa, heterodimeric protein. It requires magnesium (Mg2+) for its optimal activity. In this report we provide biochemical evidence for modulation of glyoxalase I activity by calcium/calmodulin (Ca2+/CaM). In the presence of Ca2+ glyoxalase I showed a significant (2.6-fold) increase in its activity. It also showed a Ca2+ dependent mobility shift on denaturing gels. Its Ca2+ binding was confirmed by Chelex-100 assay and gel overlays using 45CaCl2. Glyoxalase I was activated by over 7-fold in the presence of Ca2+ (25 microM) and CaM (145 nM) and this stimulation was blocked by the CaM antibodies and a CaM inhibitor, trifluroperazine (150 microM). Glyoxalase I binds to a CaM-Sepharose column and was eluted by EGTA. The eluted protein fractions also showed stimulation by CaM. The stimulation of glyoxalase I activity by CaM was maximum in the presence of Mg2+ and Ca2+; however, magnesium alone also showed glyoxalase I activation by CaM.

Brassica↗
PubMed1999

Light regulation of nitrate reductase gene expression in maize involves a G-protein.

This paper reports three lines of evidence to demonstrate the presence of heterotrimeric G-proteins in maize and their involvement in the regulation of nitrate reductase gene expression by light: (1) Southern blot analysis of maize genomic DNA using a human Ha-ras cDNA probe revealed specific bands indicating the presence of G-protein (alpha subunit) gene(s) in maize. Northern blot analysis of maize total RNA using the same probe revealed that the putative Galpha gene(s) is transcriptionally active. (2) Western blots containing purified plasma membrane proteins from maize leaves showed specific binding of gamma [35S]-labeled GTP in a red light-dependent manner, indicating the involvement of G-proteins in mediating the light signal. The size of the putative Galpha gene product (approximately 45 kDa) indicates that it may be a heterotrimeric G-protein. (3) Cholera toxin mimicked the effect of red light to enhance the transcript levels of nitrate reductase (NR), indicating that G-proteins may mediate light regulation of NR gene expression.

Blotting, Northern↗
PubMed1999

Cloning and characterization of a cDNA encoding topoisomerase II in pea and analysis of its expression in relation to cell proliferation.

We have isolated and sequenced four overlapping cDNA clones to identify the full-length cDNA for topoisomerase II (PsTopII) from pea. Using degenerate primers, based on the conserved amino acid sequences of other eukaryotic type II topoisomerases, a 680 bp fragment was PCR-amplified with pea cDNA as template. This fragment was used as a probe to screen an oligo-dT-primed pea cDNA library. A partial cDNA clone was isolated that was truncated at the 3' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of PsTopII is 4639 bp with an open reading frame of 4392 bp. The deduced amino acid sequence shows a strong homology to other eukaryotic topoisomerase II (topo II) at the N-terminus end. The topo II transcript was abundant in proliferative tissues. We also show that the level of topo II transcripts could be stimulated by exogenous application of growth factors that induced proliferation in vitro cultures. Light irradiation to etiolated tissue strongly stimulated the expression of topo II. These results suggest that topo II gene expression is up-regulated in response to light and hormones and correlates with cell proliferation. Besides, we have also isolated and analysed the 5'-flanking region of the pea TopII gene. This is first report on the isolation of a putative promoter for topoisomerase II from plants.

3' Untranslated Regions↗
PubMed1999

Glyoxalase I from Brassica juncea: molecular cloning, regulation and its over-expression confer tolerance in transgenic tobacco under stress.

Despite its ubiquitous presence, the role of glyoxalase I has not been well investigated in plants. In order to find out its physiological functions, we have cloned and characterized a cDNA from Brassica juncea encoding glyoxalase I (Gly I) and made transgenic tobacco plants harbouring Gly I in both sense and antisense orientation. The transgenic nature of the plants was confirmed by Southern blotting, and the estimated number of genes inserted ranged from one to six. The transcript and protein levels of glyoxalase I were also monitored in transgenic plants. The expression of glyoxalase I in B. juncea was upregulated in response to salt, water and heavy metal stresses. In response to a high concentration of salt, the transcript level averaged threefold higher in 72 h, and an increase in the protein was also seen by immunoblotting. The transgenic plants over-expressing glyoxalase I showed significant tolerance to methylglyoxal and high salt, as tested in detached leaf disc senescence assay. A comparison of plants expressing high and low levels of glyoxalase I showed that the tolerance to different salt concentrations was correlated with the degree of glyoxalase I expression. Our results suggest an important role of glyoxalase I in conferring tolerance to plants under stress conditions.

Adaptation, Physiological↗
PubMed1999

Roles of nitrate, nitrite and ammonium ion in phytochrome regulation of nitrate reductase gene expression in maize.

The influence of nitrate and its metabolites on the nitrate reductase (NR) gene expression and its relationship with phytochrome (Pfr) regulation of NR in etiolated maize leaves is examined. Nitrate induction and Pfr stimulation are brought about by independent signalling phenomena. Phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), mimicked the effect of red light but could not replace the nitrate requirement for the induction of NR transcript accumulation. This suggests that while PKC-type enzymes may be involved in mediating the Pfr signal, nitrate may follow an independent signalling mechanism. Experiments with 5-hydroxytryptamine (5HT) and lithium ions (Li+), which are known to modulate phosphoinositide (PI) turnover, indicated that in addition to generating Pfr-induced second messengers for PKC activation, PI cycle may also generate other signals which mediate nitrate induction of NR gene expression in the dark. The products of nitrate reduction i.e., nitrite and ammonium ion had inhibitory and stimulatory effects respectively, on NR transcript accumulation. They work mainly at the level of nitrate induction.

Gene Expression Regulation, Enzymologic↗
PubMed1999

Calnexin from Pisum sativum: cloning of the cDNA and characterization of the encoded protein.

A full-length cDNA of 1951 bp encoding a calnexin (CNX) protein was cloned from a Pisum sativum expression library. The open reading frame (ORF) within this cDNA encodes a 551-amino acid protein with a calculated molecular mass of 62.47 kDa that exhibits extensive homology with the CNX proteins from soybean (80%), Arabidopsis thaliana (70%), maize (70%), and dog (39%). The characteristic CNX signature motifs, KPEDWDE and GXW, generally found in molecular chaperones, are present in pea CNX (PsCNX), along with putative sites for Ca2+ binding and phosphorylation. In PsCNX, a signal sequence and a single transmembrane domain are also present at the N- and C-terminal ends, respectively. The PsCNX protein is expressed constitutively at the RNA level in vegetative and flowering tissues, as was evident from Northern analysis. Expression of PsCNX was light independent. In vitro translation of PsCNX cDNA yielded a 75-kDa precursor, which, in the presence of canine microsomal membranes, was cotranslationally processed into a 72.5-kDa product and was imported and localized to the endoplasmic reticulum. Trypsin treatment of the in vitro translated PsCNX in the presence of canine microsomes generated a further processed 67-kDa intraluminal form. The results with PsCNX also showed that the plant protein is a phosphoprotein containing phosphoserine residues, as evidenced by immunoprecipitation of PsCNX with anti-phosphoserine antibody. The PsCNX protein was also phosphorylated by endogenous kinases of pea microsomes.

Amino Acid Sequence↗
PubMed1998

Biochemical evidence for a calmodulin-stimulated calcium-dependent protein kinase in maize.

We provide biochemical evidence for the presence of a Ca2+-dependent calmodulin (CaM)-stimulated protein kinase (CCaMK) from etiolated maize coleoptiles. The kinase, with a molecular mass of 72.3 kDa, was purified to homogeneity by means of ammonium sulphate precipitation, DEAE-Sephacel chromatography. CaM-Sepharose chromatography and gel purification. The purified kinase required 5 mM Mg2+ for activity and had an optimum pH of 7.5. The kinase is a Ca2+-binding protein, as was evident by 45Ca2+-binding and Ca2+ mobility-gel-shift assays. 1 microM Ca2+ stimulated the kinase activity about 12-fold and was further stimulated by the addition of exogenous CaM (approximately 100 nM). Addition of Ca2+ and CaM antagonists decreased the kinase activity. Under in vitro assay conditions the kinase phosphorylated preferentially syntide-2, histone IIIS and casein. Syntide-2 and histone IIIS were phosphorylated at serine residues, showing that the kinase belongs to the serine/threonine family of protein kinases. Autophosphorylation of CCaMK occurred on threonine residue(s) and was Ca2+ dependent. Addition of exogenous CaM had no effect on autophosphorylation. The properties of the maize kinase suggests that it is a CCaMK that shows dual stimulation with Ca2+ and CaM for substrate phosphorylation and only Ca2+ requirement for autophosphorylation. Antibodies raised against the kinase cross-reacted with maize total proteins to give a single band of 72 kDa and precipitated substrate (syntide-2 and histone IIIS)-phosphorylation and autophosphorylation activities in a specific manner. Localisation studies with antibodies showed that the kinase is ubiquitous.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1998

ZmcPKC70, a protein kinase C-type enzyme from maize. Biochemical characterization, regulation by phorbol 12-myristate 13-acetate and its possible involvement in nitrate reductase gene expression.

The crucial enzyme in diacylglycerol-mediated signaling is protein kinase C (PKC). In this paper we provide evidence for the existence and role of PKC in maize. A protein of an apparent molecular mass of 70 kDa was purified. The protein showed kinase activity that was stimulated by phosphatidylserine and oleyl acetyl glycerol (OAG) in the presence of Ca2+. Phorbol 12-myristate 13-acetate (PMA) replaced the requirement of OAG. [3H]PMA binding to the 70-kDa protein was competed by unlabeled PMA and OAG but not by 4alpha-PMA, an inactive analog. The kinase phosphorylates histone H1 at serine residue(s), and this activity was inhibited by H-7 and staurosporine. These properties suggest that the 70-kDa protein is a conventional serine/threonine protein kinase C (cPKC). Polyclonal antibodies raised against the polypeptide precipitate the enzyme activity and immunostained the protein on Western blots. The antibodies also cross-reacted with a protein of expected size from sorghum, rice, and tobacco. A rapid increase in the protein level was observed in maize following PMA treatments. In order to assign a possible role of PKC in gene regulation, the nitrate reductase transcript level was investigated. The transcript level increased by PMA, not by 4alpha-PMA treatments, and the increase was inhibited by H-7 but not by okadaic acid. The data show the existence and possible function of PKC in higher plants.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1998

Biochemical and immunochemical characterization of Brassica juncea glyoxalase I.

A homogenous preparation of glyoxalase I (S-lactoylglutathione-lyase, EC 4.4.1.5) was obtained from Brassica juncea seedlings. The enzyme is a heterodimer with 27,000 and 29,000 M(r) subunits and native M(r) of 56,000. The circular dichroic spectra of the protein showed characteristics of a distinctly helical protein, and magnesium affected the secondary structure. It is a zinc metalloenzyme. Amino acid modification studies suggested the involvement of histidine residues in catalysis. Apo-glyoxalase I was reactivated by divalent cations Mn2+ (0.5 Mm) > Mg2+ (5 Mm) > Zn2+ (0.05 Mm) and Ca2+ (0.01 Mm). Monospecific, polyclonal anti-glyoxalase I antibodies were raised, which showed its presence in seeds, roots, hypocotyl, cotyledon and different flower parts. They showed varied degree of cross reactivity with the extracts from various plants, yeast, bacteria and animal system.

Blotting, Western↗
PubMed1997

Characterization of EhCaBP, a calcium-binding protein of Entamoeba histolytica and its binding proteins.

A novel calcium-binding protein (EhCaBP) has been recently identified and characterized from the protozoan parasite Entamoeba histolytica. In order to decipher the function of this protein, a few basic properties were investigated and compared with the ubiquitous Ca(2+)-signal transducing protein calmodulin (CaM). Indirect immunofluorescence and immunoprecipitation analyses using specific antibodies against EhCaBP suggest that it is a soluble cytoplasmic protein with no major post-translational modification. EhCaBP did not stimulate cAMP-phosphodiesterase activity, differentiating it from all known CaMs. Affinity chromatography of [35S]methionine-labelled proteins of E. histolytica trophozoites using EhCaBP-sepharose column showed Ca(2+)-dependent binding of a group of proteins. Radiolabelled proteins from the same extract also bound to CaM-sepharose. However, the proteins bound to the two columns were different as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. At least one of the EhCaBP-binding proteins became phosphorylated as revealed by in vivo phosphorylation analysis. The binding-proteins could not be detected in E. invadens (a species that is pathogenic in reptiles) and E. moshkovskii (which is found in the human gut but is not pathogenic), two species in which EhCaBP-like protein has not been found. Two distinct Ca(2+)-dependent protein kinases, which get activated by EhCaBP and CaM respectively, were detected in E. histolytica. These kinases require different levels of Ca2+ for their maximal activities. Affinity chromatography also showed the binding of protein kinase(s) to EhCaBP in a Ca(2+)-dependent manner. Our data suggest that there may be novel Ca(2+)-signal transduction pathway in E. histolytica mediated by EhCaBP.

Animals↗
PubMed1996

Phosphorylation/dephosphorylation steps are key events in the phytochrome-mediated enhancement of nitrate reductase mRNA levels and enzyme activity in maize.

We provide evidence to show that the increase in nitrate reductase (NR) transcript level stimulated by red light is mediated via a phosphorylation-dependent step. The light-stimulated enhancement of NR transcript level was significantly inhibited by H-7, a protein kinase inhibitor, whereas okadaic acid (OKA), a phosphatase inhibitor, had no effect. Phorbol myristate acetate (PMA), an activator of protein kinase C (PKC) enhanced the NR transcript level in dark-grown leaves. No correlation between changes in NR transcript level and NR activity (NRA) was observed. Inhibition of NRA by OKA and stimulation by H-7 indicated that NRA is increased by dephosphorylating the enzyme. We have identified a protein kinase (C type) that can phosphorylate the purified NR in vitro without the involvement of other accessory proteins. By in vivo labelling with 32P and immunoprecipitation of NR with NR antibodies it was found that in the presence of OKA most NR protein (NRP) was present in phosphorylated state, while with H-7 the reverse was seen. The red (R) and far-red (FR) light reversible experiments suggested that phytochrome (Pfr, an active form) stimulation of NRA is mediated by dephosphorylation of the enzyme, suggesting that Pfr regulates both NR transcription and NRA via phosphorylation/dephosphorylation steps controlled by separate signal transduction pathways.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗