PubMed Health⌕ Search

Biomedical subjects

R S Datla

Publications and source records attributed to R S Datla.

17 recordsLinked to original sources
PubMed2006

Genetic transformation of peanut (Arachis hypogaea L.) using cotyledonary node as explant and a promoterless gus::nptII fusion gene based vector.

We have generated putative promoter tagged transgenic lines in Arachis hypogaea cv JL-24 using cotyledonary node (CN) as an explant and a promoterless gus::nptII bifunctional fusion gene mediated by Agrobacterium transformation. MS medium fortified with 6-benzylaminopurine (BAP) at 4mg/l in combination with 0.1 mg/l alpha -napthaleneacetic acid (NAA) was the most effective out of the various BAP and NAA combinations tested in multiple shoot bud formation. Parameters enhancing genetic transformation viz. seedling age, Agrobacterium genetic background and co-cultivation periods were studied by using the binary vector p35SGUSINT. Genetic transformation with CN explants from 6-day-old seedlings co-cultivated with Agrobacterium GV2260 strain for 3 days resulted in high kanamycin resistant shoot induction percentage (45%); approximately 31% transformation frequency was achieved with p35S GUSINT in beta-glucuronidase (GUS) assays. Among the in vivo GUS fusions studied with promoterless gus::nptII construct, GUS-positive sectors occupied 38% of the total transient GUS percentage. We have generated over 141 putative T 0 plants by using the promoterless construct and transferred them to the field. Among these, 82 plants survived well in the green house and 5 plants corresponding to 3.54% showed stable integration of the fusion gene as evidenced by GUS, polymerase chain reaction (PCR) and Southern blot analyses. Twenty-four plants were positive for GUS showing either tissue-specific expression or blue spots in at least one plant part. The progeny of 15 T 0 plants indicated Mendelian inheritance pattern of segregation for single-copy integration. The tissue-specific GUS expression patterns were more or less similar in both T 0 and corresponding T 1 progeny plants. We present the differential patterns of GUS expression identified in the putative promoter-tagged transgenic lines in the present communication.

Arachis↗
PubMed2001

Phosphorylation of human N-myristoyltransferase by N-myristoylated SRC family tyrosine kinase members.

N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational and/or posttranslational transfer of myristate to the amino terminal glycine residue of a number of important proteins especially the non-receptor tyrosine kinases whose activity is important for tumorigenesis. Human NMT was found to be phosphorylated by non-receptor tyrosine kinase family members of Lyn, Fyn and Lck and dephosphorylated by the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin. Deletion of 149 amino acids from the N-terminal end resulted in the absence of phosphorylation suggesting that the phosphorylation sites are located in the N-terminal end of NMT. Furthermore, a site-directed mutagenesis study indicated that substitution of tyrosine 100 with phenylalanine served NMT as a poor substrate for the Lyn kinase. A synthetic peptide corresponding to the amino-terminal region encompassing tyrosine 100 of NMT served as a good substrate for the Lyn and Fyn kinases. Our studies also indicated that NMT was found to interact with Lyn through its N-terminal end in a phosphorylation-dependent manner. This is the first study demonstrating the cross-talk between NMT and their myristoylated protein substrates in signaling pathways.

Acyltransferases↗
PubMed2000

Increased expression of N-myristoyltransferase in gallbladder carcinomas.

BACKGROUND: Activated Src, which has intrinsic protein tyrosine kinase activity, has been found in human solid tumors such as colorectal and breast carcinomas. The Src gene encodes a cytoplasmic tyrosine kinase p60src, which attaches to the inner surface of the membrane after N-terminal myristoylation and is implicated in transduction of signals to the nucleus. N-myristoyltransferase (NMT) catalyzes the biochemical modification process called N-myristoylation. To investigate whether, through Src, NMT contributes to the pathogenesis of gallbladder carcinoma, the authors investigated expression of NMT and p53 in in situ and invasive carcinomas. METHODS: One hundred cases of documented gallbladder carcinoma were reviewed, and 30 cases were selected randomly to evaluate expression of NMT and p53 by immunohistochemistry in both in situ and in invasive tumor components. RESULTS: Eighteen cases (60%) of gallbladder carcinoma showed moderate to strong cytoplasmic positivity for NMT with increased intensity in the invasive component, and 12 cases (40%) were negative. The in situ component revealed mild to moderate cytoplasmic staining in 20 cases (67%), whereas the normal gallbladder mucosa showed weak to negative cytoplasmic staining. Moderate to strong p53 staining was observed in 17 in situ cases (63%) and 24 invasive cases (80%). The in situ staining patterns of p53 were unrelated to the clinical outcome of the tumor. However, moderate to strong staining of the invasive component as observed in 15 cases (50%) was associated with a mean survival of 8.8 months. Amplification of intron-8 in normal gallbladder mucosa and invasive carcinoma were similar in intensity, suggesting the absence of NMT gene amplification in these tumors. CONCLUSIONS: The increased expression of NMT in these tumors could be due to transcriptional activation. Tumors with increased expression of NMT and p53 were associated with poor clinical outcomes as evidenced by their mean survival times. NMT is likely to play a pathogenic role in gallbladder carcinoma.

Acyltransferases↗
PubMed2000

N-myristoyltransferase.

Myristoylation refers to the co-translational addition of a myristoyl group to an amino-terminal glycine residue of a protein by an ubiquitously distributed enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT, EC 2.3.1.97). This review describes the basic enzymology, molecular cloning and regulation of NMT activity in various pathophysiological processes such as colon cancer and diabetes.

Acyl Coenzyme A↗
PubMed2000

Identification of a CYP84 family of cytochrome P450-dependent mono-oxygenase genes in Brassica napus and perturbation of their expression for engineering sinapine reduction in the seeds.

CYP84 is a recently identified family of cytochrome P450-dependent mono-oxygenases defined by a putative ferulate-5-hydroxylase (F5H) from Arabidopsis. Until recently F5H has been thought to catalyze the hydroxylation of ferulate to 5-OH ferulate en route to sinapic acid. Sinapine, a sinapate-derived ester in the seeds, is antinutritional and a target for elimination in canola meal. We have isolated three F5H-like genes (BNF5H1-3) from a cultivated Brassica napus, whose amphidiploid progenitor is considered to have arisen from a fusion of the diploids Brassica rapa and Brassica oleracea. Two cultivated varieties of the diploids were also found to contain BNF5H3 and additionally either BNF5H1 or BNF5H2, respectively. Whereas all three are >90% identical in their coding sequence, BNF5H1 and BNF5H2 are closer to each other than to BNF5H3. This and additional data suggest that the two groups of genes have diverged in an ancestor of the diploids. B. napus showed maximal F5H expression in the stems, least in the seeds, and subtle differences among the expression profiles of the three genes elsewhere. Transgenic B. napus with cauliflower mosaic virus 35S-antisense BNF5H contained up to 40% less sinapine, from 9.0 +/- 0.3 mg in the controls to 5.3 +/- 0.3 mg g(-1) seed. F5H from Arabidopsis and a similar enzyme from sweetgum (Liquidamber styraciflua) has recently been shown to have coniferaldehyde hydroxylase activity instead of F5H activity. Thus the supply of 5-OH coniferaldehyde or 5-OH ferulate has a bearing on sinapine accumulation in canola seeds.

Acrolein↗
PubMed1999

Genomic organization of human myristoyl-CoA: protein N-myristoyltransferase-1.

Myristoylation is a biochemical modification of proteins in which the lipid myristate becomes covalently bound to various cellular, viral, and oncoproteins catalyzed by a monomeric enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). This modification is important for the biological activity of several proteins, especially the regulation of several oncoproteins involved in various types of cancers. Complementary DNA encoding human NMT-1 (hNMT-1) has been previously reported; however, the genomic organization of hNMT-1 has not been available. Attempts to amplify genomic fragments corresponding to hNMT-1 cDNA sequence yielded only one fragment. We have searched databases using both the cDNA and sequence of one of the intron sequence and this identified a human BAC clone sequence from chromosome 17. Alignment of hNMT-1 cDNA coding information on human chromosome 17 resulted in the complete structural identity of 23,960 bp of the hNMT-1 gene. The hNMT-1 gene is composed of 11 exons and 10 introns with consensus GT/AG boundaries. Finally, we show that 140 bp from the 5' end of recently reported full-length cDNA of hNMT-1 was not part of this genomic region raising the possibility for posttranscriptional modification in generating larger transcripts likely by trans splicing. Further, the availability of this genomic sequence will assist in unraveling the molecular basis for several observed NMT isoforms.

Acyl Coenzyme A↗
PubMed1998

Effects of L-histidine and its structural analogues on human N-myristoyltransferase activity and importance of EEVEH amino acid sequence for enzyme activity.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. Human NMT (hNMT) activity was found to be activated by L-histidine in a concentration-dependent manner. In contrast, two structural analogues of L-histidine, L-histidinol and histamine, inhibited hNMT activity in a noncompetitive manner with half-maximal inhibitions of 18 and 1.5 mM, respectively. The inhibition of hNMT activity by L-histidinol was reversed by a 2-fold molar excess of L-histidine, suggesting that L-histidine and L-histidinol were competing for a common site on NMT. Kinetic data indicated that whereas L-histidine enhanced the Vmax, both L-histidinol and histamine decreased the Vmax; none of these compounds altered the Km. Our studies suggest that L-histidine and its analogues may be interacting with His-293, involved in myristoyl-CoA transfer, rather than His-218, and implicated in the transfer of myristoyl-CoA to the peptide substrates. Site-directed mutagenesis of His-293, Val-291, and Glu-290 resulted in proteins with no measurable NMT activity. The most conserved region in the catalytic domain EEVEH (289-293) is critical for the myristoyl-CoA transfer in the NMT-catalyzed reactions. This region will be useful for the design of regulators of NMT function.

Acyltransferases↗
PubMed1998

Myristoyl-coA:protein N-myristoyltransferase from bovine cardiac muscle: molecular cloning, kinetic analysis, and in vitro proteolytic cleavage by m-calpain.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the attachment of myristate onto the amino terminal glycine residue of select polypeptides. Cardiac tissue expresses high levels of cAMP-dependent protein kinase whose catalytic subunit is myristoylated; however, cardiac muscle extracts were found to contain low NMT activities. Northern blot analysis of bovine heart poly(A)+ RNA probed with bovine spleen NMT cDNA revealed a 1.7-kb mRNA. Western blot analysis of cardiac muscle extracts with human NMT antibody indicated a prominent immunoreactive band with a molecular mass of 50 kDa. The expression of mRNA and protein levels in cardiac muscle is not correlated with NMT activities, suggesting the presence of regulators of the enzyme activity. We have isolated the cDNA encoding bovine cardiac muscle NMT (cNMT) by reverse transcription polymerase chain reaction. The single long open reading frame of 1248 bp of bovine cNMT specifies a protein of 416 amino acids with a predicted mass of 46,686 Da. The cDNA clone expressed in Escherichia coli resulted in the production of functionally active 50-kDa NMT. Ultrastructural and immunolocalization of NMT utilizing the immunogold labeling technique demonstrated cytoplasmic distribution with occasional mitochondrial and myofilaments localization of the NMT antibody. Cardiac muscle NMT has a higher affinity for myristoyl-CoA than toward palmitoyl-CoA. Substrate specificity indicated that cNMT has a higher affinity toward pp60src and M2 gene segment of reovirus type 3-derived peptide substrates than toward cAMP-dependent protein kinase-derived peptide. Primary translational product of cNMT sequence contained several regions rich in proline, glutamic acid, serine, and threonine, which are known as "PEST" regions. PEST-FIND analysis of the amino acid sequences indicated eight PEST regions were present in the cNMT. These PEST regions are suggested to be recognized by specific proteases, particularly Ca(2+)-dependent neutral proteases, calpains, which are responsible for the degradation of PEST-containing proteins. We have demonstrated the abolishment of NMT activity and NMT protein degradation in vitro by m-calpain. The proteolysis of cNMT by m-calpain and the abolishment of NMT activity was prevented by the calpain inhibitor, calpastatin. These observations indicate that calpains may regulate NMT activity.

Acyltransferases↗
PubMed1998

Recombinant bovine spleen myristoyl CoA: protein N-myristoyltransferase.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the co-translational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. Recently, we have isolated full length cDNA encoding bovine spleen NMT [27] the full length cDNA was cloned and expressed in E. coli, resulting in the expression of functionally active 50 kDa NMT. Using the combination of SP-Sepharose fast flow and Mono S fast protein liquid chromatography, the enzyme was purified 20-fold with a high yield. The spleen NMT (sNMT) fusion protein exhibited an apparent molecular weight of 53 kDa on SDS-PAGE. Upon cleavage by the Enterokinase the sNMT exhibited an apparent molecular weight of 50 kDa without loss of catalytic activity. The two synthetic peptide substrates based on the N-terminal sequence of pp60src (GSSKSKMR) and cAMP dependent protein kinase (GNAAAKKRR) have different kinetic parameters of Km values of 40 and 200 microM. Recombinant sNMT was also potently inhibited by Ni2+ (histidine binder) in a concentration dependent manner with a half maximal inhibition of 280 microM. The E. coli expressed sNMT was homogenous and showed enzyme activity.

Acyltransferases↗
PubMed1997

Molecular cloning and biochemical characterization of bovine spleen myristoyl CoA:protein N-myristoyltransferase.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. We have isolated full-length cDNA encoding bovine spleen NMT (sNMT). The single long open reading frame of 1248 bp of sNMT specifies a protein of 416 amino acids with a predicted mass of 46,686 Da. The protein coding sequence was expressed in Escherichia coli resulting in the production of functionally active 50-kDa NMT. Deletion mutagenesis showed that the C-terminus is essential for activity whereas up to 52 amino acids can be deleted from the N-terminus without affecting the function. One of the N-terminal deletions resulted in threefold higher NMT activity. Genomic Southern analysis indicated the presence of two strong hybridizing bands with three different restriction enzyme digests suggesting the possibility of two copies of the NMT gene in the bovine genome. RNA blot hybridization analysis of total cellular RNA prepared from bovine brain, heart, spleen, lung, liver, kidney, and skeletal muscle probed with bovine sNMT cDNA revealed a single 1.7-kb mRNA. Western blot analysis of various bovine tissues with human NMT peptide antibody indicated a common prominent immunoreactive band with an apparent molecular mass of 48.5-50 kDa in all tissues. Additional immunoreactive bands were observed in brain (84 and 50 kDa), lung (58 kDa), and skeletal muscle (58 kDa). Activity measurements demonstrated that brain contained the highest NMT activity followed by spleen, lung, kidney, heart, skeletal muscle, pancreas, and liver. It appears therefore that mRNA and protein expression do not correlate with NMT activity, suggesting the presence of regulators of the enzyme activity.

Acyltransferases↗
PubMed1997

Promoter sequences from two different Brassica napus tapetal oleosin-like genes direct tapetal expression of beta-glucuronidase in transgenic Brassica plants.

To investigate the sequences responsible for the regulated expression of tapetal-specific oleosin-like genes, ca. 2 kb of the 5'-upstream regions from two divergent genes, OlnB;4 and OlnB;13, were isolated, sequenced and fused to the reporter gene beta-glucuronidase for study in transgenic Brassica napus plants. Although the proteins encoded by these two genes are highly divergent, except for the conserved oleosin-like domain, the first 250 bp of their 5'-upstream regions was 86% identical, including a region of 150 bp upstream from the TATA box. Analysis of 42 independent transformants by histochemical and fluorometric methods showed that both promoters directed tapetal-specific expression that peaked at the 4 mm flower bud stage.

Base Sequence↗
PubMed1996

Expression of human N-myristoyltransferase in Escherichia coli. Comparison with N-myristoyltransferases expressed in different tissues.

Myristoyl CoA:protein N-myristoyltransferase catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins. Escherichia coli transformed with human NMT expression construct produced high levels of N-myristoyltransferase. Using the combination of ammonium sulfate precipitation, chromatography on SP-Sepharose fast flow and fast protein liquid chromatography on Mono-S, the enzyme was purified more than 100 fold with 40% yield. The hNMT fusion protein exhibited an apparent molecular weight of 53 kDa on SDS-polyacrylamide gel electrophoresis. Upon cleavage by the Enterokinase [(Asp)4-Lys], the hNMT exhibited an apparent molecular mass of 49 kDa without loss of catalytic activity. The hNMT activity could be greatly activated severalfold with the use of Tris, SDS, ethanol and acetonitrile. The catalytic activity of hNMT was potently inhibited in a concentration dependent manner by NIP71, a bovine brain NMT inhibitory protein with a half maximal inhibition of 31.0 nM. The E. coli expressed hNMT was homogeneous and showed enzyme activity.

Acyltransferases↗
PubMed1996

Overexpression of human N-myristoyltransferase utilizing a T7 polymerase gene expression system.

Myristoyl CoA:protein N-myristoyltransferase catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins. The gene encoding human N-myristoyltransferase (hNMT) was cloned into the overexpression vector pT7-7 which utilizes the T7 RNA polymerase gene expression system. The hNMT enzyme was purified to near homogeneity with more than 95% recovery using a single-step purification method involving SP-Sepharose fast flow column chromatography. The specific activity of the purified NMT was 220 nmol/min/mg of protein in the presence of oncoprotein-derived peptide substrate pp60src. The hNMT exhibited an apparent molecular weight of 49 kDa on SDS-polyacrylamide gel electrophoresis. Antibodies to Escherichia coli-expressed hNMT specifically recognize hNMT from crude bacterial lysates. The over-expressed hNMT was homogeneous and showed enzyme activity.

Acyltransferases↗
PubMed1994

Modification of lignin biosynthesis in transgenic Nicotiana through expression of an antisense O-methyltransferase gene from Populus.

An aspen lignin-specific O-methyltransferase (bi-OMT; S-adenosyl-L-methionine: caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase, EC 2.1.1.68) antisense sequence in the form of a synthetic gene containing the cauliflower mosaic virus 35S gene sequences for enhancer elements, promoter and terminator was stably integrated into the tobacco genome and inherited in transgenic plants with a normal phenotype. Leaves and stems of the transgenes expressed the antisense RNA and the endogenous tobacco bi-OMT mRNA was suppressed in the stems. Bi-OMT activity of stems was decreased by an average of 29% in the four transgenic plants analyzed. Chemical analysis of woody tissue of stems for lignin building units indicated a reduced content of syringyl units in most of the transgenic plants, which corresponds well with the reduced activity of bi-OMT. Transgenic plants with a suppressed level of syringyl units and a level of guaiacyl units similar to control plants were presumed to have lignins of distinctly different structure than control plants. We concluded that regulation of the level of bi-OMT expression by an antisense mechanism could be a useful tool for genetically engineering plants with modified lignin without altering normal growth and development.

Base Sequence↗
PubMed1991

A bifunctional fusion between beta-glucuronidase and neomycin phosphotransferase: a broad-spectrum marker enzyme for plants.

We have used an in vivo selection approach to isolate a gene encoding a bifunctional fusion peptide between Escherichia coli beta-glucuronidase (GUS) and neomycin phosphotransferase II (NPT-II) from transposon Tn5 in the NH2-GUS::NPT-II-COOH configuration. The fused gene is predicted to encode a fusion peptide 885 amino acids long, and was shown in E. coli to synthesize a 97-kDa GUS+ NPT-II+ gene product. Gel-filtration chromatography suggested that, while the native GUS may be active as a dimer and NPT-II as a monomer, the elution profile of the fusion protein is consistent with that of a trimer. The fusion marker has been produced and defined in transgenic Nicotiana tabacum plants, where both the chimeric gene and the gene product were stable. The bifunctional gene enabled direct KmR selection at the callus stage and enzymatic or histochemical assessment of the steady-state production of GUS activity in regenerated plants. In addition to allowing structure-function determination for the GUS and NPT-II domains of the fusion peptide, the gus::npt-II gene simplifies vector constructs where both marker domains are desired.

Amino Acid Sequence↗