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M K Reddy

Publications and source records attributed to M K Reddy.

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

Isolation and expression analysis of salt stress-associated ESTs from contrasting rice cultivars using a PCR-based subtraction method.

Salt stress adversely affects the growth of rice plants. To understand the molecular basis of salt-stress response, four subtracted cDNA libraries were constructed employing specific NaCl-stressed tissues from salt-tolerant (CSR 27 and Pokkali) and salt-sensitive (Pusa basmati 1) rice cultivars. An efficient PCR-based cDNA subtraction method was employed for the isolation of the salt-stress responsive cDNA clones. In all, 1,266 cDNA clones were isolated in the course of this study, out of which 85 clones were end-sequenced. Database search of the sequenced clones showed that 22 clones were homologous to genes that have earlier been implicated in stress response, 34 clones were novel with respect to their function and six clones showed no homology to sequences in any of the public database. Northern analysis showed that the transcript expression pattern of selected clones was variable amongst the cultivars tested with respect to stress-regulation.

Blotting, Northern↗
PubMed2002

Cloning and characterization of a cell cycle-regulated gene encoding topoisomerase I from Nicotiana tabacum that is inducible by light, low temperature and abscisic acid.

We have cloned a full-length 2874-bp cDNA coding for tobacco topoisomerase I, with an ORF of 2559 bp encoding a protein of 852 amino acids with a calculated molecular mass of 95 kDa and an estimated pI of 9.51. The deduced amino acid sequence shows homology to other eukaryotic topoisomerases I. Tobacco topoisomerase I was over-expressed in Escherichia coli, and the purified recombinant protein was found to relax both positively and negatively super-coiled DNA in the absence of the divalent cation Mg(2+)and ATP. These characteristic features indicate that the tobacco enzyme is a type I topoisomerase. The recombinant protein could be phosphorylated at (a) threonine residue(s) by protein kinase C. However, phosphorylation did not cause any change in its enzymatic activity. The genomic organization of the topoisomerase I gene revealed the presence of 8 exons and 7 introns in the region corresponding to the ORF and one intron in the 3' UTR region. Transcript analysis using RT-PCR showed basal constitutive expression in all organs examined, and the gene was expressed at all stages of the cell cycle--but the level of expression increased during the G1-S phase. The transcript level also increased following exposure to light, low-temperature stress and abscisic acid, a stress hormone.

Abscisic Acid↗
PubMed2002

Global amplification of cDNA from limiting amounts of tissue. An improved method for gene cloning and analysis.

In this study we present an improved polymerase chain reaction (PCR)-based methodology to generate large amounts of high-quality complementary DNA (cDNA) from small amounts of initial total RNA. Global amplification of cDNA makes it possible to simultaneously clone many cDNAs and to construct directional cDNA libraries from a sequence-abundance-normalized cDNA population, and also permits rapid amplification of cDNA ends (RACE), from a limited amount of starting material. The priming of cDNAs with an adapter oligo-deoxythymidine (oligo-dT) primer and the ligation of a modified oligonucleotide to the 3' end of single-stranded cDNAs, through the use of T4 RNA ligase, generates known sequences on either end of the cDNA population. This helps in the global amplification of cDNAs and in the sequence-abundance normalization of the cDNA population through the use of PCR. Utilization of a long-range PCR enzyme mix to amplify the cDNA population helps to reduce bias toward the preferential amplification of shorter molecules. Incorporation of restriction sites in the PCR primers allows the amplified cDNAs to be directionally cloned into appropriate cloning vectors to generate cDNA libraries. RACE-PCR done with biotinylated primers and streptavidin-coated para-magnetic particles are used for the efficient isolation of either full-length coding or noncoding strands.

Base Sequence↗
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

Plasma drug levels compared with DNA incorporation of 3'-azido-3'-deoxythymidine (AZT) in adult cynomolgus (Macaca fascicularis) monkeys.

Zidovudine (3'-azido-3'-deoxythymidine, AZT), widely used for the therapy of the Human Immunodeficiency Virus-1 (HIV-1), is a nucleoside analog of thymidine that becomes phosphorylated and incorporated into nuclear and mitochondrial DNA. Levels of AZT incorporation into DNA of humans, monkeys, and mice are highly variable and suggest interindividual variability in phosphorylation pathways. In addition, studies in rhesus monkeys (1) have shown a lack of correlation between levels of unbound AZT in plasma and tissue AZT-DNA. However, the correlation between plasma AZT and tissue AZT-DNA has not been previously examined in the same primate. Here we examine the relationship between AZT-DNA incorporation in leukocytes and multiple organs, and levels of the drug circulating in plasma of adult female cynomolgus (Macaca fascicularis) monkeys. Three monkeys were dosed with 40.0 mg of AZT/day for 30 days by naso-gastric intubation. The average daily dose of 9.9 mg of AZT/kg/body wt was similar to the approximately 8.6 mg of AZT/kg/body wt (600 mg/day) given to adult HIV-1-infected patients. In all three monkeys, at the time of sampling, values for AZT concentrations in plasma were similar and values for AZT incorporation into leukocyte DNA (86.1, 100.0, and 114.1 molecules of AZT/10(6) nucleotides) were also similar. AZT-DNA incorporation was detected in liver, uterus, spleen, and kidney from the three AZT-exposed animals, with values for positive samples ranging from 5.8 to 97.4 molecules of AZT/10(6) nucleotides. In brain cortex and lung DNA from AZT-exposed animals, AZT incorporation was undetectable. The data suggest that organ-specific differences in AZT uptake and/or metabolism may contribute to AZT phosphorylation and subsequent drug incorporation into DNA. In addition, AZT-DNA levels in monkey organs were similar to or lower than values observed in peripheral leukocytes of adult AIDS patients.

Animals↗
PubMed2000

A DNA helicase from Pisum sativum is homologous to translation initiation factor and stimulates topoisomerase I activity.

DNA helicases play an essential role in all aspects of nucleic acid metabolism, by providing a duplex-unwinding function. This is the first report of the isolation of a cDNA (1.6 kb) clone encoding functional DNA helicase from a plant (pea, Pisum sativum). The deduced amino-acid sequence has eight conserved helicase motifs of the DEAD-box protein family. It is a unique member of this family, containing DESD and SRT motifs instead of DEAD/H and SAT. The encoded 45.5 kDa protein has been overexpressed in bacteria and purified to homogeneity. The purified protein contains ATP-dependent DNA and RNA helicase, DNA-dependent ATPase, and ATP-binding activities. The protein sequence contains striking homology with eIF-4A, which has not so far been reported as DNA helicase. The antibodies against pea helicase inhibit in vitro translation. The gene is expressed as 1.6 kb mRNA in different organs of pea. The enzyme is localized in the nucleus and cytosol, and unwinds DNA in the 3' to 5' direction. The pea helicase interacts with pea topoisomerase I protein and stimulates its activity. These results suggest that pea DNA helicase could be an important multifunctional protein involved in protein synthesis, maintaining the basic activities of the cell, and in upregulation of topoisomerase I activity. The discovery of such a protein with intrinsic multiple activity should make an important contribution to our better understanding of DNA and RNA transactions in plants.

Adenosine Triphosphatases↗
PubMed1999

In vitro reconstitution of the bacteriophage T4 clamp loader complex (gp44/62).

The clamp loader complex (CLC) of bacteriophage T4 is essential for viability and has analogs in both prokaryotes and eukaryotes. The gp44 and gp62 subunits of the T4 CLC, in a 4:1 ratio, tightly associate such that the two proteins co-purify. Using transformed Escherichia coli, we were able to demonstrate for the first time purification of the unique protein gp62 in the absence of gp44. We experimentally determined the isoelectric point for the individual subunits. An in vitro physical interaction could be observed between the native subunits, which resulted in a reconstituted CLC that displayed the signature pattern of the ATPase functions of native CLC. Thus we demonstrate that the CLC forms via a self-assembly pathway rather than through a translational capture mechanism.

Bacteriophage T4↗
PubMed1999

Comparison of the assembly of the bacteriophage T4 clamp loader complex (gp44/62) expressed in a cis versus trans genomic configuration.

Proper formation of the bacteriophage T4 DNA polymerase holoenzyme requires a wide spectrum of protein-protein and protein-DNA interactions among the DNA polymerase gp43, the sliding clamp gp45, and gp44/62, the clamp loader complex (CLC). The 44 and 62 proteins associate to form a tight complex maintained in a 4:1 ratio. The 44 and 62 genes are adjacent to each other on the T4 genome, are cotranscribed, and are translationally coupled. It has been suggested that translational coupling may play a role in the formation of the clamp loader complex and may control its stoichiometry. To examine the effect of coupling on the assembly of the complex, expression in trans of genes 44 and 62 was accomplished by cotransforming Escherichia coli with compatible, inducible plasmid vectors. A gp44/62 complex could be purified from such cells. The complex assembled in trans exhibited stoichiometry and ATPase activity identical to native complex. Burst sizes were determined to gauge the efficiency of clamp loader complex formation. When gp44 was supplied by a plasmid and gp62 was supplied by the T4 genome, complex formation was as efficient as in wild-type virus. However, when gp62 was supplied by plasmid and gp44 was supplied by the T4 genome, efficiency of complex formation was decreased. This decrease in the efficiency of complex formation was temperature dependent, being more pronounced at higher temperatures. At higher temperatures, a larger proportion of gp62 expressed from the plasmid was found to be present in an insoluble form. The decrease in efficiency of complex formation correlated to a decrease in solubility of the gene 62 protein.

Bacteriophage T4↗
PubMed1999

Heparin-surface-modified intraocular lenses in pediatric cataract surgery: prospective randomized study.

PURPOSE: To evaluate the performance of heparin-surface-modified (HSM) intraocular lenses (IOLs) in pediatric eyes after cataract surgery. SETTING: L.V. Prasad Eye Institute, Hyderabad, India. METHODS: This prospective, randomized, double-masked, controlled clinical trial comprised 90 children aged 2 to 14 years with cataract. The patients were consecutively randomized to receive an HSM (Group 1) or an unmodified (Group 2) poly(methyl methacrylate) (PMMA) IOL. Extracapsular cataract extraction (ECCE) with IOL implantation was performed in children 8 years and older and ECCE with primary posterior capsulotomy, anterior vitrectomy, and IOL implantation in children younger than 8 years. Outcome parameters were inflammatory cell deposits on the IOL surface, posterior synechias, and anterior chamber reaction. RESULTS: Follow-up data were available for 73, 70, 60, and 68 patients at 1 week, 1 month, 3 months, and 6 months, respectively. Significantly fewer cell deposits were noted in Group 1 at 1, 3, and 6 months (P < .001). Synechia formation and anterior chamber reaction were comparable in the 2 groups. CONCLUSION: The lower incidence of inflammatory cell deposit formation in eyes with HSM PMMA IOLs indicates that these IOLs have greater bicompatibility than unmodified IOLs in pediatric cataract surgery.

Adolescent↗
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

Co-occurrence of pneumococcal keratitis and dacryocystitis.

PURPOSE: To evaluate the specific risk factors responsible for the development of pneumococcal keratitis. METHODS: In a retrospective analysis of 383 patients of culture-proven bacterial keratitis, seen between 1991 and 1995, 139 had monobacterial isolates on culture. We analyzed the various predisposing factors in these patients, with special emphasis on the patency of nasolacrimal drainage system. RESULTS: Of the 139 patients with monobacterial keratitis, 48 (group 1) grew Streptococcus pneumoniae in culture. In the remaining 91 patients (group 2), Pseudomonas (18), coagulase-negative Staphylococci (15), Staphylococcus epidermidis (23), Staphylococcus aureus (16), Corynebacterium species (12), and others (seven) were isolated. Trauma was found to be a predisposing factor in 12 patients of group 1 and 27 patients of group 2 (p = 0.5601). Twelve (25%) patients of group 1 revealed chronic dacryocystitis, and nine of them underwent sac excision. On the contrary, only three patients of group 2 demonstrated dacryocystitis (p = 0.0003). CONCLUSION: These results underscore the importance of assessing the patency of lacrimal drainage system in patients with infectious keratitis, especially of pneumococcal origin.

Adolescent↗
PubMed1999

Contact lens-induced peripheral ulcers with extended wear of disposable hydrogel lenses: histopathologic observations on the nature and type of corneal infiltrate.

PURPOSE: Contact lens-induced peripheral ulcer (CLPU), a sudden-onset adverse event observed with extended wear of hydrogel lenses, is characterized by a single, small, circular, focal anterior stromal infiltrate in the corneal periphery or midperiphery. The condition is always associated with a significant overlying epithelial loss and resolves in a scar. The aim was to determine, by using histopathologic techniques, the nature and type of the corneal infiltrate of these events. METHODS: Three CLPUs observed in three patients using disposable hydrogel lenses on an extended-wear schedule were examined. The eye was topically anesthetized, and a corneal section including all of the infiltrate was taken. A small triangular piece of conjunctiva immediately adjacent to the infiltrate was sectioned. The tissue was immediately fixed, processed, stained using hematoxylin and eosin and periodic acid-Schiff stains, and examined by using light microscopy. RESULTS: The diameter of these three corneal infiltrates varied from 0.3 to 0.6 mm. Histopathology of the corneal sections revealed a focal epithelial loss corresponding to the infiltrated stroma in all three patients. The adjacent epithelium was thinned. Bowman's layer was intact in two patients and had a localized area of loss in the remaining patient. The anterior stroma was densely infiltrated with polymorphonuclear leukocytes and had focal areas of necrosis. The infiltration was most dense in the region immediately underlying Bowman's layer. No other infiltrative cell type was seen in any of the sections. Histopathology of the conjunctiva revealed features consistent with normal conjunctival tissue. CONCLUSIONS: On histopathology of CLPU, distinctive features (i.e., focal corneal epithelial loss, an intact Bowman's membrane, and a localized infiltration of the anterior stroma with polymorphonuclear leukocytes) were seen. These features suggest that the event is an acute inflammatory process and probably noninfective in nature.

Adolescent↗
PubMed1998

Familial subepithelial corneal amyloidosis (gelatinous drop-like corneal dystrophy): exclusion of linkage to lactoferrin gene.

PURPOSE: Because corneal tissue with familial subepithelial corneal amyloidosis (FSCA; gelatinous drop-like dystrophy of the cornea) contains lactoferrin the possibility that the FSCA gene was the human lactoferrin (hLF) gene was investigated. Due to contradictory published information we also mapped the hLF gene. METHODS: We mapped the hLF gene using a genomic clone of the entire hLF gene as a probe by fluorescence in situ hybridization (FISH). Utilizing PCR primers that are specific to the hLF gene, we also mapped the hLF via radiation somatic cell hybrid analysis. Linkage of the FSCA gene to the hLF gene was evaluated by genetic linkage analysis using polymorphic markers within and in the vicinity of the hLF gene. RESULTS: The hLF gene mapped to the short arm of chromosome 3 at 3p21. Linkage analysis using polymorphic markers for hLF and haplotype analysis of the 3p21 loci indicates that the FSCA gene is not linked to the 3p21 locus. CONCLUSIONS: The gene for FSCA is not the hLF gene in these families.

Amyloidosis↗
PubMed1998

Watermelon bud necrosis tospovirus is a distinct virus species belonging to serogroup IV.

The nucleocapsid protein gene of a tospovirus infecting watermelon in India was cloned and sequenced. Sequence analyses showed that the gene was most closely related to those of watermelon silver mottle tospovirus (WSMV) from Taiwan and peanut bud necrosis tospovirus (PBNV) from India, the two definitive species of serogroup IV. Amino acid sequence similarity was 84% and 82% with WSMV and PBNV, respectively. On the basis of the sequence divergence and the previously determined host range differences, the watermelon tospovirus, designated as watermelon bud necrosis tospovirus, should be considered as a distinct species belonging to serogroup IV.

Amino Acid Sequence↗
PubMed1998

Microbial keratitis in children.

OBJECTIVE: Microbial keratitis is a major cause of corneal blindness worldwide. This problem is particularly relevant to children, because most of their visual life is ahead of them, and they are uniquely at risk for irreversible ocular deficits, such as those resulting from amblyopia. The objective of this study was to determine the etiologic agents and predisposing factors in childhood infectious keratitis and to examine the outcome of treatment in terms of structure and visual acuity. DESIGN: The study design was a retrospective cases series. PARTICIPANTS: The authors studied 113 eyes in 107 children 16 years of age and younger who were treated for (nonviral) microbial keratitis at the LV Prasad Eye Institute in Hyderabad, India, during the 4.5-year period between February 1, 1991, and June 30, 1995. INTERVENTION: The patients who met the following criteria were included in the study: (1) corneal stromal infiltrate was present on slit-lamp examination; and (2) a corneal scraping was taken at the time of examination for suspected microbial keratitis. MAIN OUTCOME MEASURES: Etiologic micro-organisms, predisposing factors, treatment method, structural treatment outcome, and visual acuity treatment outcome of the infectious keratitis episode were measured. RESULTS: The principal predisposing factors identified in this study were trauma (21.2%), ocular disease (17.7%), systemic disease (15.9%), and prior penetrating keratoplasty in the same eye (8.8%). Vitamin A deficiency was an important factor within the category of severe systemic disease, and contact lens wear was not involved in any of the cases. A total of 85 organisms were isolated in cultures of corneal scrapings from 64 (56.6%) of the 113 cases. Staphylococcus species (43.7%), Streptococcus pneumoniae (18.8%), and fungi (17.2%) were the most common isolates. Eighteen eyes (15.9%) required surgery, and 28 (36.4%) of the 77 patients on whom visual acuity was assessed at last follow-up achieved an unaided visual acuity of 20/60 or better at last follow-up. CONCLUSION: This work represents the largest recent study on childhood (nonviral) microbial keratitis, its management, and treatment outcomes. In this study, amblyopia is highlighted as a potentially significant sequela of childhood microbial keratitis. Identification of the appropriate predisposing factors, etiologic microbial organisms, and treatment outcome from this study may aid in early recognition and treatment of microbial keratitis in children.

Adolescent↗