Biomedical subjects

A Chandrashekar

Publications and source records attributed to A Chandrashekar.

9 recordsLinked to original sources

PubMed2006

Evaluation of PCR-based methods for isolating flanking regions of genes.

Several polymerase chain reaction (PCR)-based methods are available for isolation of unknown genomic fragments. In the present study, a comparative evaluation of a few methods of ligation-mediated PCR methods and a ligation-independent one were made by isolating promoter fragment for N-methyltransferase gene involved in the caffeine biosynthetic pathway of Coffea canephora. The benefits of tertiary PCR and the effects of a 4-base cutting restriction endonuclease on the size of the PCR products obtained were demonstrated in one of the ligation-mediated PCR methods. The methods adopted in this study differed in the sizes of the 5'-flanking regions obtained. The efficiencies of various methods used reflect the inherent limitations of the PCR-based methods for isolation of unknown flanking regions.

5' Flanking Region↗

PubMed2005

Stable transformation and direct regeneration in Coffea canephora P ex. Fr. by Agrobacterium rhizogenes mediated transformation without hairy-root phenotype.

A system for genetic transformation of Coffea canephora by co-cultivation with Agrobacterium rhizogenes harbouring a binary vector has been developed. The objective of the present study was the genetic transformation and direct regeneration of transformants through secondary embryos bypassing an intervening hairy root stage. Transformants were obtained with a transformation efficiency up to 3% depending on the medium adjuvant used. A. rhizogenes strain A4 harbouring plasmid pCAMBIA 1301 with an intron uidA reporter and hygromycin phosphotransferase (hptII) marker gene was used for sonication-assisted transformation of Coffea canephora. The use of hygromycin in the secondary embryo induction medium allowed the selection of transgenic secondary embryos having Ri T-DNA along with the T-DNA from the pCAMBIA 1301 binary vector. In addition transgenic secondary embryos devoid of Ri-T-DNA but with stable integration of the T-DNA from the binary vector were obtained. The putative transformants were positive for the expression of the uidA gene. PCR and Southern blot analysis confirmed the independent, transgenic nature of the analysed plants and indicated single and multiple locus integrations. The study clearly demonstrates that A. rhizogenes can be used for delivering transgenes into tree species like Coffea using binary vectors with Agrobacterium tumefaciens T-DNA borders.

Blotting, Southern↗

PubMed2005

Isolation of promoter for N-methyltransferase gene associated with caffeine biosynthesis in Coffea canephora.

N-Methyltransferases (NMTs) catalyze the three SAM dependent sequential methylation of xanthosine, producing caffeine in Coffea species. In the present work, a PCR based genome walking method was adopted to isolate and clone the promoter for the NMT gene. Inspection of the promoter sequence revealed the presence of several motifs important for the regulation of the gene expression. The whole fragment was fused to the beta-glucuronidase (gus) reporter gene and used in Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum. GUS assays proved that the isolated promoter was able to direct the expression of the reporter gene in transgenic tobacco. Based on the promoter sequence, primer was designed and the genomic fragment comprising the promoter and its corresponding gene was amplified and cloned. Sequencing of one of the genomic clones revealed the presence of four exons and three introns in NMT gene. The differences in the restriction pattern among the genomic clones were studied using PCR-RFLP. This is the first report of cloning of the promoter for a gene involved in caffeine biosynthetic pathway and it opens up the possibility of studying the molecular mechanisms that regulate the production of caffeine.

Agrobacterium tumefaciens↗

PubMed2004

PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize.

Contamination of food and feedstuffs by Aspergillus species and their toxic metabolites is a serious problem as they have adverse effects on human and animal health. Hence, food contamination monitoring is an important activity, which gives information on the level and type of contamination. A PCR-based method of detection of Aspergillus species was developed in spiked samples of sterile maize flour. Gene-specific primers were designed to target aflR gene, and restriction fragment length polymorphism (RFLP) of the PCR product was done to differentiate Aspergillus flavus and Aspergillus parasiticus. Sterile maize flour was inoculated separately with A. flavus and A. parasiticus, each at several spore concentrations. Positive results were obtained only after 12-h incubation in enriched media, with extracts of maize inoculated with A. flavus (101 spores/g) and A. parasiticus (104 spores/g). PCR products were subjected to restriction endonuclease (HincII and PvuII) analysis to look for RFLPs. PCR-RFLP patterns obtained with these two enzymes showed enough differences to distinguish A. flavus and A. parasiticus. This approach of differentiating these two species would be simpler, less costly and quicker than conventional sequencing of PCR products.

Aspergillus↗

PubMed2003

Identification of polyhydroxyalkanoate (PHA)-producing Bacillus spp. using the polymerase chain reaction (PCR).

AIMS: The aim of the work was to develop efficient method to identify polyhydroxyalkanoate (PHA)-producing species of Bacillus from numerous soil isolates of bacteria. Identification of the isolates and characterization of the PHA produced by strains positive on the polymerase chain reaction (PCR) was envisaged. METHODS AND RESULTS: Different bacteria isolated from soil were screened by PCR using two sets of primers designed for Bacillus megaterium. Amongst 23 isolates examined, the DNA of 12 isolates reacted positively with the primers giving amplicons identical in size to that obtained from B. megaterium. The isolates which were identified as strains of B. sphaericus, B. circulans, B. brevis and B. licheniformis, produced 11- 41% of PHA in biomass, in sucrose-containing medium, over a growth period of 24-72 h. The nature of the PHA thus produced was analyzed by Fourier transform infrared spectroscopy, gas chromatography and by nuclear magnetic resonance (NMR) and found to contain polyhydroxy butyrate and polyhydroxyvalerate. CONCLUSIONS: The results indicate that most of our isolates from different species contained the B. megaterium type of PHA synthase. Bacillus licheniformis appeared to belong to another group as it did not react with both sets of primers. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the universality of the B. megaterium type of PHA synthase in soil isolates of Bacillus. Some variations were also found.

Acyltransferases↗

PubMed2003

Staphylococcal accessory gene regulator (sar) as a signature gene to detect enterotoxigenic staphylococci.

AIMS: To evaluate the use of a staphylococcal accessory gene regulator (sar) as a means of detecting enterotoxigenic staphylococci. METHODS AND RESULTS: SarA gene-specific primers were designed and applied in PCR, which resulted in the detection of 49 sar-positive isolates from a total of 67 natural food isolates of staphylococci. Colony hybridization using PCR-generated Digoxigenin (DIG)-labelled sarA probe tested in spiked samples of khoa (a traditional heat-concentrated milk product) comprising a mixed microflora ensured the specificity of the probe. Validation experiments with the commercial samples of khoa also demonstrated the specificity of the probe. PCR characterization for enterotoxins A-D revealed the presence of at least one of the toxin-encoding genes in all the sarA-positive isolates tested. CONCLUSION: The study indicated that sarA gene could be an ideal marker gene either in colony hybridization or in PCR, for an effective detection of potentially enterotoxigenic strains of staphylococci in a food system. SIGNIFICANCE AND IMPACT OF THE STUDY: As an alternative to targeting the individual toxin genes, a regulatory gene responsible for controlling the synthesis of various virulence factors may be a suitable target gene for screening potentially toxigenic staphylococci in food system using nucleic acid-based methods.

Animals↗

PubMed2002

Detection of Bacillus cereus in foods by colony hybridization using PCR-generated probe and characterization of isolates for toxins by PCR.

Isolates of Bacillus cereus from traditional Indian foods were detected by colony hybridization using the PCR-generated phospholipase (PL-1) probe. In all, 29 isolates picked up by the probe were confirmed as B. cereus by conventional cultural and biochemical characteristics. All the isolates reacted positively in PCR with phospholipase (PL-1) primers. Among the native isolates, 11 of them showed the discontinuous pattern of haemolysin BL activity in gel diffusion assay. Though 14 isolates reacted positively in PCR with primers (Ha-1) specific to the B gene of haemolysin BL, only four of them showed both the presence of gene and haemolysin BL activity. More than 50% of the isolates indicated their potential enterotoxigenicity by reacting positively with primers specific for the BceT gene encoding for diarrhoeal enterotoxin. PCR with primers for different inverse (IS) repeat elements revealed that isolates carrying transposon IS 231-P 231-1 did not carry IS 240-P 240. Some of the isolates were devoid ofthese IS elements. The study demonstrated the potential of using of a PCR-generated labelled PL-1 probe for the direct detection of B. cereus in food samples and PCR for characterizing the toxigenic isolates.

Bacillus cereus↗

PubMed2001

Antifungal proteins and other mechanisms in the control of sorghum stalk rot and grain mold.

Research on antifungal proteins and other mechanisms that provide the biochemical basis for host-plant resistance to stalk rot and grain molds is reviewed in this paper. Stalk rot caused by Fusarium species leads to substantial yield loss due to poor grain filling and/or lodging. A transgenic sorghum expressing high levels of chitinase exhibited less stalk rot development when exposed to conidia of F. thapsinum. Grain mold of sorghum is associated with warm humid environments and results from colonization by several fungi (F. thapsinum, Curvularia lunata, and Alternaria alternata) of the developing caryopsis. The roles of several biochemical mechanisms (tannins, phenolic compounds, red pericarp, proteins, hard endosperm, and antifungal proteins) on grain mold resistance are discussed. Resistance mechanisms related to these compounds appear to be additive, and pyramiding of genes is a feasible approach to limit grain deterioration. Several experimental approaches are proposed to extend current findings.

Antifungal Agents↗

PubMed2000

Lactobacillus farciminis MD, a newer strain with potential for bacteriocin and antibiotic assay.

A native isolate Lactobacillus farciminis MD isolated from fermenting mushroom exhibited a high degree of sensitivity to the majority of the bacteriocins produced by strains of lactobacilli, leuconostoc and pediococci. Also, the efficacy of Lact. farciminis MD as a sensitive strain for antibiotic assay was established against different antibiotics including ampicillin, cefazoline, chloramphenicol and nitrofurantoin at concentrations of 30 microg each, showing an inhibition zone of 30 mm diameter. The high degree of sensitivity towards bacteriocins and antibiotics provide potential for the exploitation of Lact. farciminis MD in establishing very well-defined bacteriocin producers.

Agaricales↗