Browsing by Author "Kesarwani, Meenu"

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Cloning and characterization of 5'-flanking region of oxalate decarboxylase gene from Flammulina velutipes
(Biochemical Society, 2002) Azam, Mohammad; Kesarwani, Meenu; Chakraborty, Subhra; Natarajan, Krishnamurthy; Datta, Asis
The oxalate-degrading enzyme, oxalate decarboxylase (OXDC), was purified and characterized from Flammulina elutipes, a basidiomycetous fungus [Mehta and Datta (1991) J. Biol. Chem. 266, 23548–23553]. The cDNA cloning and analyses revealed that OXDC transcription was induced by oxalic acid. However, in this report, we show that OXDC transcription is induced by low pH, not by oxalate. To understand the regulatory mechanism of OXDC expression, we have cloned and analysed a 580-bp genomic fragment from the 5h-flanking region of the OXDC gene. Sequence analysis showed the presence of several eukaryotic transcription factor binding motifs within the k580 bp of the upstream region. Electrophoretic-mobility-shift assays with partially purified cell extracts revealed specific binding of a factor in acid-induced, but not in uninduced, extracts. Furthermore, DNase I protection assays using the partially purified fraction from oxalic acid-induced extract revealed a footprint of a 13-bp sequence 5hGCGGGGTCGCCGA3h, termed low pH responsive element (LPRE), corresponding to the k287 to k275 bp region of the OXDC promoter. Our results suggest that in F. elutipes cells, activation of OXDC transcription in response to low pH is mediated by the binding of a novel transcription factor through the LPRE site in the OXDC promoter.
Oxalate decarboxylase from Collybia velutips: molecular cloning and its over expression to confer resistance to fungal infection in transgenic tobacco and tomato
(The American Society for Biochemistry and Molecular Biology, Inc., 2000) Kesarwani, Meenu; Azam, Mohammad; Natarajan, K; Mehta, Anuradha; Datta, Asis
Oxalic acid is present as nutritional stress in many crop plants like Amaranth and Lathyrus. Oxalic acid has also been found to be involved in the attacking mecha- nism of several phytopathogenic fungi. A full-length cDNA for oxalate decarboxylase, an oxalate-catabolizing enzyme, was isolated by using 5؅-rapid amplification of cDNA ends-polymerase chain reaction of a partial cDNA as cloned earlier from our laboratory (Mehta, A., and Datta, A. (1991) J. Biol. Chem. 266, 23548 –23553). By screening a genomic library from Collybia velutipes with this cDNA as a probe, a genomic clone has been isolated. Sequence analyses and comparison of the genomic sequence with the cDNA sequence revealed that the cDNA is interrupted with 17 small introns. The cDNA has been successfully expressed in cytosol and vacuole of transgenic tobacco and tomato plants. The transgenic plants show normal phenotype, and the transferred trait is stably inherited to the next generation. The recombinant enzyme is partially glycosylated and shows oxalate decarboxylase activity in vitro as well as in vivo. Transgenic tobacco and tomato plants ex- pressing oxalate decarboxylase show remarkable resistance to phytopathogenic fungus Sclerotinia sclerotio- rum that utilizes oxalic acid during infestation. The result presented in the paper represents a novel approach to develop transgenic plants resistant to fungal infection.
A secretion signal is present in the Collybia velutipes oxalate decatboxylase gene
(Elsevier B.V., 2001) Azam, Mohammad; Kesarwani, Meenu; Natarajan, Krishnamurthy; Datta, Asis
The oxalate decarboxylase (OXDC) gene from Collybia velutipes is overexpressed as an active form in Schizosaccharomyces pombe. The recombinant enzyme shows similar pH optima and stability, while substrate kinetic analysis shows a ninefold decrease in K m value with respect to native OXDC. Most of the expressed protein was present in periplasm and remained firmly bound to cell-wall materials. However, 20% of enzyme expressed was secreted out into the medium suggesting the presence of a secretion signal (C. velutipes) in the oxalate decarboxylase gene. This secretion signal is associated with the N-terminal of OXDC as is evident by secretion of nonsecretory genes AmA1 and Beta-galactosidase. An expression vector using this signal is constructed for expression and secretion of heterologous proteins in S. pombe.