Molecular cloning and characterization of a heat-shock induced calmodulin binding protein gene and cDNAs encoding glutamate decarboxylase from tobacco

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dc.contributor.authorDharmasiri, M.A. Nihal
dc.date.accessioned2009-07-15T17:24:05Z
dc.date.available2009-07-15T17:24:05Z
dc.date.issued1995
dc.description.abstractTwo individual heat shock induced/enhanced tobacco (Nicotiana tabacum L. cv Wisconsin-38) CaMBP cDNAs, pTCB48 and pTCB15, were characterized. Both of these clones were partial cDNAs. The 5' end of pTCB48 was obtained using 5' RACE technique. The ORF of this cDNA codes for a protein with a molecular weight of 50 kD. Using several deletions coupled with CaM gel overlay, CaM binding domain of pTCB48 was localized to the very end of the C-terminus of the protein. Structural prediction of this domain revealed a possibility of forming a BAA structure but an alternative β-strand and β-turn structure could not be excluded. Polyclonal antibody was raised against purified TCB48 fusion protein and used in Western analysis. The gene (TG48) corresponding to the pTCB48 cDNA was isolated and characterized. TG48 contains a total of 6149 bp including 1064 bp of 5' flanking region and 364 bp of 3' flanking sequence. The TG48 gene is consists of 6 exons and 5 introns. The 5' flanking sequence contains common promoter elements such as TATA and CAAT boxes, and 5 putative HSEs, characteristic of heat shock induced genes. These HSEs may be involved in heat shock induction of this gene. The structure of both TG48 gene and full length cDNA indicate that of two heat shock induced mRNAs, the 1.8 kb transcript is encoded by this gene. Northern analysis indicates this gene is subjected to tissue specific, developmental and environmental regulation. Sequence analysis of pTCB15 indicated that it encodes a CaM binding glutamate decarboxylase (GAD). Two full length GAD clones were isolated by screening a N. tabacum L. cv Xanthi cDNA library. Both clones had similar nucleotide sequences except that GAD9 had a shorter 3' UTR. CaM binding domain of the tobacco GAD may be located at C-terminus and possibly a BAA structure. Results presented here indicate GAD transcript level is subjected to developmental, environmental and tissue specific regulation.
dc.description.degreePh.D.
dc.identifier.urihttp://hdl.handle.net/10125/9480
dc.languageeng
dc.publisherUniversity of Hawaii at Manoa
dc.relationTheses for the degree of Doctor of Philosophy (University of Hawaii at Manoa). Botanical Sciences (Plant Physiology); no. 3249
dc.rightsAll UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
dc.subjectHeat shock proteins
dc.subjectTobacco--Physiology
dc.subjectCalmodulin
dc.subjectTobacco--Genetics
dc.titleMolecular cloning and characterization of a heat-shock induced calmodulin binding protein gene and cDNAs encoding glutamate decarboxylase from tobacco
dc.typeThesis
dc.type.dcmiText

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Ph.D. - Botanical Sciences (Plant Physiology)