Transgenic tobacco containing a rat glutathione peroxidase gene: Studies on the mechanism of incorporation of selenocysteine into plant protein

Gaitry Ramakrishnan Iyer, Purdue University

Abstract

Selenium is an essential constituent of several enzymes in bacteria and mammals. It is found within the active site of these enzymes as selenocysteine, that is inserted cotranslationally, making selenoenzymes a unique group of proteins. Some of the signals directing selenoprotein synthesis in Escherichia coli as well as mammals have been identified. Selenocysteine is incorporated cotranslationally at a specific, in-frame UGA codon, utilizing a unique selenocysteine-charged tRNA containing the appropriate UCA anticodon. Recoding of UGA from nonsense to selenocysteine depended upon a specific sequence in the mRNA, which was reported to form a stem-loop structure. In E. coli, this sequence is present immediately downstream from UGA. In mammals however, a 200 nucleotide "selenocysteine insertion sequence" in the 3$\sp\prime$ utr has been shown to be critical. A biochemical function for Se has not yet been established in higher plants. The objective of this study was to determine if the specific components of the translation machinery essential for the incorporation of the unusual amino acid selenocysteine, are present in higher plants. Tobacco plants were transformed with the coding region of Se-dependent GPx cDNA. GPx enzyme activity (above the background) was not detected in the transgenic plants. A heat sensitive, hydrogen peroxide-dependent enzyme activity was detected in wild type tobacco. It appears to be a Se-independent activity as it was not inhibited by mercaptosuccinate, an inhibitor of GPx. These results can be explained by the fact that, only the coding region of the GPx cDNA was used to transform tobacco. The sequences in the 3$\sp\prime$ utr of the mammalian selenoproteins are required for the recognition of UGA as a selenocysteine codon. Therefore, new constructs were made in which the 3$\sp\prime$ utr was also included. As cysteine mutants of selenoenzymes are shown to be enzymatically active, tobacco transformations were done using a cysteine mutant of GPx. If the plants lack the ability to incorporate selenocysteine into a polypeptide, then the cysteine mutant of GPx would be expressed in the plants. The transgenic plants transformed with the new constructs, were not obtained and therefore, their analysis could not be included in this study.

Degree

Ph.D.

Advisors

Mason, Purdue University.

Subject Area

Molecular biology|Biochemistry|Plant propagation

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