Electronic Theses and Dissertations

Identifier

531

Date

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biology

Committee Chair

Steven Donald Schwartzbach

Committee Member

Carlos Enrique Estrano

Committee Member

Judith Cole

Committee Member

Charles Lessman

Abstract

The Euglena plastid is thought to have evolved from a phagocytosed eukaryotic algae residing within a vacuolar compartment. Critical to the conversion of endosymbiont into a plastid was development of a mechanism to return nuclear encoded plastid proteins back to the chloroplast. The chloroplast protein targeting bipartite presequence contains an ER targeting signal peptide and a plastid targeting transit peptide. Since only some Euglena plastid targeted proteins contain a transit peptide hydrophobic domain, the evolutionary origin and function of this domain remains unclear. Targeting sequence function is evolutionarily conserved as evidenced by the in vitro co-translational translocation of Euglena chloroplast preproteins into canine microsomes and pea chloroplasts. Taking advantage of evolutionary functional conservation, Euglena presequence-GFP deletion and insertion constructs were expressed in mammalian cells to determine the function and evolutionary origin of the bipartite presequence regions. The presence of the transit peptide hydrophobic domain confers signal peptide functionality to the transit peptide. The addition of the Euglena hydrophobic domain to Arabidopsis transit peptides makes them functional signal peptides. This suggests that transit peptides evolved from signal peptides.A nuclear Genome transformation protocol has been developed for the Euglena gracilis. A transformation cassette encoding the selectable zeocin resistant marker was constructed by inserting the Sh ble gene conferring zeocin resistance between the 5' and 3' end of the Euglena LHCPII gene. Zeocin resistant cells were isolated after electroporation and three rounds of zeocin selection. PCR demonstrated the presence of the Sh ble gene linked to the 5' and 3' end of the Euglena LHCPII gene in transformed cells. Expression of the Sh ble protein in cells maintained for one year under nonselective conditions under control of the LHCPII promoter was demonstrated by western blotting. The presence of zeocin resistant gene and Sh ble protein was demonstrated by PCR and western blotting indicating that stable transformation of Euglena has been achieved.

Comments

Data is provided by the student.

Library Comment

Dissertation or thesis originally submitted to the local University of Memphis Electronic Theses & dissertation (ETD) Repository.

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