Title page for ETD etd-07052007-000931


Type of Document Dissertation
Author Ynalvez, Ruby Agno
Author's Email Address rynalv1@lsu.edu
URN etd-07052007-000931
Title Isolation and Characterization of Two Closely Related Beta-Carbonic Anhydrases of Chlamydomonas reinhardtii
Degree Doctor of Philosophy (Ph.D.)
Department Biological Sciences
Advisory Committee
Advisor Name Title
James V. Moroney Committee Chair
David J. Longstreth Committee Member
John C. Larkin Committee Member
Sue G. Bartlett Committee Member
Bin Chen Dean's Representative
Keywords
  • carbon dioxide concentrating mechanism
  • carbonic anhydrases
  • insertional mutant
  • Chlamydomonas reinhardtii
Date of Defense 2007-06-14
Availability unrestricted
Abstract
Aquatic photosynthetic organisms such as the green alga, C. reinhardtii respond to low carbon dioxide conditions by inducing a carbon dioxide concentrating mechanism (CCM). Important components of the CCM are carbonic anhydrases (CAs), zinc metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. C. reinhardtii now has nine carbonic anhydrases, three alpha-CAs and six beta-CAs. This dissertation describes the identification and characterization of two closely related beta-CAs, CAH7 and CAH8. These CAs are 63% identical.

CAH7 and CAH8 encode proteins of 399 and 333 amino acids, respectively. Both of these CAs are constitutively expressed at the transcript and protein level. Preliminary results of immunolocalization studies localized CAH7 in the chloroplast while CAH8 was localized in the periplasm. Both the CAH7 and CAH8 open reading frames (ORF) were cloned in the overexpression vector pMal-c2x and expressed as recombinant proteins. Activity assay demonstrated that CAH7 and CAH8 are active carbonic anhydrases. The proposed roles for CAH7 and CAH8 are discussed.

Previously, insertional mutants were generated to be able to isolate bicarbonate-transporters and other proteins that might be essential for a functional CCM. One of the generated insertional mutants is slc211, a mutant that requires high carnon dioxide for optimum growth. The mutant slc211 had an insertion in the novel gene designated as CIA7. RNA interference was successfully used to reduce the expression of CIA7. The resultant transformants had a growth phenotype similar to slc211 requiring high carbon dioxide for optimum growth. These results suggest that CIA7 is a gene that facilitates growth in C. reinhardtii under low carbon dioxide conditions. The possible functions of CIA7 are discussed.

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