Date of Award

1996

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

James V. Moroney

Abstract

At low CO$\sb2$ conditions, C. reinhardtii, like many other green algae, induces a CO$\sb2$ Concentrating Mechanism (CCM) to raise the internal Ci concentration. During the induction, at least five new polypeptides and six different genes are upregulated and some of the corresponding mRNAs or proteins are absent in high CO$\sb2$ requiring mutants. However the identities and functions of these genes are unknown. In this dissertation, I partially characterized four low CO$\sb2$ inducible genes and one low CO$\sb2$ inducible protein LIP-36. The complete cDNA sequences of clones 11I3 and 2I5 are 1311 bp and 985 bp, respectively. Neither of these two clones has any significant homology to known genes. Investigation of their expression in three high CO$\sb2$-requiring mutants suggested that 11I3 might play some roles in the CCM, but clone 2I5 might not be directly involved. The full-length cDNA of a third gene, clone 4I29, is 2552 bp long encoding a putative protein of 521 amino acids. Homology studies suggested it encodes an alanine $\alpha$-ketoglutarate aminotransferase. This is supported by enzyme activity assays and Western blot analysis. Further physiological studies using an aminotransferase inhibitor indicated that it is involved in the efficient operation of the C$\sb2$ cycle to minimize the carbon loss due to photorespiration. To investigate the possible functions of LIP-36 in CCM, LIP-36 protein was purified and partial amino acid sequences were obtained. Based on the peptide sequences, the two genes encoding the LIP-36 were cloned. LIP-36 G1 is 2025 bp with an ORF of 358 amino acids. The complete cDNA sequence for LIP-36 G2 is 2068 bp encoding a protein of 355 amino acids. One interesting discovery is that the DNA sequence of LIP-36 G1 is identical to that of another low CO$\sb2$ inducible clone 4I7. The two genes were both upregulated by low CO$\sb2.$ These two genes have been over-expressed in E. coli, and both gene products were recognized by LIP-36 antibodies. Sequence analysis suggests that LIP-36 is a transporter protein and shares 30-35% homology with mitochondrial carrier proteins.

ISBN

9780591289008

Pages

173

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