Major
Biological Sciences
Advisor
Roberts, Alison, W
Advisor Department
Biological Sciences
Date
5-2012
Keywords
Cellulose; Moss; Molecular; Zinc; CESA
Abstract
Understanding the Importance of the zinc binding domain in the cellulose synthase complex: Some Assembly Required
Alfred Schupp
Sponsor: Alison Roberts, Biological Sciences
Cellulose microfibrils are vital components of the cell wall. A microfibril is made up of multiple strands of glucose chains, and these cellulose microfibrils are produced by protein complexes in the plasma membrane called cellulose synthase complexes, or CSCs for short. Each complex is made up of 36 subunits called Cellulose Synthase proteins, or CESAs. Each CESA produces a glucose polymer made from beta-1,4 glucan linkages. It is known that CESA proteins aggregate to form CSCs, and that different CESA proteins come together to make the primary or secondary cell wall, but exactly how the proteins associate is unknown.
Previous studies in cotton suggest that the beginning of the CESA protein contains a zinc binding motif with specific amino acid residues that form strong covalent bonds following oxidation. This senior project explores what would happen if this motif were completely deleted and not simply mutagenized as previous studies have done. I looked at CESA5, which is required for the formation of leafy gametophores in the moss Physcomitrella patens. If DNA encoding a zinc binding domain deficient CESA5 cannot rescue a CESA5 knockout, it suggests that this motif is essential for proper protein association in the CSCs involved in the formation of leafy shoots. It does not explain which CESAs specifically interact, but it would show the importance of the zinc binding domain for protein interaction.
If a CESA5 knockout could not be rescued, I wanted to know if a coiled-coil, another type of protein association, could restore the normal phenotype in the zinc binding domain deficient CESA5.
The project was carried out using PCR, restriction digestion and ligation enzymes to produce the gene with the deleted zinc binding motif, and transformation techniques to introduce the altered gene into the moss plant.
Keywords: Cellulose, Moss, Molecular, Zinc, CESA,