| Expression
Knockdown of RSP7, an EF-Hand Protein in Chlamydomomas, Using RNAi
Leo
Studach
Marquette
University
Summer
Mentor: Dr. Pinfen Yang
Diverse
evidence indicates that ciliary and flagellar motility is regulated by
radial spokes. However, the exact mechanism remains to be revealed. RSP7
is postulated to be an integral part of this molecular mechanism. Analysis
using SMART and Blast indicate the presence of seven EF-Hand domains which
are believed to bind calcium. The N-terminus also contains a RIIa domain.
We hypothesize that this RIIa domain of RSP7 interacts with RSP3 through
an AKAP domain. Through this interaction these two proteins are believed
to be critical in mediating calcium induced waveform changes. RNA interference
(RNAi) will be used to test this hypothesis. Expected is the normal assembly
of the radial spoke. However, transformants will predictably fail to display
photoresponse that is induced by calcium
A PCR
approach was used to build an RNAi construct. cDNA was used as template
to create a hairpin structure with an inverted repeat region and loop.
This construct was cloned into an expression vector for transformation
into Chlamydomonas. Transformants were selected through a double
selection process of first paromomycin and then L-tryptophan and 5-fluroindole.
After the double selection several clones were eliminated indicating the
absence of the genes necessary for survival and the RNAi construct.
Preliminary
results show abnormal phenotype in selected strains compared to wild type.
We are currently evaluating motility defects in these strains and will
analyze the level of RSP7 compared to wild type in the radial spoke. |
