Date of Award
Doctor of Philosophy
Genetics, Development and Cell Biology
Ted W. Huiatt
The objective of this project was to construct and identify a cDNA probe for the striated muscle protein titin. RNA was isolated from skeletal and cardiac muscle of 16 day chicken embryos using a guanidinium isothiocyanate procedure. The RNA thus isolated was both intact (as shown by agarose gel electrophoresis) and transcribable in vitro; additionally, it was possible to obtain very large RNA molecules in this manner. cDNA was then constructed utilizing random oligodeoxynucleotides for priming of the first strand synthesis reaction, instead of the more common oligo-dT priming. Random priming was also utilized for second strand synthesis. As with the RNA isolation procedures, it was possible to construct very large (>25 kb) cDNA molecules by this method;Four libraries were constructed: one derived from poly-A[superscript]+ skeletal muscle RNA, one from total skeletal muscle RNA, and two from total heart RNA. After addition of EcoRI linkers, the libraries were inserted into the expression vector [lambda]gt11 for subsequent screening with rabbit antibodies prepared against chicken skeletal muscle titin. Three of the libraries were inserted into [lambda]gt11 without additional modification, but one of the heart libraries was digested with three endonucleases prior to insertion into the vector to reduce the size of the passenger DNA. The libraries contained an average of 94% recombinants and ranged from 2.6 x 10[superscript]5 to 1.8 x 10[superscript]6 total plaque-forming units;Three of the four libraries were screened with a polyclonal titin antiserum, and 20 putative positive clones were identified. One of these, from the nuclease-digested cardiac muscle library, showed a stronger reaction than the others. This clone was purified and the DNA isolated. The presence of an insert, approximately 200 base pairs in length, was demonstrated by agarose gel electrophoresis of an EcoRI digest of the purified DNA. Western blot analysis of plaque lysates from the positive clone demonstrated the presence of a fusion protein recognized by both anti-[beta]-galactosidase and anti-titin antibodies. The insert was subcloned into the phagemid pUC118 for sequencing. Sequence analysis demonstrated the presence of an open reading frame. Finally, insert was radiolabeled and shown to hybridize with a high-molecular-weight RNA species in heart, but not brain, RNA by Northern blotting. Thus, this clone, isolated from a cardiac muscle cDNA library, appears to contain a cDNA insert coding for titin.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Greg Robert Sater
Sater, Greg Robert, "Isolation of a chicken cardiac muscle titin cDNA probe " (1990). Retrospective Theses and Dissertations. 9887.