Structural Analysis of Dna Complementary to Bovine Parathyroid Hormone Messenger-Rna and Restriction Endonuclease Mapping of the Parathyroid Hormone Gene
Gordon, David Frederick
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https://hdl.handle.net/2142/71416
Description
Title
Structural Analysis of Dna Complementary to Bovine Parathyroid Hormone Messenger-Rna and Restriction Endonuclease Mapping of the Parathyroid Hormone Gene
Author(s)
Gordon, David Frederick
Issue Date
1982
Department of Study
Physiology and Biophysics
Discipline
Physiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Animal Physiology
Abstract
To facilitate studies on the structure and regulated expression of the gene for parathyroid hormone (PTH), full length single and double stranded DNA complementary (cDNA) to bovine PTH mRNA was synthesized by reverse transcriptase and E. coli DNA polymerase I. Single stranded cDNA migrated as a single discrete band of 750 nucleotides by denaturing gel electrophoresis. Hybridization of the cDNA to excess template mRNA revealed one rapidly hybridizing component consisting of 50% of the cDNA. Although second strand cDNA synthesis yielded incomplete transcripts at 15(DEGREES)C, incubations between 20-37(DEGREES)C produced one major cDNA species of 1500 nucleotides with a hairpin loop. The loop was specifically cleaved with S1 nuclease to produce a 700bp double stranded species. A restriction map was constructed of the population of cDNA molecules. Fragments corresponding to the 5' terminus of the sense strand were identified by comparing the fragment sizes obtained before and after cleavage of the hairpin loop.
PTH cDNA was recombined with plasmid pBR322 DNA by dC/dG homopolymer extension techniques and was amplified in bacteria. Plasmids with the largest inserts were selected by agarose gel electrophoresis and those containing PTH cDNA were detected by the release of a 380bp SstI fragment as predicted from the map of uncloned cDNA.
The complete nucleotide sequence of the largest plasmid, pPTHi4, was determined by analysis of 12 overlapping restriction fragments by the chemical method. The PTH cDNA insert contained 92bp in the 5' noncoding region, 348bp coding for bovine preproPTH, and 224bp in the complete 3' noncoding region in addition to 52bp of the polyA tail. The consensus sequence 5'AAUAAA3' is present about 20bp from the polyA tract as in nearly all eucaryotic mRNAs sequenced so far. The internal organization of the bovine PTH gene was examined by Southern blotting. Single major bands for a variety of enzymes suggested only one PTH gene. Comparison of restriction endonuclease maps of PTH cDNA and the PTH gene revealed the presence of at least one intervening sequence of 1500bp in the 5' region.
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