Synthesis, restriction analysis, and molecular cloning of near full length DNA complementary to bovine parathyroid hormone mRNA

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Parathyroid hormone (PTH) is crucial for calcium and phosphate homeostasis.
• Understanding PTH gene expression requires methods to isolate and analyze its messenger RNA (mRNA).

Purpose of the Study:

• To synthesize and characterize complementary DNA (cDNA) of bovine parathyroid hormone (PTH).
• To generate a restriction map of the PTH cDNA for clone identification.

Main Methods:

• Synthesis of cDNA from bovine parathyroid hormone mRNA using avian myeloblastosis viral reverse transcriptase.
• Characterization of cDNA using S1 nuclease digestion and RNA hybridization.
• Construction of double-stranded cDNA and subsequent restriction endonuclease digestion.
• Identification of restriction fragments using S1 nuclease to cleave hairpin loops.

Main Results:

• Synthesized bovine PTH cDNA of approximately 750 bases, highly sensitive to S1 nuclease.
• Identified a rapidly hybridizing component in the mRNA preparation.
• Generated a double-stranded PTH cDNA amenable to cleavage by Alu I, Mbo II, Sau 3A, Sst I, and Taq I.
• Constructed a partial restriction map of the PTH cDNA.

Conclusions:

• The synthesized PTH cDNA is suitable for identifying bacterial clones containing the gene.
• The restriction map facilitates the selection of recombinant plasmids with full-length PTH cDNA inserts.
• This work provides a foundation for further studies on PTH gene regulation and expression.