Identification and cell-free translation of mRNA coding for a precursor of parathyroid secretory protein.
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Biomedical subjects
Publications and source records attributed to H M Kronenberg.
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We have cloned in Escherichia coli a DNA copy of mRNA coding for bovine preproparathyroid hormone. Double-stranded DNA was inserted into the Pst I site in plasmid pBR322 by using the poly(dG)-poly(dC) homopolymer extension technique to join the DNA molecules. Recombinant plasmids coding for preproparathyroid hormone were identified by the plasmid's ability to arrest specifically the translation of preproparathyroid hormone mRNA. The nucleotide sequence of the largest recombinant was determined by using both chemical and enzymatic techniques. The parathyroid insert contains 470 nucleotides--102 nucleotides from the 5' noncoding region of the mRNA, 345 nucleotides representing the entire coding region, and 23 nucleotides from the 3' noncoding region. The coding sequence clarifies the hormone's amino acid sequence, which has been disputed. Codon usage is discussed.
The precursor of bovine proparathyroid hormone was synthesized by translation of parathyroid mRNA in a wheat-germ cell-free system. The amino acid sequence of the NH2-terminal extension (the pre sequence) was determined by repetitive Edman degradation of the polypeptide labeled with radioactive amino acids (radiosequencing). The pre sequence of pre-proparathyroid hormone is (formula: see text) which is followed by the sequence of proparathyroid hormone. It is significant that 20 of the 25 amino acids in the sequence are hydrophobic. This high hydrophobicity is consistent with the proposed role of the pre sequence as a membrane-penetrating peptide. The precursor-specific sequence of 31 amino acids was snythesized chemically by the solid-phase technique. This synthetic peptide was shown to bind to the microsomal fraction of homogenates prepared from extracts of parathyroid glands, a finding consistent with the proposed role of the precursor peptide in the attachment of the nascent chain--mRNA--ribosome complex to the endoplasmic reticulum.
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DNA complementary in sequence to the messenger RNA for pre-proparathyroid hormone was synthesised using reverse transcriptase. In a linked transcription-translation system using RNA polymerase and cell-free extract from wheat germ, the DNA directed the synthesis of a protein identified as pre-proparathyroid hormone by N-terminal sequencing and by electrophoretic and immunologic criteria.
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