Nucleotide sequence of part of the gene for human chorionic somatomammotropin: purification of DNA complementary to predominant mRNA species

A novel purification procedure for DNA complementary to individual mRNA species has been developed by using restriction endonuclease cleavage of cDNA transcribed from a complex mixture of mRNAs. This procedure has allowed us to isolate and analyze DNA fragments complementary to the mRNA coding for the human peptide hormone chorionic somatomammotropin (HCS). The mRNA for this hormone is a major constituent of placental polyadenylated RNA as shown by in vitro translation of placental RNA and by nucleic acid hybridization using HCS cDNA as a specific probe.

The purification of HCS cDNA was achieved by Hae III and Hha I restriction endonuclease cleavage of single-stranded cDNA synthesized in vitro from total polyadenylated placental RNA. Polyacrylamide gel electrophoresis of the products allowed detection and purification of discrete DNA fragments. A comparison of the nucleotide sequence of these fragments with that predicted from the amino acid sequence of HCS demonstrated that the fragments are transcripts of HCS mRNA, containing most of the translated and 3′ untranslated regions. The latter region is characterized by a UAG termination codon immediately adjacent to the translated region (a second in phase UAG occurs 9 nucleotides away) and a palindromic sequence (GUGACCCCUCCCCAGUG) centered 27 nucleotides from the termination codon.

The purification scheme outlined for HCS cCNA should be applicable to DNA sequences complementary to mRNA species which represent at least 2% of any polyadenylated RNA preparation. This was demonstrated by restriction endonuclease cleavage of cDNA synthesized from a mixture of purified rabbit globin and total polyadenylated human placental RNAs.