NUCLEOTIDE AND AMINO-ACID-SEQUENCE OF LYMPHOCYTE-DERIVED CORTICOTROPIN - ENDOTOXIN INDUCTION OF A TRUNCATED PEPTIDE

To determine the degree of similarity between pituitary and lymphocyte proopiomelanocortin, the lymphocyte mRNA was reverse transcribed, cloned, and sequenced. Murine lymphocyte mRNA was first purified by oligo(dT)-cellulose affinity chromatography and was reverse transcribed by using a selective 3′ antisense oligonucleotide primer directed at the boundary between the translated/nontranslated region on the 3′ end of exon 3. This cDNA was then amplified in a polymerase chain reaction with selective primers containing Sal I and Kpn I restriction endonuclease sites. Amplified cDNA was then directionally ligated into M13mp18 and M13mp19 bacteriophage and was sequenced. The nucleotide sequence encoding this peptide was identical to that of mouse pituitary corticotropin (ACTH). Elevated levels of lymphocyte immunoreactive ACTH were then induced with bacterial lipopolysaccharide and the peptide(s) was purified by antibody affinity chromatography and reverse-phase high-performance liquid chromatography. The predominant immunoreactive ACTH species was ≈3 kDa and its sequence was identical to pituitary ACTH(1-25). These results conclusively demonstrate that lymphocytes produce authentic ACTH and harbor its mRNA.