Molecular cloning of newt prolactin (PRL) cDNA: Effect of temperature on PRL mRNA expression

A partial prolactin (PRL) cDNA was specifically PCR amplified from a cDNA l ibrary constructed from pituitary mRNAs of the newt (Cynops pyrrhogaster) a nd cloned into plasmid vectors. One clone thus obtained contained a 739-bp insert encoding the C-terminal amino acid sequence of the mature hormone mo lecule. Using this clone as a probe, the full-length newt PRL cDNA was scre ened from the cDNA library. The PRL cDNA clone thus obtained consisted of 1 024 bp encoding the entire sequence of the mature PRL molecule in addition to its signal peptide. The amino acid sequence of newt PRL deduced from its nucleotide sequence showed higher homologies with those PRL sequences of t etrapod animals than with those of teleosts. Northern blot analysis reveale d the newt PRL mRNA size to be approximately 1 kb. In situ hybridization us ing the newt PRL cDNA as a probe revealed that the pituitary region express ing PRL mRNA corresponded to that immunoreactive with antiserum against PRL . PRL mRNA levels in the pituitary of newts subjected to room and low tempe ratures were determined by Northern analysis employing the PRL cDNA as a pr obe. PRL mRNA levels were significantly higher in the pituitaries of newts subjected to 10 degrees than in those of newts kept at 23 degrees. Likewise , immunoassayable plasma PRL levels were higher in animals subjected to 10 degrees than in those kept at 23 degrees. (C) 2001 Academic Press.