Cloning and characterization of mouse growth hormone-releasing hormone (Grh) complementary dna: Increased grh messenger rna levels in the growth hormone-deficient lit/lit mouse

We have isolated and cloned the full length cDNA for mouse GH-releasing hormone (mGRH) from mouse hypothalamus using a recently described strategy involving the polymerase chain reaction technique (PCR). Degenerate oligonucleotide primers were selected based on short (six amino acids) conserved regions in the human and rat GRH peptides that would recognize DNA sequences encoding similar amino acids regardless of codon usage. Primer-extended cDNA was amplified by PCR on cDNA templates prepared by reverse transcribing total mouse hypothalamic RNA. After cloning and sequencing the initial product, the 3′ and 5′ ends of mGRH were generated using a separate PCR strategy (RACE protocol). The mGRH cDNA encodes a 103-amino acid reading frame, structurally similar to the human and rat GRH genes, containing a signal sequence, a 42-residue GRH peptide, and a 31-residue C-terminal region. Although the structures of mouse and rat GRH are highly conserved in the signal peptide and C-terminal region, there is considerable diversity in the GRH region, which exhibits nearly comparable homology with the rat (68%) and human (62%) structures. Differences between mouse and rat GRH were also found in the amino acid cleavage sites at the 5′ and 3′ ends of the mature peptide and at the polyadenylation signal. Northern blot analysis of hypothalamic RNA revealed a single band of approximately 750 bases that was visible using total RNA from one or poly(A)⁺ RNA from 1.5 hypothalami. A single band of similar size was also detected using 3 µg placental total RNA. Northern blotting of hypothalamic RNA from little (lit/lit) mice with isolated GH deficiency revealed a 3-fold increase in hypothalamic GRH mRNA levels, providing evidence for an inhibitory role of GH on GRH gene expression.