Leptin is a 167-amino acid hormone produced chiefly by adipocytes. It plays an important role in regulation of food intake, energy metabolism and reproduction in mammals. However, a leptin gene homologue has yet to be cloned in a non-mammalian vertebrate. The aim of this thesis was to establish the existence of a leptin gene homologue in the domestic chicken (Gallus gallus) genome, and to determine the degree of sequence identity with mammalian leptin genes, and with a putative chicken leptin sequence published during the course of the thesis work. An initial attempt was made to
clone the chicken leptin gene by heterologous RT-PCR using degenerate primers to conserved regions of mammalian leptin sequences. However, no leptin-like products were amplified from chicken adipose tissue and liver cDNAs,or from genomic DNA. RTPCR was also used to test the existence of a published chicken leptin cDNA sequence that shares 95% identity with mouse leptin at the nucleotide level. When PCR primers identical to the mouse and published chicken leptin sequences were used, no PCR product sharing close similarity to the mouse leptin sequence were generated from any chicken templates, whereas amplification of mouse leptin leptin sequences was consistently obtained from control mouse templates. Following the failure to clone the
chicken leptin by RT-PCR, evidence for the existence of a mammalian-like leptin in the chicken genome was sought by Southern analysis. Southern blots under low stringency
hybridization and washing conditions revealed hybridization of a mouse leptin probe to chicken genomic DNA. With high stringency washing, the chicken signal disappeared,
while those from sheep and mouse genomic DNA remained. Screening of a chicken adipose tissue cDNA library, and chicken genomic DNA and cosmid libraries with the
same mouse probe failed to isolate a chicken leptin homologue. Collectively, these results indicate that if a chicken leptin homologue exists in the chicken genome, it is likely to be of low homology to mammalian leptin sequences. The results do not support the existence of a mouse-like leptin sequence in the chicken genome, an assertion supported
by theoretical analysis of the molecular evolution of leptin based on the rate of synonymous substitution. This analysis indicated that the probability that the chicken and mouse leptin sequences are 95% identical, is less than one in a million.