Sequence and characterization of cDNA encoding the motilin precursor from chicken, dog, cow and horse. Evidence of mosaic evolution in prepromotilin.

This research aimed to isolate and sequence the cDNA encoding the motilin precursor from different species including chickens, dogs, cows, and horses. Motilin is a crucial component in the regulation of fasting motility patterns in humans and dogs. However, its role functions differently in other species. The findings present evidence of ‘mosaic evolution’ showing that different regions of the motilin precursor have evolved at varying rates.

Research Approach

• The research began by isolating the total RNA from the duodenal mucosa of chickens, dogs, cows, and horses.
• Then, single-stranded cDNA was synthesized. Motilin cDNA fragments were subsequently amplified using PCR, a molecular biology technique for creating multiple copies of DNA.
• These amplified fragments were then ligated into a plasmid and cloned. Clones that tested positive after they were screened with a radioactively labelled probe were then chosen to undergo sequencing.
• The ‘Rapid Amplification of cDNA Ends’ (RACE) method was used to determine the 5′- and 3′- ends of the cDNA.

Findings

• Analysis of the cDNAs revealed an open reading frame coding for either 115 (in chickens and cows) or 117 (in dogs and horses) amino acids.
• The entire sequence consists of a 25 amino acid signal peptide, the motilin, and a motilin associated peptide (MAP) made up of either 68 (in chickens and cows) or 70 (in dogs and horses) amino acids.
• Similar to all motilin precursors sequenced before (man, monkey, pig, and rabbit), an endoproteinase cleavage site was found present at Lys(23)-Lys(24).
• Although there was significant similarity in the amino acid and nucleotide sequence of the signal peptide and motilin across all the species, the MAPs demonstrated substantial differences, both in length and in sequence.

Conclusion

• The research contributes valuable insights into the evolution of motilin precursors by highlighting that different regions have evolved at different rates – a demonstration of ‘mosaic evolution’.
• The findings also indicate the sequence similarity and sequence divergence among different species, thus furthering our understanding about the function and evolution of motilin in different species.