Abstract
Background/Aim: Core clock genes exert influence over a spectrum of behavioral consequences and physiological processes. In mammals, distinct transcription-translation feedback loops oscillate approximately every 24 hours, aligning peripheral tissues with the dark-light cycle. Consequently, species manifest diverse activity patterns in their daily routines; for instance, diurnal species exhibit sleep during dark periods and heightened activity during light periods. Over the course of evolution, subtle changes in protein sequences can lead to divergent phenotypic traits. This study aims to scrutinize core clock protein residues to identify significant amino acid sites that might underlie diurnal or non-diurnal tendencies from an evolutionary standpoint.
Methods: We performed multiple sequence alignment on coding sequences of vertebrate core clock protein, CRY1, orthologs obtained from the NCBI database. This was achieved using the Constraint-based Multiple Alignment Tool (COBALT). Vertebrate species were classified into diurnal (n=127) and nocturnal (n=92) categories. Statistical methods were applied to analyze the non-random distribution of amino acid residues between the two groups.
Results: Our analysis of the CRY1 protein provides initial insights into specific amino acid residues exhibiting non-random associations with both nocturnal and diurnal behaviors. Notably, we identified five residues subject to purifying selection and 21 residues under positive selection within CRY1 orthologs. These residues span across exons 1, 4, and 9-12, predominantly concentrated in the tail region.
Conclusion: This preliminary investigation highlights the importance of particular amino acids within core clock proteins, offering valuable insights into their functional significance. The observed discrepancies in conservation patterns between diurnal and nocturnal groups underscore the potential disruption of essential folding processes or post-translational mechanisms by mutations in these regions. This research marks a pioneering advancement in understanding the phenotypic outcomes of core clock proteins and their direct correlation with behavioral traits within evolutionary contexts, potentially linking to sleep disorders.

Key words: Circadian Rhythm, CRY1 Protein, Behavioral Traits, Chronotype