미꾸라지(Misgurnus mizolepis) alpha actin 유전자의 isoform별 구조 및 발현 특징 분석

Abstract

Three alpha actin isoform genes were isolated and characterized from mud loach (Misgurnus mizolepis). Mud loach alpha actin isoforms [i.e., α-skeletal (ACTA1), α-cardiac (ACTC1) and α-smooth (ACTA2)] shared a significant homology one another at both coding nucleotide sequence and amino acid sequence levels; also they revealed highly conserved features typically known for other vertebrate counterparts with a considerable degree of sequence homology in the vertebrate lineage. At amino acid level, several amino acid residues are isoform specific, allowing each isoform type to be distinguished from other isoforms. Molecular phylogenetic analysis of alpha actin genes revealed that vertebrate alpha actin genes formed a monophyletic group. Overall, ACTA1, ACTC1 and ACTA2 are divergently visualized in the phylogenetic tree. Within each isoform group, the tree topology was in accordance with the patterns of known taxonomic appraisal. Genomic isoform genes of mud loach alpha-actins showed an identical structure in terms of the presence of a non-translated exon, 8 translated exons and 8 introns including one following the non-translated exon. Conserved gt/ag rule was consistent in every exon-intron boundary region. From the bioinformatic analysis of the regulatory upstream region of each actin isoform, various putative motif sites targeted by different transcription factors were predicted. They included sites for ATF-1, C/EBP, GKLF, MyoD, MEF-2, and SMAD3, which have been known to be involved in cell differentiation and growth. In addition, motifs for stress/immune-response factors such as AP1, AHR, GR, MTF-1, HIF-1 and IRFs as well as potential binding sites for some factors related with lymphocytes such as E2A, LEF-1 and TCF-1 were also predictable. In spite of very high sequence identity among actin isoforms, their mRNA expression patterns were highly isoform-dependent and the patterns of tissue distribution were generally in congruent with their proposed functions. Mud loach ACTA1 mRNA was observable in wide array of tissue types including gonadic tissues; however, in a quantitative term, its basal expression level was highly predominant in skeletal muscle. In case of ACTC1, mRNA expression was detectable in less number of tissues than was ACTA1 with the highest expression level in heart. On the other hand, ACTA2 showed more ubiquitous distribution of mRNA expression across tissues in which highest expression was found in intestine/spleen. During embryonic development ACTA1/ACTC1 mRNA expression began to appear at the time of myotome formation, peaked at the period from hatching to yolk sac absorption and reduced afterward the first feeding (3 days post hatching). On the other hand, ACTA2 mRNA expression was not clearly observable during pre-hatching period. Onset expression was apparent from hatching, peaked at the period from 2 to 4 days after hatching, and then reduced afterward. In summary, findings from this study suggest that the three actin isoforms from mud loach be regulated highly in an isoform-specific manner in adult tissues and developing embryos, in spite of their extremely high structural homology. Data from the present study could be a good basis for future study not only to gain a deeper insight into the functional diversification or neofunctionalization of alpha actin isoforms in vertebrate lineage and also to utilize the actin regulators for genetic manipulation with various theoretical and practical research purposes.