Characterization Of The 5'-to-5' Linked Adult α-and β-globin Genes From Three Sciaenid Species And Their Cis-regulatory Elements

In most bony fish and higher vertebrate species, hemoglobin (Hb) is a tetramericmolecule that consists of twoα-globin and twoβ-globin chains. Each of the fourglobin chains is coordinated with a heme group that functions directly for oxygentransport. As the most primitive group among the vertebrates, fish provides anexcellent model for studying the diversity and evolution of globin genes.Sciaenidae is widely distributed throughout the world and is one of the largestfamilies in the order Perciformes, with approximately 70 genera and 300 species. Fishfrom this family were popularly known as croakers or drums due to the sound theyproduced using muscles associated with the swim bladder. Large yellow croaker(Pseudosciaena croeea), red drum (Sciaenops ocellatus) and cuneate drum (Nibeamiiehthioides) are the members of the family Sciaenidae, which are the major fish inartificial breeding in the Southeast Chinese shore. However, there are only a fewmolecular biological studies on these species. To understand the process of globingene duplication and identify the regulatory elements present in the globin genes, wecloned oneα-and twoβ-globin genes from the P. crocea , and oneα-and twoβ-globin genes from the S. ocellatus. When comparing with humanα-andβ-globins,three important alterations in the structural regions of the two sciaenid globins can benoted:α39 Thr→Gln,α123 His→Tyr andβ117 His→Lys. Theα-andβ-globin genesare all composed of three exons separated by two introns, and the positions of theintrons conform to the vertebrate norms for globin genes. Furthermore, the intronicsplice junctions follow the GT/AG rule.Linkage analysis revealed that theα-andβ₂-globin genes from the P. croceawere oriented head-to-head relative to each other. The orientation of theα-andβ₁-globin genes from the S. ocellatus was also head-to-head. The three sciaenid globingene complexes, which include the linkedα-andβ-globin genes and the intergenicregion between the translation initiation codons of them were identified and compareddetailedly. The three sciaenid globin gene complexes revealed a remarkable degree of sequence identity. Theα-globin genes in the complexes all possessed the insertion ofGGG(A) at the second exon in contrast to the other fishα-globin genes. We concludedthat it was the common feature of the species in family Sciaenidae. Although the firstintrons of theα-globin genes differ in size, they contain high identical regions. Themajor difference of the sciaenidα-globin genes was focused on the second introns.The difference of the second introns was found not only in interspecies but also inlarge yellow croaker obtained from different areas. A repeat sequence (35bp)occurring midway in the second intron was observed in the large yellow croakerobtained from Zhoushan. This repeat could be the result of an inversion that tookplace in theα-globin gene during the evolution. The sciaenid intergenic regionsbetween the linkedα- andβ-globin genes were the smallest in reported fish globincomplexes to date. Many evolutionary conserved sequences such as TATA box,CCAAT box, CACCC motif and GATA motif were found in the intergenic region.We also found TATA box, CACCC and GATA motifs in the first introns of thesciaenidβ-globin genes.We constructed the expression vector pInter/EGFP with ligating the intergenicregion from large yellow croaker globin complex. The promoter function of the P.crocea intergenic region was tested by transient expression of EGFP in Vero cells. Toexplore the three sciaenid intergenic promoter strength, we cloned the three intergenicregions into the luciferase-reporter vector pGL3-Basic and transfected the constructspRedrum, pCroaker and pCuneate into the Vero cells respectively. The luciferaseactivity with the pRedrum was slightly elevated (no statistically significant difference),compared to the pCroaker and pCuneate constructs.To identify the regulatory elements present in the intragenic regions of the P.crocea globin complex, different fragments of the intergenic region alone or togetherwith theβ-globin gene first intron were cloned into the luciferase-reporter vectorpGL3-Basic respectively. The reporter gene constructs were transiently transfectedinto the primary erythrocytes of crucian carp respectively. The intergenic regions cannot support high-level expression of luciferase. However the promoter activity of theintergenic region was stimulated by the regulatory elements located in theβ-globin gene intron 1. To determine whether the intron 1 contained a classical enhancer, theintron 1 was inserted into the construct pInterIntron at 3' to the luciferase in bothsense and antisense orientations. Low levels of the luciferase activity were obtainedfrom the plnterInron. Thus, although it had a strong positive effect on the promoteractivity of the intergenic region, the intron 1 only functioned in its physiologicposition, and did not contain a classical enhancer.