International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 2(9), 59-68, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 59 Identification and Characterization of the YGHL1 Gene from Yellowtail fish Seriola quinqueradiata) and the evolution of the YGHL1/HIG1 family in VertebratesAbe Shunnosuke* Ravi Nagganatha Suganthan Sahi Vaidurya Pratap and Morita Eugene HayatoLaboratory of Molecular Cell Physiology, Faculty of Agriculture, Ehime University,3-5-7 Tarumi, Matsuyama 790-8566, JAPANAvailable online at: www.isca.in Received 3rd June 2013, revised 15th June 2013, accepted 21st July 2013Abstract A 1529bp full length cDNA of the yellowtail growth hormone like-1 (YGHL1) from Seriola quinqueradiata was cloned, and its structure, genomic organization and expression were analyzed. The yellowtail YGHL1 gene is composed of three coding exons and one 5' non-coding exon, with putative transcription factor binding sites present in upstream of the transcription site. The YGHL1 in yellowtail fish was highly expressed in brain, gill, heart, and kidney, while no appreciable expression was observed in liver and skeletal muscle. This study also shows the phylogeny and evolution of the YGHL1/HIG1 orthologs in mammals and other vertebrates, by comparing their sequences and syntenic context. According to the deduced peptide sequence alignment, the “YGHL1/HIG1 exon 3 domain” peptide seems to be well conserved in the YGHL1/HIG1 gene family. Our data also suggest that an ancestral locus similar to YGHL1 in Ciona intestinalis underwent duplications to create orthologous loci of the YGHL1/HIG1 family in the vertebrates. The regions encoding the YGHL1/HIG1 paralogs in human and mouse were close to the regions where some homeostatically important genes are clustered. These clusters are prominent in human 3p25-22 and 17q11-12 and their orthologous region in mouse 6D1-6E3 and 11D, showing a concerted evolution between them. Keywords:YGHL1HIG1 exon 3 domain yellowtail evolution vertebrate lineage. Introduction The generation of multiple copies of diverse eukaryotic genes has been attributed to polyploidization in ancestral vertebrates. Following such duplication, a multitude of mechanisms have rearranged the relative positions of different genes and genomic regions, causing diversification of the metabolic pathways that ultimately affect the morphology and function of the organism1-6. Taylor et al reported twenty seven groups of orthologous duplicated fish genes in their study of genome duplication of ray finned fishes which included genes from human, mouse, chicken, frog and zebrafish; and phylogenetic analysis of these genes suggested that genomic duplications took place during the evolution of fish, 300 to 450 million years ago. Genomic studies involving a novel RING zinc finger protein, belonging to the MKRN family8,9, have also provided an insight into the functional linkage and evolution of human chromosome region 3p21-25, in which apparently unrelated orthologous loci in fish, YGHL2MKRN2, RAF1, and the HIGD1Aare present10. These works also illustrate that syntenic analysis of flanking genes is valuable for elucidation of the concerted evolution of gene families. The yellowtail growth hormone like-1 (YGHL1) cDNA (GenBank: D85880) was obtained on screening a pituitary cDNA library of Seriola quinqueradiata (Yellowtail fish or Japanese Amberjack) using the antibody for Salmon growth hormone in 1996. Later, a cDNA encoding a 91aa protein homologous to the yellowtail YGHL1 was identified in liver tissue of a euryoxic fish, Gillichthys mirabilis, as Hypoxia-Inducible Gene 1, HIG1 (GenBank: AF266206), which has shown to be induced after prolonged hypoxia in liver11. Based on the report of Gracey et al11, many members of the HIG1gene family were identified. Based on the genomic organization and extensive conservation of nucleotide and protein sequences orthologous to the yellowtail YGHL1, we propose to call the HIG1 gene family as YGHL1/HIG1 family. In the present study, the yellowtail YGHL1 was cloned and characterized. Its genomic structure including the 5' upstream region and its expression were studied. We compared these sequences with their orthologous loci in vertebrates and invertebrates to understand the evolution of the extensive YGHL1/HIG1 family. We also analyzed their flanking genes and showed that multiple vertebrate paralogs have evolved from a single ancestral gene through concerted evolution along with their flanking genes. Material and Methods Isolation and analysis of genomic DNA: Testis of yellowtail fish (0.5g) was homogenized in 5 volumes of extraction buffer [10mM Tris-HCl (pH.8), 25mM EDTA (pH.8), 100mM NaCl, 0.25% SDS, 100 µg/ml of Protease-K] and incubated at 55C for 90min, the mixture then was extracted three times with an equal volume of TE saturated phenol, followed by three times International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(9), 59-68, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 60 with an equal volume of phenol/chloroform/isoamyl alcohol, 25:24:1 (v/v/v). The aqueous phase was purified at least twice with an equal volume of chloroform/isoamyl alcohol, 24:1 (v/v). The extracted genomic DNA was precipitated from the aqueous phase with 2.5 volumes of ethanol at 20C. The pellet was collected by centrifugation and dissolved in TE buffer (10mM Tris-HCl [pH7.5], 1mM EDTA [pH7.5]). The exon/intron boundary structure was analyzed by means of the polymerase chain reaction (PCR) using oligonucleotide primers based on the cDNA sequence. The fragments obtained on PCR were subcloned using a pMOSBlue blunt ended cloning kit (GE Healthcare, USA). Isolation of total RNA, determination of cDNA extremities, and cDNA analysis: A two to three years old yellowtail was obtained from a local fishery and total RNA was immediately extracted from various organs using an RNA Extraction Kit (GE Healthcare, USA). The 5'-cDNA extremity was determined by the rapid amplification of cDNA ends (RACE) method. Poly (A+) RNA was purified from total RNA (100 µg) using an mRNA Purification kit (GE Healthcare, USA). After annealing 1 µg of poly (A) RNA with 10 pmoles of a gene specific reverse primer (5'-gtggagataggcacagttgat-3') derived from the partial cDNA sequence, the first strand cDNA was synthesized by incubation with ReverTraAce reverse transcriptase (Toyobo, Japan) for 1 hour at 42C. Then, the blunt ended second strand was synthesized by simultaneous inclusion of DNA polymerase I (9U/µl), E. coli DNA ligase (6U/µl, Takara, Japan), and T4 DNA polymerase (9U/µl) in the presence of RNase H (1U/µl) in a reaction buffer composed of 33.4mM Tris-HCl (pH 8.5), 4.6mM MgCl, 10mM (NHSO, 6.6mM 2-mercaptoethanol, 0.06mM EDTA, 0.005% BSA, 100mM KCl, and 0.15mM NAD at 16C for 90min., and the double-stranded cDNA obtained (5µl) was ligated with 12.5 pmoles of an adaptor consisting of complimentary oligo DNA, (5'-gtaatacgactcactatagggcac gcgtggtcgacggcccgggctggt-3') and (5'-accagcccggg-3'), using 4 U/µl T4 DNA ligase at 16 oC overnight. T4 DNA ligase, DNA polymerase I, and RNase H were obtained from Toyobo, Japan. The ligated cDNA fragments were 5'-RACE amplified by using an adapter primer (5'-gtaatacgactcactatagggc-3') with gene specific primers in 25 µl volume reaction mixtures using XL-PCR kit (PE Applied Biosystems), and then subcloned into the pT7blue vector. We also identified and sequenced EST clones homologous to YGHL1 from Danio rerio and Xenopus leavis. Putative transcription factor binding sites were identified manually based on the consensus sequences reported. The CRE sequences were found manually and confirmed with CREB target gene database (http://natural.salk.edu/CREB/) DNA sequencing and analysis: Subcloned DNA was isolatedand purified using a FlexiPrep kit (GE Healthcare, USA), and sequenced using a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Invitrogen) and an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems). Northern blotting analysis: Total RNA was isolated from various freshly harvested yellowtail organs: heart, kidney, brain, muscle, liver, and gill, using a RNA Extraction Kit (GE Healthcare, USA). Each sample (20µg) was loaded on to a 1% agarose gel containing 5% formaldehyde and then capillary-transferred to a nylon membrane, Hybond-XL, (GE Healthcare, USA). The membrane was hybridized with a [32P]-labelled yellowtail YGHL1 cDNA probe. Sequence data analysis: Nucleotide sequences were compared with those in the NCBI database using the basic local alignment search tool (BLAST) algorithm (National Center for Biotechnology Information). Amino acid sequences, deduced from cDNA sequences, were analyzed by means of the CLUSTALW multiple sequence alignment tool12 and tree generation was done using neighbour-joining algorithm13 in MEGA 5.114. The bootstrap analysis was done with 1000 replications. Results and Discussion Structure, genomic organization and expression analysis of the yellowtail YGHL1: Screening of the pituitary gland cDNA library of yellowtail fish for growth hormone related sequences resulted in a cDNA of yellowtail growth hormone like-1 in 1996 (GenBank: D85880). The 5’ and 3’ RACE experiments yielded the full length cDNA of YGHL1 (GenBank: AB073641), which encoded a short peptide of 92aa(figure 1. A). The 1529bp cDNA has a coding sequence of 279bp, preceded by a 127bp5’ UTR and followed by a 1123bp 3’UTR (figure 1. B). The genomic organization of the Yellowtail YGHL1 was analyzed by comparing the cDNA sequence with the 6658bp genomic sequence of the yellowtail YGHL1, (GenBank: AB094665). The gene consists of 4 exons and 3 introns with exon/intron boundaries having the canonical GT/AG splice sites. The first exon is a non-coding exon, whereas the remaining exons share the coding sequence. The total RNA samples isolated from various tissues of yellowtail (figure 2. A) were hybridized with a probe derived from the ORF of the yellowtail YGHL1 cDNA (figure 2. B). The northern blot showed a discrete transcript of 1.8kb in gill, brain, heart, and kidney. The highest level of expression was detected in heart, while no transcript was detected in muscle or liver. There seems to be a differential expression of the YGHL1/HIG1orthologs in liver of fishes11,15, which can be attributed to the euryoxic ability of the fish species. Gracey et al. proposed that HIG1 may be regulated by the HIF- mediated transcriptional mechanism, requiring the presence of hypoxia response element (HRE) in the promoter region. We found 5 HRE sites in the 2066 bp upstream sequence of the yellowtail YGHL1 based on the HRE consensus sequence16, 17. Olive flounder fYGHL1 is reported to have HRE sites in its promoter region15. Based on the consensus recognition sequence search, we also found putative binding sites for some specific International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(9), 59-68, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 61 transcription factors in the yellowtail YGHL1 (figure 3). The putative sites are TPA Response Elements (TRE) for Activator Protein-1, binding sites for CCAT/Enhancer Binding Proteins and cAMP Response Elements (CRE) for cAMP Binding Proteins18-20 Presence of these transcription factor binding sites may be a key for understanding the expression and physiological roles of the yellowtail YGHL1. The stabilized and activated, HIF heterodimer binds to the consensus HRE in the HIF target genes to promote survival in low-oxygen conditions. HIF-1 also interacts with other transcription factors, for instance, HIF-1 cooperates with ATF-1 and CREB-1 to activate the lactate dehydrogenase. A gene21, 22 or with AP-1 binding factors to activate the vascular endothelial growth factor gene23. C/EBP family transcription factors together with other transcription factors are known to have pivotal roles in numerous cellular responses, including the control of cellular growth and differentiation, immune and inflammatory processes and various diseases24. However, presence of both CRE and HRE sites in the promoter region of yellowtail YGHL1 might allow the gene to express in both aerobic and anaerobic conditions. Generally, under normoxic conditions, the CREB protein family is principal mediators of gene expression in response to cAMP following phosphorylation by cAMP-dependent protein kinase (PKA) which is necessary for many living organisms and life processes. Amino acid alignment and Phylogenetic tree construction ofthe YGHL1/HIG1 homologs: The YGHL/HIG1 orthologous to the yellowtail YGHL1 are present in other fishes, amphibians, birds and mammals. The deduced amino acid sequence ofthe yellowtail YGHL1(GenBank: BAB91154.1) was aligned with other YGHL1 and HIG1 polypeptides using CLUSTALW program. The alignment result showed high conservation between the amino acid sequences (figure 4). The yellowtail YGHL1 protein showed 72-82% identity with other fish sequences; 63% identity with human HIGD1A and 56% with mouse Higd1a. The alignment also showed a highly similar 46aa polypeptide present in all the sequences at the N-terminus, starting and ending with a glycine. The polypeptide is encoded by the 135bp third exon of the Yellowtail YGHL1 which is also well conserved in other YGHL1/HIG1 orthologs. Therefore, we propose a nomenclature for the conserved domain as YGHL1/HIG1 Exon 3 domain” which is also called as HIG-1- N domain. The well-conserved structure of the YGHL1/HIG1exon 3 domain suggests that it is either quite stable or sensitive to alterations that would impair its function. In mouse, a chimeric transcript is formed between UbiE2 and Yghl1-4, a non-homologous but tandem gene, by the intergenic splicing event (Genbank: AB110950). The chimeric transcript retains the YGHL1/HIG1 exon 3 domain, which strongly indicates that the exon 3 domain is functionally important for the YGHL1/HIG1 family. A phylogenetic tree was constructed based on amino acid sequences ofYGHL1 and HIG1 genes from various organisms using neighbour joining method (figure 5). The tree showed that the yellowtail YGHL1 is more closely related to the orthologous sequences in fishes. The sequences of human, mouse and other organisms form a separate group from the fishes but still related altogether. The Ciona intestinalis YGHL1/HIG1 seems to be the ancestral source and very closely related to the human HIGD2Aand mouse Higd2a. Evolution of the YGHL1/HIG1 gene family by genomic duplication: The phylogenetic and the genomic analyses show the presence of the YGHL1/HIG1 genes in both the ray finned fishes and the mammals. This fact shows that the YGHL1/HIG1gene family might have evolved 450 mya ago by events of genomic duplication taken place between the emergence of vertebrates and the divergence between tetrapods and ray-finned fish as that of MKRN gene family8,9,25. An YGHL1/HIG1homolog was also found in the Ciona intestinalis, which shows that an ancestral YGHL1/HIG1 was present in Chordata before urochordates and euchordates diverged, approximately 500 mya. This follows a trend, which is that gene families with several vertebrate paralogs often have only one homolog in the urochordate Ciona intestinalis26, 27. Altogether, we propose a schema depicting the evolution of the YGHL1/HIG1 paralogs in human and mouse (figure 6). Though we consider Ciona intestinalis to be the origin for the YGHL1/HIG1 gene family, there is a possibility of a more primitive eukaryote to have hosted the YGHL1/HIG1. The torafugu YGHL1 represents the YGHL1/HIG1 homologs in the fish genome from which the paralogs in human and mouse are derived. In human, HIGD1A, HIGD2A, HIGD1C and CLST11240 were mapped to 3p22, 5q35, 12q13, and 17q21 respectively. In mouse, Higd1a, Higd2a,Yghl1-4 and Higd1b were mapped to 9F4, 13B1, 15F1, and 11D, respectively. The human HIGD1A (3p22), HIGD2A(5q35) and mouse higd2a (13B1) seems to be derived from the original source after the genomic duplications. The present chromosomal position of the other YGHL1/HIG1 paralogs appears to be the result of further duplications during the mammalian lineage. Duplication of a chromosomal segment leads to groups of paralogs. All the neighboring genes in such segment are duplicated at the same event. The number of duplicate genes depends on the size of the segment. Duplication of large chromosomal segments is often called block or segmental duplication, and the resulting duplicate segments with several linked paralogs are often denoted as a paralogon or paralogous region28. According to our genomic analysis performed previously for 3p25 synteny in human chromosome 310, the HIGD1A seems to be clustered with some significant members of the nuclear receptor (NR) superfamily, the WNT and STATfamilies. Paralogs of these clustered genes are distributed together with the other paralogous loci of the YGHL1/HIG1. The human GH1, GH2 and mouse Gh genomic regions are also adjacent to the CLST11240 in 17q24 (Human) and Higd1b in 11D (Mouse). The synteny associated with YGHL1/HIG1paralogs may pose strong evidence that this gene family International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(9), 59-68, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 62 underwent concerted evolution with these homeostatically and developmentally important genes in vertebrates, especially mammals (figure 6). This theorizes that the YGHL1/HIG1 gene family might function or be under control of hormonal and signal pathways, in which these genes are involved29-34. Figure-1 Sequence and structural analysis of yellowtail YGHL1[A] Nucleotide sequence and deduced amino acid sequence of yellowtail YGHL1. The initiation codon (atg) is underlined and the stop codon (tga) is indicated by an asterisk. The shaded box indicates the Kozak initiation motif (annatgg)35. The putative polyadenylation signal (AATAAA) is shown in bold and underlined. The conserved YGHL1/HIG1 exon 3 domain sequence is shown in bold. [B] Genomic structure of yellowtail YGHL1. The exons are shown by boxes, where shaded region show the region encoding the ORF and unshaded region indicate untranslated regions. Introns are shown as lines Figure-2 Northern blot analysis of yellowtail YGHL1 in Seriola quinqueradiata organs [A] Total RNA was isolated and electrophoresed on a 1% agarose/formaldehyde gel from the gill (G), muscle (M), brain (B), heart (H), liver (L) and kidney (K). Hind III digest of DNA was used as marker (M). L and S with arrows to the left indicate ribosomal large and small RNAs, respectively. [B] A northern blot of the gel from panel A was hybridized with a probe derived from yellowtail YGHL1 cDNA. Lanes are as mentioned in panel A. The position of the yellowtail YGHL1 transcript (approximately 1800 bp) is indicated by an arrow mentioning YGHL1 to the right. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(9), 59-68, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 63 Figure-3 Nucleotide sequence of the 5’ upstream region of the yellowtail YGHL1 gene Putative binding sites of the transcription factors are shown underlined and bold. A line is drawn to separate the CRE and HRE regions at end of the sequence Conclusion We cloned, sequenced and characterized the yellowtail YGHL1gene and its promoter region in Seriola quinqueradiata. The well conserved YGHL1/HIG1 Exon 3 domain was identified by multiple polypeptide analysis of sequences orthologous to yellowtail YGHL1. The ancestral homolog of YGHL1/HIG1 has been the source of this extensive YGHL1/HIG1 family. The synteny of YGHL1/HIG1 paralogs along with linked flanking genes in human and mouse are results of genomic duplication events. Further empirical studies are required to reveal the function of yellowtail YGHL1 and to determine the role of transcription factor binding sites in yellowtail YGHL1 and the YGHL1/HIG1 family. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(9), 59-68, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 64 Figure-4 Alignment of the deduced amino acid sequences of YGHL1/HIG1 from Seriola quinqueradiata and other organismsThe deduced amino acid sequences of the yellowtail YGHL1is aligned with YGHL1/HIG1 homologs of other organisms using CLUSTALW alignment tool (Thompson et al. 1994). Asterisks mark absolutely conserved residues, and two dots or one dot represent highly or moderately similar amino acids, respectively. Dashes represent gaps inserted by the program to optimize the alignment. The red coloured region shows the highly conserved YGHL1/HIG1 exon 3 domain. GenBank accession numbers of theYGHL1/HIG1 amino acid sequences are as follows: Seriola quinqueradiata YGHL1 (GenBank: BAB91154.1), Gillichthys mirabilis HIG1 (GenBank: AAG13326.1), Takifugu rubripes YGHL1 (GenBank: BAD98705.1), Danio rerio YGHL1 (GenBank: BAC82438.1), Paralichthys olivaceus YGHL1 (GenBank: AEP34037.1), Xenopus laevis YGHL1 (GenBank: BAC75823.1) Homo sapiens HIGD1A (GenBank: NP_054775.2), Homo sapiens HIGD2A (GenBank: NP_620175.1), Homo sapiens CLST11240 (GenBank: NP_057522.1), Homo sapiens HIGD1C (GenBank: NP_001103089.1), Mus musculus Higd1a (GenBank: BAE92116.1), Mus musculus Higd2a (GenBank: NP_080209.1), Mus musculus Higd1b (GenBank: AAH05568.1), Mus musculus Yghl1-4 (GenBank: BAE92117.1), Gallus gallus HIGD1A(GenBank: NP_001026128.1), Ciona Yghl1/HIG1 (GenBank: XP_002130406.1) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(9), 59-68, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 65 Figure-5 Phylogenetic analysis of the deduced amino acid sequences of YGHL1/HIG1 from Seriola quinqueradiata and other organisms A Phylogenetic tree of YGHL1/HIG1 amino acid sequences from Seriola quinqueradiata and other organisms was constructed using the neighbor-joining algorithm in MEGA version 5.1 (Tamura et al., 2011). Bootstrap values supporting the branches were determined with 1000 repetitions and indicated at the corresponding nodes. The tree illustrates the evolutionary relationships among YGHL1/HIG1 in various organisms International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(9), 59-68, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 66 Figure-6 Evolution of YGHL1/HIG1 paralogs in mouse and human A schema showing the possible origin of primitive YGHL1/HIG1 in Ciona intestinalis , its duplication and location in human and mouse chromosomal positions as YGHL1/HIG1 paralogs. The genes flanking the YGHL1/HIG1 paralogs show the syntenic relationships in human and mouse. 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