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Comparative Genomics of T-complex protein 10 like in Humans and Chimpanzees Comparing 231 genes on chimpanzee chromosome 22 with their orthologous on human chromosome 21, we have found that 15 orthologs have indels within their coding sequences. It was rather surprising that significant number of genes have changed by indel, despite the shorter time since their divergence and led us hypothesize that indels and structural changes may represent one of the major mechanism of proteome evolution in the higher primates. Human T-complex protein 10 like (TCP10L) is a representative having indel within its coding sequence. Gene structure of human TCP10L compared with chimpanzee TCP10L gene showed 16 base pair difference in genomic DNA. As a result of the indel, frame shift mutation occurs in coding sequence (CDS) and human TCP10L express longer polypeptide of 21 amino acid residues than that of chimpanzee. Our prediction found that the indel may affect to dramatic change of secondary protein structure between human and chimpanzee TCP10L. Especially, the structural changes in the C-terminal region of TCP10L protein may affect on the interacting potential to other proteins rather than DNA binding function of the protein. Through these changes, TCP10L might influence gene expression profiles in liver and testis and subsequently influence the physiological changes required in primate evolution. Recently, as an international collaborative project, we reported the entire DNA sequence of the euchromatic region of chimpanzee chromosome 22 (PTR22) and compared the whole chromosome with human chromosome 21 (HSA21) (Watanabe et al., 2004). We found that PTR22 and HSA21 differ at approximately 1.44% of their 33 million aligned nucleotides. In addition we reported nearly 68,000 insertion or deletions (indels). The number of indels is approximately one seventh the numbers of point mutations accumulated over the time since human and chimpanzees diverged. Since each indel involves the change of several nucleotides, this result confirms Britten’s estimates (Britten, 2002) that indels are a major soruce of sequence divergence between human and chimpanzees. Indeed comparing 231 orthologous genes on the chromosome, we found that 15 orthologs have indels within their coding sequences and 32 have changes in the start codon or stop codon, which would potentially lead to gross structural differences between human and chimpanzee protein products. These results led us hyperthesize that indels and structural changes may represent one of the major mechanisms of proteom evolution in the higher primates. Among 15 kinds of indels between human and chimpanzee orthologous genes, TCP10L is a representative having indel within its coding sequence (Watanabe etal., 2004). The TCP10L showed 16 base pair differences in genomic DNA sequence between human and chimpzanzee. The primary structure of human TCP10L contains the representative character of the leucine zipper motif, with a leucine residue in every seventh position. The leucine zipper motif has been observed in a number of proteins thought to function as eukaryotic transcription factors (Amati et al., 1993; Perez et al., 2001; Sellers etal., 1989). Especially, the gene in human is known to be specifically expressed in the liver and testis (Chen etal., 2003) but not in 28 other tissues, and the protein is accumulated in nucli (Chen et al., 2003). The subcellular localization and the leucine zipper structure of the protein suggested that it functions as a transcription factor. Indeed, human TCP10L has an activity of transcription repressor and the leucine zipper is indispensable to the biological functions of the transcription factors (Chen etal., 2003). It has long been argued that changes in gene regulation may be more important to morphological and functional evolution than overall genomic divergence (Cherty et al., 1978). To study this old issue, transcription factors can be a good candidate because the change of transcription factors could result in a global change in gene regulation. In this study, we analyze and compare genomic structure of TCP10L gene between human and chimpanzee and consequently discuss its implication in liver physiology and primate evolution. Genomic organization of human TCP10L gene was reported by Chen et al. previously (Chen etal., 2003). The TCP10L is localized at chromosome 21 p22.11, and its genomic size is 8955 bp. As shown Fig. 1, human TCP10L was composed of five exons and four introns. To understand the genomic characters of chimpanzee TCP10L, we predicted genomic structure of chimpanzee TCP10L using SIM4 program (Florea et al., 1998) that run by matching human Ref.Seq as a query sequence to chimpanzee genomic DNA sequence. The chimpanzee TCP1OL showed genomic size of 8615 bp and similar exon and intron structure with human TCP10L. DNA sequence similarity of the TCP10L gene between human and chimpanzee was 97.8 % in total and similarities for each exons are shown in Table 1. Next, we tried to speculate the changes in CDS of human TCP10L caused by the genomic change. As shown in Fig. 2, human TCP10L has 9 base substitution and 16 bp indel in exon 4. The 16 bp indel in exon 4 resulted in frame shift of protein coding structure and also inactivation of stop codon. As a result, the human TCP10L showed longer CDS of 63 base pair than that of chimpanzees. Comparison of TCP10L CDS sequence between human and chimpanzee found that the human TCP10L deduced to be 215 amino acids polypeptide that is longer of 21 amino acid residues than that of chimpanzees (Fig. 3). We further predicted their secondary structure using PSIPRED server (McGuffin et al., 2000) and found that the 16 base pair indel resulted in dramatic changes on the secondary structure between them. As shown in Fig. 4, the typical leucine zipper motif exits in the middle of peptides in both human and chimpanzees, but another helical structure in the chimpanzee TCP10L exist on the N-terminus of the typical leucine zipper. Dramatic changes of protein structure found out on the C-terminal region of the TCP10L. In spite of Chimpanzee TCP10L has two helixs and two beta-sheets in secondary protein structures, human TCP10L showed one helix and 4 beta-sheets structure on the C-terminal region. These dramatic changes on the secondary structure might influence on three dimensional structures and also in their biological activities of the TCP10L between two species. In this study, we show that human TCP10L has 16 base pair difference in genomic DNA compare to chimpanzee ortholog. As a consequence, frame shift mutation occurs in CDS of human TCP10L and affects to human express as longer protein of 21 amino acid residues than that of chimpanzee. Our further analysis found that the changes of primary structure of human TCP10L resulted in dramatic changes in secondary structure prediction. Functional study showed that human TCP10L is deduced to be 215 amino acids polypeptide and a typical leucine zipper motif exists in the middle of the peptide (Chen et al., 2003). The leucine zipper is DNA binding motif and indispensable to the biological functions of these transcription factors through dimerization. Based on these facts, the structural changes in the C-terminal region of TCP10L protein may not affect the DNA binding function of this transcription repressor. More likely the C-terminal changes would affect some other function of human TCP10L such as interacting potential to other transcription factors or stability of the protein. It has been known that the TCP10L interact with MAD as a transcription factor (Jiang et a!., 2004). More recently, Yu et al. reported that TCP10L is expressed in nucleus of spermatogenic cell and bind to death associated protein kinase-3 (DAPK-3) (Yu et al., 2005). Base on this, they suggested that TCP10L might play crucially important roles in spermatogenesis through its interaction with DAPK-3. However, it is not clear that C-terminal region of TCP10L is responsible for interaction with DAP-3. In this point, our finding through comparative study of chimpanzee and human TCP10L can supply new approach method to understand the roles of TCP10L in spermatogenesis. The human TCP10L gene is specifically expressed in the liver and testis. In the female, TCP10L only expresses in the liver. In previous report, Enard et al. showed that gene expression patterns in human liver are more similar to those of the chimpanzee than to those of macaques (Enard et al. 2002). These results coincide with the evolutionary relationship of the species suggesting that gene expression patterns in liver reflect the evolutionary relationship in the primates. The liver is an essential organ playing a central role in metabolism and detoxification of poisonous substances. Therefore, these changes in liver physiology might be required to adapt new environment such as changes of food and xenobiotics. The various genes participating in these metabolic reactions are regulated on transcription revel by many transcription factors (Schiaffonati etal., 1997). The implication of the changes in transcription factors to human and chimpanzee evolution are unclear. However, these changes in TCP10L gene might influence gene expression profiles in liver and testis and subsequently influence physiological changes required in primate evolution. Further molecular biological studies on the TCP10L may help us gain insights into the molecular implication of the changes in the physiological functions of liver and primate evolution.