Reiko Tanuma

Whole-genome sequencing and analysis of the Malaysian cynomolgus macaque (Macaca fascicularis) genome

BackgroundThe genetic background of the cynomolgus macaque (Macaca fascicularis) is made complex by the high genetic diversity, population structure, and gene introgression from the closely related rhesus macaque (Macaca mulatta). Herein we report the whole-genome sequence of a Malaysian cynomolgus macaque male with more than 40-fold coverage, which was determined using a resequencing method based on the Indian rhesus macaque genome.ResultsWe identified approximately 9.7 million single nucleotide variants (SNVs) between the Malaysian cynomolgus and the Indian rhesus macaque genomes. Compared with humans, a smaller nonsynonymous/synonymous SNV ratio in the cynomolgus macaque suggests more effective removal of slightly deleterious mutations. Comparison of two cynomolgus (Malaysian and Vietnamese) and two rhesus (Indian and Chinese) macaque genomes, including previously published macaque genomes, suggests that Indochinese cynomolgus macaques have been more affected by gene introgression from rhesus macaques. We further identified 60 nonsynonymous SNVs that completely differentiated the cynomolgus and rhesus macaque genomes, and that could be important candidate variants for determining species-specific responses to drugs and pathogens. The demographic inference using the genome sequence data revealed that Malaysian cynomolgus macaques have experienced at least three population bottlenecks.ConclusionsThis list of whole-genome SNVs will be useful for many future applications, such as an array-based genotyping system for macaque individuals. High-quality whole-genome sequencing of the cynomolgus macaque genome may aid studies on finding genetic differences that are responsible for phenotypic diversity in macaques and may help control genetic backgrounds among individuals.

Large-scale analysis of Macaca fascicularis transcripts and inference of genetic divergence between M. fascicularis and M. mulatta

BackgroundCynomolgus macaques (Macaca fascicularis) are widely used as experimental animals in biomedical research and are closely related to other laboratory macaques, such as rhesus macaques (M. mulatta). We isolated 85,721 clones and determined 9407 full-insert sequences from cynomolgus monkey brain, testis, and liver. These sequences were annotated based on homology to human genes and stored in a database, QFbase http://genebank.nibio.go.jp/qfbase/.ResultsWe found that 1024 transcripts did not represent any public human cDNA sequence and examined their expression using M. fascicularis oligonucleotide microarrays. Significant expression was detected for 544 (51%) of the unidentified transcripts. Moreover, we identified 226 genes containing exon alterations in the untranslated regions of the macaque transcripts, despite the highly conserved structure of the coding regions. Considering the polymorphism in the common ancestor of cynomolgus and rhesus macaques and the rate of PCR errors, the divergence time between the two species was estimated to be around 0.9 million years ago.ConclusionTranscript data from Old World monkeys provide a means not only to determine the evolutionary difference between human and non-human primates but also to unveil hidden transcripts in the human genome. Increasing the genomic resources and information of macaque monkeys will greatly contribute to the development of evolutionary biology and biomedical sciences.

Cynomolgus monkey testicular cDNAs for discovery of novel human genes in the human genome sequence

BackgroundIn order to contribute to the establishment of a complete map of transcribed regions of the human genome, we constructed a testicular cDNA library for the cynomolgus monkey, and attempted to find novel transcripts for identification of their human homologues.ResultThe full-insert sequences of 512 cDNA clones were determined. Ultimately we found 302 non-redundant cDNAs carrying open reading frames of 300 bp-length or longer. Among them, 89 cDNAs were found not to be annotated previously in the Ensembl human database. After searching against the Ensembl mouse database, we also found 69 putative coding sequences have no homologous cDNAs in the annotated human and mouse genome sequences in Ensembl.We subsequently designed a DNA microarray including 396 non-redundant cDNAs (with and without open reading frames) to examine the expression of the full-sequenced genes. With the testicular probe and a mixture of probes of 10 other tissues, 316 of 332 effective spots showed intense hybridized signals and 75 cDNAs were shown to be expressed very highly in the cynomolgus monkey testis, but not ubiquitously.ConclusionsIn this report, we determined 302 full-insert sequences of cynomolgus monkey cDNAs with enough length of open reading frames to discover novel transcripts as human homologues. Among 302 cDNA sequences, human homologues of 89 cDNAs have not been predicted in the annotated human genome sequence in the Ensembl. Additionally, we identified 75 dominantly expressed genes in testis among the full-sequenced clones by using a DNA microarray. Our cDNA clones and analytical results will be valuable resources for future functional genomic studies.

Prediction of unidentified human genes on the basis of sequence similarity to novel cDNAs from cynomolgus monkey brain

BackgroundThe complete assignment of the protein-coding regions of the human genome is a major challenge for genome biology today. We have already isolated many hitherto unknown full-length cDNAs as orthologs of unidentified human genes from cDNA libraries of the cynomolgus monkey (Macaca fascicularis) brain (parietal lobe and cerebellum). In this study, we used cDNA libraries of three other parts of the brain (frontal lobe, temporal lobe and medulla oblongata) to isolate novel full-length cDNAs.ResultsThe entire sequences of novel cDNAs of the cynomolgus monkey were determined, and the orthologous human cDNA sequences were predicted from the human genome sequence. We predicted 29 novel human genes with putative coding regions sharing an open reading frame with the cynomolgus monkey, and we confirmed the expression of 21 pairs of genes by the reverse transcription-coupled polymerase chain reaction method. The hypothetical proteins were also functionally annotated by computer analysis.ConclusionsThe 29 new genes had not been discovered in recent explorations for novel genes in humans, and the ab initio method failed to predict all exons. Thus, monkey cDNA is a valuable resource for the preparation of a complete human gene catalog, which will facilitate post-genomic studies.

Genomic structure and chromosome location of RPL27A/Rpl27a, the genes encoding human and mouse ribosomal protein L27A

The intron-containing genes encoding human and mouse ribosomal protein (r-protein) L27A were cloned and sequenced. The human r-protein L27A gene (RPL27A) shared an identical exon/intron structure with the mouse r-protein 27A gene (Rpl27a). The translational start codon ATG was separated from the main reading frame by the first intron sequence in both genes. An approximately 200-bp sequence upstream of the translational start site of both genes displayed remarkable similarity, and contained the putative promoters lacking canonical TATA, but harbored Sp1 binding sites and a short stretch of pyrimidine cluster, similar to other r-protein genes. Transcriptional regulatory elements, Box-A and GABP, found in the promoters of some other r-protein genes were also conserved in both genes. These structural features were included in the typical CpG island identified in the 5′-end sequences, suggesting that RPL27A/Rpl27a cloned here are authentic and transcriptionally active. Fluorescence in situ hybridization (FISH) analysis localized the mouse intron-containing Rpl27a to chromosome 7E2–F1 syntenic to human chromosome 11p15, where human RPL27A was located.

Cloning, expression analysis and chromosome mapping of human casein kinase 1 γ1 (CSNK1G1): Identification of two types of cDNA encoding the kinase protein associated with heterologous carboxy-terminal sequences

Casein kinase 1 γ1(CK1 γ1) is known to be involved in the growth and morphogenesis of eukaryotic cells. We have isolated two types of cDNA for human casein kinase 1 γ1 (hCK1 γ1). One of them (hCK1 γ1S) was found to encode a polypeptide consisting of 393 amino acids, which is highly homologous with already reported rat CK1 γ1 (rCK1 γ1). The other type of cDNA (hCK1 γ1L) encodes a polypeptide consisting of 422 amino acids, which is quite identical in the kinase domain, but different in the C-terminal sequence from hCK1 γ1S. Namely, hCK1 γ1L has a characteristic sequence of 50 amino acids at the C-terminal end and this motif was shown to be shared by the casein kinase γ2 and γ3 from rat and human, suggesting that it is a signature sequence of the γ-isoforms. In this sense, newly isolated hCK1 γ1L might be the original form of CK1 γ1 subspecies rather than rCK1 γ1 and hCK1 γ1S. RT-PCR analysis revealed that hCK1 γ1S mRNA is predominantly present in the testis, whereas the abundance of hCK1 γ1L mRNA was nearly the same in the twelve tissues examined. These results suggest that novel hCK1 γ1L may have a unique functional role different from that of hCK1 γ1S and rCK1 γ1. The human hCK1 γ1 gene (CSNK1G1) was mapped to chromosome 15q22.1→q22.31 by fluorescence in situ hybridization.

Cloning and chromosomal localization of a paralog and a mouse homolog of the human transaldolase gene

A sequence homologous to the transaldolase gene (TALDO) was identified in a polymorphic cosmid DNA mapped on human chromosome 11p15 by exon trapping with pSPL3. Analysis of lambda clones contiguous to the cosmid clone showed that the related gene (TALDOR) consists of 8 exons spanning approximately 19kb from the translation start site to the polyadenylation signal. The exon sequence of TALDOR was almost identical with that of TALDO localized on 1p33-34. 1, but its exons corresponding to exons 4 and 5 of TALDO were found to be split by 4 introns in TALDOR. To examine the evolutionary conservation of two genes for transaldolase, we have isolated the cDNA for its mouse homolog and determined the nucleotide sequence covering the complete coding region. Fluorescence in situ hybridization using the cDNA as a probe showed that the mouse transaldolase gene (Taldo) is localized on chromosome 7 F3-F4 as a single copy gene. This chromosomal region is known to be syntenic to human chromosome 11p15 rather than to 1p33-p34.1, suggesting that TALDOR is the ancestral form. The existence of TALDOR implies a duplication of the mammalian transaldolase gene after divergence of rodent and primate.

Cloning and chromosome mapping of the human casein kinase I γ3 gene (CSNK1G3)

Casein kinase I (CKI) is a ubiquitous Ser/Thr protein kinase found in the nuclei, cytoplasm and membrane fractions of eukaryotic cells. The enzyme exists as a monomeric protein ranging in size from 25 to 55 kDa. It preferentially phosphorylates acidic substrates using ATP as a phosphate donor. CKI comprises a multigene family. In vertebrates, seven CKI isoforms (·, ß, Á1, Á2, Á3, ‰ and Â) have been reported and the genes for ·, Á2, ‰ and  isoforms have been isolated from human cells (Fish et al., 1995; Kitabayashi, et al., 1997; Kusuda, et al., 1996; Tapia, et al., 1994; Zhai, et al., 1995). CKI homologues were also identified in both Saccharomyces cerevisiae (Hrr25+, Yck1+, Yck2+ and Yck3+) and Schizosacchcaromyces pombe (Hhp1+, Hhp2+, Cki1+ and Cki2+). The yeast CKI gene, HRR25 is essential for growth and viability and is known to be involved in the regulation of DNA repair (Hoekstra, et al., 1991). Human genes encoding CKI ‰ (CSNK1D) and  (CSNK1E), which are quite similar to HRR25, complement the repair function of this yeast mutant (Fish et al., 1995). In addition, the expression of rat CKI Á1 and Á3 can restore growth and normal morphology to a yeast mutant carrying a disruption of YCK1 and a temperature-sensitive allele of YCK2 (Zhai et al., 1995). On the other hand, rat CKI Á2 has been shown to be associated with the adopter protein Nck via Src homology domain 3 (SH3). Nck protein also binds to the SH2 domain of the receptor protein kinase or its substrates such as insulin-like substance 1, suggesting that CKI Á2 could be involved in the insulin signal transduction pathway (Lussier et al., 1997). To complete the catalog of human CKI isoform genes and to establish their chromosome locations, we isolated cDNA for the CKI Á3 gene (CSNK1G3) from a human testis cDNA library. The cDNAs obtained were sequenced and characterized. CSNK1G3 was mapped to 5q23 by radiation hybrid panel analysis and FISH.

cDNA cloning and chromosome mapping of the mouse casein kinase I epsilon gene (Csnk1e).

Casein kinase I (CKI) is a family of serine/threonine protein kinases found in all eukaryotes, first described over 20 years ago as having the ability to phosphorylate preferentially acidic proteins like casein and phosvitin using ATP, but not GTP. To date, seven isoforms of CKI, termed ·, ß, Á1, Á2, Á3, ‰ and Â, have been identified in higher eukaryotes (Fish et al., 1995; Graves et al., 1993; Kitabayashi, et al., 1997; Kusuda et al, 1996, 1998; Rowles et al., 1991; Zhai et al., 1995). CKI has been implicated in a number of unrelated important biological functions: mouse CKI · (mCKI ·) regulates the progression from interphase to mitosis during the first cell cycle (Gross et al., 1997); human CKI Â (hCKI Â) complements the repair function of a yeast mutant, hrr25, which is defective in a CKI homologue (Hoekstra et al., 1991; Fish et al., 1995); rat CKI Á1 (rCKI Á1) and Á3 (rCKI Á3) can restore growth and normal morphology to a yeast mutant with a disruption of the CKI homologue, YCK1 and a temperature-sensitive allele of YCK2 (Zhai et al., 1995); rat CKI Á2 (rCKI Á2) might play a role in the insulin signal transduction pathway by binding to the SH3 domain of adaptor protein, Nck (Lussier, et al., 1997). Recently, one of the Drosophila clock-related genes, doubletime (dbt) has been cloned and its gene product was shown to be a homologue of human CKI Â (Kloss et al., 1998). The DBT protein (dCKI Â) phosphorylates PERIOD protein (PER), resulting in a reduction of the stability of PER. The phosphorylation of PER is believed to affect the interaction of PER with TIMELESS protein (TIM) and transfer of the PER-TIM complex to the nucleus. Two mutants of the DBT gene, dbtS and dbtL have either a shortened or lengthened period of behavioral rhythm, and null mutant, dbtP shows no rhythmical oscillation. Analysis of the mutant genes revealed that dbtS and dbtL each contain one base missense mutation in the coding exon of dCKI Â, and dbtP has an insertion of a P-element in the first intron of the dCKI Â gene, which causes the failure in its transcription. As the first step in examining whether CKI Â is involved in the mammalian circadian clock core, we identified the complete coding sequence of the mouse CKI Â gene (Csnk1e) in a cDNA clone, the partial sequence of which was already registered in a DNA database as an expressed tag sequence (EST). The insert in this clone was completely sequenced and the CKI Â gene was also localized on a mouse chromosome by fluorescence in situ hybridization (FISH) using the cDNA as a probe.

Assignment of a human autoimmune antigen, p80-coilin gene to chromosome 17q21-q23 and of its possible pseudogene to chromosome 14

In order to determine the chromosomal locations of an autoimmune antigen, the coilin gene and its pseudogene, we amplified the segments of the two genes by the polymerase chain reaction (PCR) and screened a panel of somatic cell hybrids for the presence of the gene products. The results indicate that the human coilin gene and its pseudogene can be assigned to chromosome 17 and chromosome 14, respectively. Further analysis of cell hybrids bearing chromosome 17 with various deletions localized the coilin gene to the region q21–q23.