Briefings in Functional Genomics and Proteomics Advance Access originally published online on February 20, 2006
Briefings in Functional Genomics and Proteomics 2006 5(1):4-7; doi:10.1093/bfgp/ell005
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Special Issues Papers |
Kazusa mammalian cDNA resources: towards functional characterization of KIAA gene products
Corresponding author. Osamu Ohara, PhD, Department of Human Gene Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan. Tel: +81-438-52-3913; Fax: +81-438-52-3914; E-mail: ohara{at}kazusa.or.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONAcknowledgementsReferences |
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The Kazusa cDNA project pioneered an extensive sequencing project of human cDNAs in their entirety and focused sequencing efforts particularly on large cDNAs encoding large proteins. More than 2000 human genes, referred to as ‘KIAA’ genes, were initially identified through this cDNA project. Since many KIAA genes still remain functionally uncharacterized, our current focus is to determine their biological functions in vivo. In this review, we describe the current status of the Kazusa mammalian cDNA resources and the future direction of the functional characterization of KIAA genes.
Keywords: cDNA analysis, ORF cloning, antibody production, recombinant protein production, database
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONAcknowledgementsReferences |
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Characterization of mammalian (particularly human and mouse) cDNAs has been extensively conducted by many groups and has revealed a considerable number of novel and unexpected findings about the mammalian transcriptome [1, 2]. However, we must also consider the fact that a huge number of entirely sequenced cDNA clones has become available as a result of these cDNA projects, which serve as an indispensable resource for experimental exploration of the functions of mammalian gene products. Having considered this approximately 10 years ago, we pioneered an extensive sequencing project of human cDNAs in their entirety to predict the coding sequences of unidentified human genes [3, 4]. The novel human genes identified by our cDNA project were systematically designated as ‘KIAA’ plus a four-digit number, and are thereby known as KIAA genes. The most unique feature of our cDNA project is that we have focused our sequencing efforts on long cDNAs (>4 kb) that encode large proteins (>1000 amino acid residues) [5]. It should be noted that our cDNA project aims to predict unknown protein sequences through cDNA analysis rather than to simply identify and characterize transcripts from the human genome. After the human genome sequence was completed, discovery of new human genes has obviously ended. Consequently, we considered that it was time to end the first phase of the Kazusa human cDNA project and advance beyond the identification of transcribed sequences. From our perspective, the functional characterization of KIAA genes and their products is the main issue in the second phase of our cDNA project. In this review, we describe the current status and the future direction of the Kazusa mammalian cDNA project.
The current status of Kazusa cDNA projects
Since the discovery rate of unknown human cDNAs encoding large proteins greatly reduced after the human genome was completely sequenced, the number of KIAA genes became almost saturated. However, to allow functional characterization of KIAA genes, we have continued to collect cDNA clones that encode homologues of the KIAA genes and determine their complete sequence since functional comparison of these homologues would be expected to offer some insight into the potential functions of KIAA genes. Presently, more than 5000 human cDNA clones have been entirely sequenced at the Kazusa DNA Research Institute. In parallel with the complete sequencing of additional human cDNA clones, we have converted these cDNA clones into ‘ORF’ clones as described by other groups [6]. In addition to the collection of ‘ORF’ clones, we have also undertaken the collection of mouse KIAA (mKIAA) cDNAs and antibodies against their encoding proteins since 2001. To manage the large amounts of data produced by these Kazusa cDNA projects, we have constructed several databases conveying many lines of information regarding KIAA and/or mKIAA cDNAs. The Kazusa mammalian cDNA resources that have been constructed to date are described in detail subsequently and an overview is shown in Figure 1.
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ORF clones
A cDNA clone is not always an authentic copy of a mature mRNA. This is because cDNA clones sometimes carry artificial coding interruption(s) and/or truncation(s) due to errors in reverse transcription, copying of the immature mRNA or incorrectly spliced mRNA [7]. Since incorrect prediction of the coding sequences inevitably causes serious problems in downstream functional analyses of the gene products, we carefully examined the integrity of each of our cDNA clones by making use of existing sequence information [8]. After performing this manual process, we prepared ‘ORF’ clones harbouring the complete protein coding sequences with or without a termination codon. The latter ORF clones were used to generate clones encoding KIAA proteins fused to an appropriate reporter protein, such as green fluorescent protein (GFP). To expedite this process, we developed a method based on homologous recombination in Escherichia coli and applied it to generate ORF clones for KIAA and mKIAA genes [9]. Presently, in excess of 2000 ORF clones for KIAA and mKIAA cDNAs have been generated. The resultant ORF clones have been used for in vitro protein production, functional analysis of transiently expressed gene products in mammalian cells and subcellular localization analysis of GFP-fused KIAA proteins (Figure 1). In addition, we are also generating stable cell lines expressing KIAA or mKIAA proteins under the control of a tetracycline-dependent regulation system.
Mouse homologous cDNAs for KIAA genes and antibodies against their gene products
Since we are most interested in functional analyses of KIAA proteins in vivo, it is reasonable to take advantage of animal models for this purpose. Consequently, since 2001, with support from the Japan Science and Technology Agency, we have been conducting a project to isolate the mouse counterparts of the KIAA (mKIAA) cDNAs [10] and to produce a set of high-affinity and specific capture molecules, such as antibodies against mKIAA gene products [11]. We consider that this alternative approach is critical for the functional characterization of KIAA gene products, since the use of antibodies is the only method currently available to identify and characterize rare proteins, such as many of the KIAA proteins in vivo. The resulting antibodies were evaluated by enzyme-linked immunosorbent assays, immunoblotting experiments and immunohistochemistry. The antibodies that show high specificity were used for further studies. This approach is expected to provide us with data that complements the results of the in vitro functional analyses of KIAA genes. Furthermore, the resultant set of antibodies will serve as indispensable reagents for an antibody array, which may enable us to comprehensively monitor KIAA protein levels in vivo [12]. These antibodies are unique and critical reagents produced by the Kazusa mammalian cDNA projects.
Databases
Over the course of the Kazusa mammalian cDNA projects, we have accumulated large amounts of information regarding KIAA/mKIAA genes at the mRNA and the protein levels. To make this information accessible to the public, we have launched several databases at http://www.kazusa.or.jp. These databases also serve as a gateway to our cDNA resources. The HUGE and ROUGE databases are based on sequence information of KIAA and mKIAA cDNAs, respectively. Tissue expression patterns of KIAA genes and protein–protein interaction data can be retrieved from the HUGE database [13]. In contrast, the InGaP and InCeP databases contain numerous fields of information obtained by experiments using mKIAA antibodies [14]. In addition to these databases, a database of ORF clones will become available soon.
Future direction
Over the past 10 years, we have constructed a collection of mammalian cDNA resources as outlined above. Our current objective is to further develop these cDNA resources in a more biologically oriented manner. Among many interesting and important biological issues, we plan to demonstrate the usefulness of these cDNA resources in a collaborative framework, with immunologists being one of the main targets. However, we wish to emphasize that the use of our cDNA resources is not limited to immunology. Since these cDNA resources are intrinsically versatile, they can be used to address a wide variety of biological issues. Attempts to address real-world biological problems using these cDNA resources have provided us with important insights into what information is absent from our cDNA projects. For example, although the availability of the mammalian cDNA resources has extended considerably beyond cataloguing cDNA clones and the proteins they encode, the analysis platforms where these resources are loaded require further development. In particular, the analysis platforms for gene function at the protein level are still in their infancy. We are actively committed to developing various analysis platforms suitable for the functional characterization of KIAA gene products since we consider it one of the primary responsibilities of our cDNA projects.
Key Points
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Acknowledgements |
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TOPABSTRACTINTRODUCTIONAcknowledgementsReferences |
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We would like to thank Dr R Kikuno, Dr M Nakayama, Dr H Yamakawa, Dr D Nakajima, Dr N Okazaki and Dr R Ohara, for their great contributions to the Kazusa cDNA projects. Without their collaboration we could not have accomplished our work. In addition, excellent technical support by many qualified technicians involved in the Kazusa cDNA projects is also gratefully acknowledged. The Kazusa cDNA projects were supported by the following grants: the CREATE Program (Collaboration of Regional Entities for the Advancement of Technological Excellence) from JST (Japan Science and Technology Agency); a grant from the Special Coordination Funds and a grant from the Organized Research Combination System of the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government; and a grant from the Kazusa DNA Research Institute.
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FOOTNOTES |
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Dr Takahiro Nagase obtained his PhD in pharmaceutical sciences at Science University of Tokyo in 1991. After having studied regulatory mechanism of gene expression at RIKEN as a termed scientist (1988-1992) he studied as a special researcher of Kazusa DNA Research Institute (KDRI) at Institute of Gerontology, Nippon Medical School and finally moved to KDRI in 1994 as a researcher. He has been making efforts to isolate and analyze cDNAs for human genes previously unidentified in the last decade to accelerate molecular biological studies. He is continuously studying functions of the gene products by cell biological approach at KDRI as head of the First Laboratory for Human Gene Research. He is also acting as a visiting professor of laboratory of Genome Structure and Function in graduate School of Life Sciences Tohoku University.
Dr Hisashi Koga is the team leader of the CREATE (Collaboration of Regional Entities for the Advancement of Technological Excellence) program from JST (Japan Science and Technology Agency) and senior researcher (head of the mouse cDNA bank section) of the Kazusa DNA Research Institute (KDRI). He is also a lecturer at the Brain Research Institute at Niigata University. He obtained his MD from the University of the Ryukyu Faculty of Medicine (1988) and his PhD from the Graduate School of Medical Sciences, Kumamoto University (1998). After one and a half years of postdoctoral work in the Department of Tumor Virology at Heinrich-Pette-Institute (Hamburg, Germany), he returned to Japan and joined the Helix Research Institute in 2000. At the end of 2001 he moved to KDRI. The main focus of his present research is the use of genomic and proteomic resources to design a suitable platform for chemical genetics.
Dr Osamu Ohara obtained his PhD in biophysics at Kyoto University in 1983. After having worked for a pharmaceutical company in Japan for 11 years, he moved to Kazusa DNA Research Institute (KDRI) in 1994. He has been involved in human cDNA sequencing project at KDRI and currently acts as department head of Human Gene Research. One of his research goals is to fill the gap between transcriptome and proteome through cDNA analysis while he is interested in development of various analysis platforms of gene functions. He also combines with group director of Immunogenomics group at RIKEN research center for Allergy and Immunology and visiting professor of pharmacogenomics laboratory in graduate School of Pharmaceutical Sciences of Chiba University.
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