International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)

D6. Comprehensive Analysis of Uncharacterized Human cDNAs that Encode Large Proteins in Brain

Manabu Nakayama, Takahiro Nagase, Reiko Kikuno, Makoto Hirosawa and Osamu Ohara
Department of Human Gene Research, KAZUSA DNA Research Institute

It is known that "large proteins" frequently play a key role in development of the nerve system, brain functions, and neurological disorders. We are thus interested in comprehensive characterization of these "large proteins" in brain, and have conducted a cDNA sequencing project focused on human brain cDNA clones which encode "large proteins" during past five years. In order to comprehensively identify unknown human genes which are overlooked by conventional methods, we have taken the following two approaches. The first approach is based on random sampling of long cDNA clones which produce proteins larger than 50 kDa in the in vitro transcription/translation system. More than 1500 cDNA sequences have been thus determined and deposited to the public databases by us. The data obtained so far indicate: (1) The predicted functions of "large proteins" (>50 kDa) are classified into either cell communication/signaling (e. g., receptor, adhesion molecule, and GTPase activating protein), or cell structure/motility (e.g., cytoskeletal proteins, membrane skeletal proteins and motor proteins), or nucleic acid management(e.g. DNA binding protein); (2) paralogous genes are efficiently isolated by this approach although they are difficult to identify by conventional methods; (3) a number of unknown large proteins without any functional clues are predicted from the cDNA sequences. The second approach is to systematically identify genes encoding proteins with a particular domain of interest. The epidermal growth factor (EGF)-like domain is thought to play an important role in extracellular events including cell adhesion and receptor-ligand interactions. Since the amino acid sequences of EGF-like domains in various known proteins have little in common except for the conserved location of cysteine residues, genes coding for EGF-like domain-containing proteins could not be systematically identified either by colony hybridization under relaxed conditions or by PCR method using degenerated primers. We have developed a computer-assisted method, called motif-trap screening, for this purpose and succeeded in identification of many large proteins with multiple EGF-like motifs. For example, MEGF1 protein, one of proteins thus identified, is a 480-kDa transmembrane protein containing 2 EGF-like motifs and 34 cadherin motifs and this gene is specifically expressed in cerebellar granule cells. Our results suggested that an enormous extracellular part of MEGF1 protein protruded from axons and fasciculated parallel fibers of the granule cells in the molecular layer of the cerebellum. Taking this gene as an example, plausible functions of large proteins in the nervous system will be discussed. Although human cDNAs had been our targets so far, we are also interested in mouse cDNAs because they would be indispensable reagents for functional studies of genes encoding large proteins in the future.

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