Cloning of Chromosomal Band Specific cDNA - cDNA related with neural development-

Park, Sun Hwa; Kim, Chang Mi; Jung, Hye Sun; Jeon, Yong Hyuck; Kim, Hyun

Abstract

Recently, surmountable amounts of genes are being cloned without information about them and it has become neccessary to develop new techniques for discovering genes with more informaiion like as chromosomal location and possible functions. We have developed one such a method and applied it to search for genes that may be related with the neural development. The mRNAs were extracted from cerebral cortex of 18 week old human fetus, cDNAs were made by reverse transcription from these mRNAs and Uni-amp cDNAs having Uni-amp adapters at both ends were made for subsequent PCR. To observe the distribution of the Uni-amp cDNAs on the chromosome, fluorescent in situ hybridization was performed with biotin labeled Uni-amp cDNAs. Among the chromosome bands showing strong hybridization with the cDNAs, 7q22 was microdissected from the chromosome hybridized with unlabeled Uni-amp cDNAs and amplified by PCR with Uni-amp primers. These amplified cDNA fragment were subcloned to vectors and the nucleic acid sequences were analysed. As a result, 46 different clones were obtained. They were categorized as 12 clones of well characterized genes, 14 clones showing low homology with known genes, 13 clones of simply registered uncharacterized human cDNAs, 7 clones of unknown genes. In situ hybridization histochemistry of 34 novel genes, except 12 known genes, were performed on developing and adult rat tissue sections to see the tissue specificity and developmental expression of these genes. The expression of several novel genes were restricted to the nervous system. From these results, it may be suggested that our technique is very useful to clone the genes expressed in the developing human braine with confirmed chromosomal location. In addition, this cloning technique can be used to discover the new genes related with neural development in combination with functional screening methods.