A Novel Member Of SH₂ Signal Protein Family: Cloning And Characterization Of SH₂A Gene At Human Chromosome 8p22

IntroductionPositional cloning of disease genes and research of protome become very important in the post-genome period after completion of human genome project( HGP). Positional cloning is a new mean with the developing of HGP. Its virtue is to hold the relationship between phe-notype and gene. It is difficult to be replaced by other means because the gene decides the phenotype.The most classic positional cloning is " cDNA-dependent" , including cDNA selection, different display and subtractive hybridization. Their limitation is to have known the tissue specification of and developing period ,of candidate gene.The exon trapping, an " cDNA-indepentent" technology, has been used for several years. Its virtue is to know the position of the exon, exclude the others sources and eliminate the problems of the putative positive or reselection. So, exon trapping is one of the most important means for isolating the disease gene at susceptible sites.8p22 of human chromosome is an region with abundant disease-related genes. It is said that it is susceptible loci of hypertension and schizophrenia. The region is also the loci of oncogene and tumor su-pressor gene. So, positional cloning is used at chromosome 8p22 by exons trapping and exon linking. An opening reading frame ( ORF) including three exons of HC8 exB13,exB10 and exB18 was found at chromosome 8p22. The sequence is homologous with the putative gene AK024799 in Genbank. The structure, position and .function of AK024799 are not reported yet. So, we analyzed the characterization. It consists of 2. 8kb cDNA with 1362bp ORF and codes 454 amino acids with only a SH2 domain. The gene was named as SH2A at chromosome 8p22. We study its structure by BLAST homologous analysis and its expression by RT-PCR and Northern Blot.Materials1. Human Liver cDNA2. Exon Trapping System3. Molecular Biology Reagents of Gene Cloning4. Reagents relating to Northern Blot and RT-PCR5. Normal tissues and tumor tissuesMethodsA gene was cloned by exon trapping and exon linking technique. Homologous analysis of the gene was researched by BLAST. Then we explored the gene expression pattern in various human tissues by Northern Blot and RT-PCR . β-actin was as a control at the same time.ResultAn opening reading frame (ORF) of 510bp containing of 3 exons was found by exon trapping and exon linking at chromosome 8p22. The sequence is homologous with the putative gene AK024799 which consists of 2880bp cDNA with 1362bp ORF and codes 454 amino acids with only a SH2 domain. The gene was named as SH2A at chromosome 8p22.SH2 A is a novel docking protein of SH2 signaling protein family, which plays an important role in cellular signal transduction pathway. Northern Blot and RT-PCR analysis show that SH2A gene is ubiquitously expressed in many tissues with three transcripts. The aberrant expression of SH2A gene in some cancers was found. It suggests that SH2A gene relates to tumors.Discussion1. Structure and Expression of SH2A Gene ;An opening reading frame of 510bp was found through HC8exB13,HC8exB10,HC8exB13. The sequence is homologous with the putative gene AK024799. The structure and function of the putative gene AK024799 are not reported. So, we analysed its characterization. It consists of 2. 8kb cDNA with 1362bp ORF and codes 454 amino acids with only a SH2 domain. The gene was named as SH2A(-SH2A alone). It is at chromosome 8p22. The molecular weight of SH2A protein is 57 ,726Da, pi is 8.09. SH2A is a novel docking protein of SH2 signaling protein family, which plays an important role incellular signal transduction pathway. It is ubiquitously expressed in many tissues with three transcripts.2. Signal Protein of SH2 Domain;SH2 domain is a conservative sequence containing of 100 amino acids. Similar sequences were found in many proteins. They become a family of proteins with SH2 domain. They play an important role in signal transductions by tyrosine protein kinase. SH2 domain is considered as "Protein Recognize...