Bioinformatic Analysis Of Cloned Rat Myoc Gene On Its Function Related To The Pathogenesis Of Glaucoma

Glaucoma is a kind of ophthalmologic disease with characteristic optic neuropathy induced by pathologic intraocular hypertension. Primary open - angle glaucoma (POAG) is a principal and common subtype of it and people tend to consider that the hereditary factor is one of the major reasons.Myocilin ( MYOC ) gene is the first candidate gene of POAG found in 1993. Up to date, the researches focused mostly on if there are the mutations in those suffered families in different areas, the functions, however, of MYCO gene and its expression product in the glaucomatous pathogenesis are still unclear. We cloned rats Myoc gene including total coding domain sequence (CDS) by RT - PCR, and run the analysis of bioinformatics on the cloned cD-NA of MYOG gene to find the aligned amino acid composition and the potential protein domains that might indicate the way of MYOC gene in the pathogenesis of glaucoma.Materials and methods1. Experimental animalSelect 26 healthy Wister rats, weigh 280 -320g; extract bilateral tissues including cornea, sclera, uvea, lens and so on, as one group; store them at -76 ℃ freezer.2. Methods2. 1 RNA extraction: deal with the collected tissues with TRIpure, isolated rat total RNA;2. 2 RT - PCR: get cDNA of target mRNA, detect the amplification results with 1.2% agarose gel electrophoresis;2. 3 Cloning:firstly, purify objective band from agarose gel electrophoresis, linkage the objective DNA into PG - T plasmid vector, transform the PG - T into DH5 - a competent B. Ecoli cell.secondly, detect the objective gene in monoclonal bacteria liquid by PCR and agarose gel electrophoresis; thirdly, digest extracted plasmid with double restriction endonuclease EcoR I and Hind III, once again agarose gel electrophoresis detect the objective segment, the positive band implied the successful cloned bacteria strain.2. 4 Sequencing: sequence the successfully cloned monoclonal bacteria liqid.2. 5 Bioinformatic analysis:DNA into Protein: with the Translate tool of Swiss - Prot from EXPASY proteomics server (http://au. expasy. org/) of Swiss Institute of Bioinformatics ( SIB ) , the DNA sequence of cloned MYOC gene was virtually translated into a-mino acid alignment, then potential functional domains of the protein was searched by InterPro Scan from European Bioinformatics Institute ( EBI) of EM-BL( www. ebi. ac. uk/InterProScan/) .ResultsAfter specified amplification by RT - PCR, a nearly 2.0Kb cDNA objective band of was shown on the Agarose gel electrophoresis. With identified cloned Ecoli bacterial liquid, the sequence of objective Myoc gene including whole coding domain segments of 1509 base pairs was successfully proved by DNA sequencing analysis. The bioinformatic analysis to the base sequence of cloned Myoc gene provided a virtual protein composition of 502 amino acids at the same alignment as Myocilin in NCBI database with 4 main functional domains including Olfactomedin - like domain.DiscussionUp to now, there are more than ten glaucomatous candidate virulence genes that have been reported. But MYOC gene, trabecular meshwork - induced glu-cocorticoid response protein (TIGR) gene, is the only one that named formally by Human Genome Organization ( HUGO) Genome Database Nomenclature Committee.The present conditions is that studies about candidate virulence gene are mostly focused on the mutation /variants and/or single nucleotide polymorphism ( SNP) , and emphasize the abnormality of typical gene sequence, however, there are few reports on the study about the total gene, its qualified expression and function.Based on the analysis of bioinformatics for protein function domain, and the relation of the functional roles of domain to the occurrence of glaucoma, three possible linked functional groups of Myoc were pointed out, olfactomedin - like protein, molecular chaperones kindred ( Prefoldin) and G - protein - coupled receptor (GPCR).1. There is an Olfactomedin - like domain in the c - terminal gene of MYOC gene. The majority of MYOC mutations are clustered in this domain, whose homogeneity with Olfactomedin is 40%. Olfactomedin is a main component in extracellular matrix (ECM) of olfactory nerve epithelial cells, thus people suspect that MYOC gene may also express a kind of component in ECM, thus its mutations will make ECM change in its expression regions, inducing the abnormal function of the cell in these regions.2. According to the function of molecular chaperones kindred - Prefoldin and pathogenesis molecular chaperones , we can suppose that when MYOC mutations happen in coding Prefoldin region, the unfolding actins cannot be pursued by the Prefoldin that has mutated, and interact each other; the proteins connect and fold in error. These proteins cannot degrade thoroughly and form irresolvable deposits. This disturbs the normal function of the trabecular meshwork seriously, and pathologic hypertension occurs.