| IntroductionWNK (with no lysine kinase) kinases belong to a novel serine/threonine protein kinase family . The members of this family lack the conserved catalytic lysine in subdomain II of kinase domain that is crucial for binding to ATP. Cys-teine is in place of lysine at the usual conserved location but WNK kinases still have kinase activity, while for other protein kinases, the residue has frequently been mutated to eliminate the catalytic activity. However, overexpression of WNK1 in HEK293 cells exerts no detectable effect on the activity of known substrates which indicates that WNK kinases belong to a novel signal pathway.Rat WNK1 gene was the first mammalian member of this family cloned and characterized. In 2001, Wilson and his colleagues cloned another two members - human WNK1 gene and WNK4 gene which were located on chromosome 12 and 17, respectively. The two genes are disease - causing genes responsible for a Mendelian hypertension. The disease - causing mutations are the large deletions in the first intron in WNK1 gene, causing increased expression in leukocytes. Mutations in WNK4 are missense mutations in the coding sequence near the coiled -coil domains. Northern blotting indicates that WNK1 is widely expressed in different tissues and has two transcripts, whereas WNK4 is expressed primarily in kidney.Pseudohypoaldosterism type II ( PHA II , OMIM#145260) , also known as Gordon syndrome, is an autosomal dominant disease characterized by severe hypertension, hyperkalemia and metabolic acidosis. It is chloride -dependent and all the abnormalities can be corrected by thiazide diuretics which is the specific antagonist of the Na - Cl cotransporter( NCCT). All these indicate that the disease should be relative to the ion transportation. WNK kinases belong to a newsignal pathway, their upstream regulators and downstream molecular targets are presently unknown. It showed by immunofluorescence that WNK1 protein was located within several epithelial involved in Cl flux which meant it could regulate Cl transportation in several tissues. WNK4 is part of the tight conjunction complex. Wild type WNK4 can inhibit the function of NCCT whereas WNK1 can e-liminate the negative regulation. These results show that WNK kinases may interact with the ion channels and regulate the ion transportation thus cause hypertension.Herein, we used cloning, sequencing, Northern blotting, in situ hybridization , in vitro translation and co - immunoprecipitation , aimed to investigate the expression of WNK genes and the function of WNK4 kinase.Materials and Methods1. Materials and reagentsHuman kidney tissues were got from the traumatic patients in the hospital. Mouse kidney tissues were from the Balb/c mice. pGEM - T vector system, JM109 competent cells, reverse transcription kit and in vitro translation kit were purchased from promega company. pcDNA3. 1 vertor, DH5a competent cells and long template expanded PCR kit were from invitrogen company. Bigdye was from applied biosystem. Co - immunoprecipitation kit and multiple tissue membranes were from Clontech company.2. Clone human WNK4 full length cDNA into pGEM -T vectorHuman kidney total UNA was extracted and used as template to amplify WNK4 full length cDNA with the forward primer (7 -27) and reverse primer (3808 -3833) with the long template expanded PCR system kit. Then the PCR product was purified, ligated into pGEM - T vector by TA cloning. The ligation products were transfected to JM109 competent cells. The positive clones were confirmed by PCR, then the positive plasmids were minipreped, digested and sequenced. The sequencing results were analysed with Navigator 4. 0 software and the plasmid with correct sequences was maxipreped and stored in -201.3. Search the SNPs in WNK4 genomic sequencesDesign the primers to get the unique PCR products of WNK4 gene which cover the full length genomic sequences. Purify the PCR products and then do sequencing with ABI 3700 autosequencer , analyse the results with navigator 4. 0 software and do s...
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