Genetic Study for G-Protein Coupled Receptor from Saccharomyces Cerervisiae and From Sera of Patients with Heart Thrombosis

Abstract

Aim of the present study is Identification of specific gene for GPCR using specific primers .and identification of difference in PCR analysis in patients with heart thrombosis  and compared with healthy, Sequencing of PCR product regarding GPCR compared for all three subject, Identification  the similarity of human GPCR with local strain of yeast fifty healthy control and fifty patients with thrombosis which diagnosed medically with cardiac specific troponin t, troponin 1 levels and electro myocardiogram ECG. The aged for all subjects ranged (39-75) years   patients were lying in cardiac care unit at Ibn- al- Nafees teaching hospital and  Sheikh  Zayed  teaching  hospital. Genomic DNA of whole blood was extracted from buffy coat and cell cultured handbook protocol using Bioneer- kit and  Genomic DNA fungus/yeast  kit was used in isolation  and purification of DNA. patients divided  into three  groups according  to their age: group A (60-75) years , group B (50-59) years ,  group C (39-49) years the  results of genomic  DNA  isolation  from blood cells extracted in pure  form which ensured by the absorbance ratio (260/280 ) was (1.6 – 1.9 ) with a concentration of 50µg/ml and one DNA band with high resolution in gel electrophoresis. The result of  genomic DNA extracted from  the local strain of  S. cerevisiae showed that DNA   extracted with  high   purity   because   the  absorbance  ratio  (260 /280 )was (1.7 to 2.0) with a concentration of 60 µg/ml  and presence one DNA band with high resolution  in gel electrophoresis. primers were designed  depending on the sequence of the gene responsible for the production of GPCR on the chromosome 11 , GPCR  contain  three exons which covered  with six primers to detect a defect in gene sequence among. Results of gel electrophoresis are showed that primer  GPRX1 gave one band for (Control , A,B ) groups but absent amplified band in the patient eight and nine from group C. with molecular weight of this band is 1000 bp. The  GPRX2  primer  used to amplify second exon in the GPCR gene ,the molecular weight of amplified bands are  400 bp were present in all control samples and three groups of thrombosis patientsand yeast. GPRX2A primer that designed to amplify  part two from second exon of GPCR gene by PCR gave one band for all samples which include control and patient, the molecular weight of this band is 500 bp. PCR analysis  showed one amplify band for all control and patients group with molecular weight 500 bp  for GPRX3 primer and 400 bp for GPRX3A ,300 bp for GPRX3B. The specific primers which  designed to covering  GPCR gene used to amplification  genomic DNA  of the local strain  S.cerevisiae by PCR technique. Results showed all six primers which gave  one band with difference molecular weight  for each primer. All samples demonstrate identity planned sizes to control and local strain of S. cerevisiae samples except patient number 8 and 9 in the group(C) that  showed non specialist bands in specific primer  with  first  exon (GPRX1) .So the genetic sequence analysis of these two case based on the sequence  of the  remainder exons to detect the genetic defect in these case. The sequence of the first part for the second exon (X2) was identity standard sequence found on the NCBI web site for case( 8). The  case (9) showed  identity with  the sequence present in the human gene bank but some difference in the first of sequence which neglected because it is in the place link of primer. The  results for  case  8  showed some mutation for Exon X2(part2). but case (9) demonstrate one  deletion and one substitution. The results, also, illustrated that the ether48 thrombosis patients didn't appeared any mutation despite the positive results for ( Troponin) that gives strong indication of thrombosis. The conclusion Primer  GPRX1 gave one band for (Control , A,B ) groups but absent amplified band in the patient eight and nine from group C. The molecular weight of this band is 1000 bp. The amplified band with molecular weight 400 bp were present in all control samples and three groups of thrombosis patients with primer GPRX2  and 500 bp with primer GPRX2A.PCR analysis  showed one amplify band for all control and patients group with molecular weight 500 bp for GPRX3 primer.,400 bp with GPRX3A and 300 bp for primer GPRX3B.Similarity between results given by healthy group and local strain of yeast. Genetic study  showed that there are  only two case of  patients  eight and nine demonstrated mutation in nucleic location on exon two and three from GPCR gene

Keyword: GPCR, Genetic Study, Saccharomysis  cerevisiae, Patient with Thrombosis.