Why the Met-Glu-Glu-Gly-Met-Met-Val-Leu-His Amino Acid Can Be Dangerous - Lab Report Example

1. We can use the given part of the G6Pase protein to determine the entire sequence and also to find the similar sequence in the human. We can use this information to convert it into oligo nucleotides and then use it for the determination of the entire gene in the library. The Met-Glu-Glu-Gly-Met-Met-Val-Leu-His amino acid sequence contains 9 triplet codons and 27 codes. For the construction of a probe 20 codons are enough as we have 27 codes it is an easy task. The second thing that we must take into account is that the synthesis of the oligo nucleotides is based on the known peptide sequence. An amino acid may be encoded by several triplets; the use of the different oligo nucleotide probes is often required. For the methionine the codon is AUG. So the probe constructed must have the starting code as TAC. The Glutamic acid has the two triplet codons. GAA and GAG. The Glycine has four triplet codons. GGU, GGC, GGA and GGG. The valine has four triplet codons GUU, GUC, GUG and GUA. The leucine has six triplet codons namely CUU, CUC, CUG CUA, UUA and UUG. Histidine has two triplet codons namely CAU and CAC. Therefore the peptide sequence Met-Glu-Glu-Gly-Met-Met-Val-Leu-His corresponds to ( 1 x 2 x 2 x 4 x 1 x 1 x 4 x 6 x 2 = 768 ) 768 possible DNA sequences. After the designing of the probe, this is a part of the DNA or RNA that is used to detect specific nucleic acid sequences by the hybridization technique. The probes are radioactively labeled and these hybridized probes can be identified by using the autoradiography. Autoradiography is used for analyzing the extend to which the hybridization with the radio labeled probes occurs and to monitor the in-situ hybridization level. (Tenover, 1989). The human genomic library (Clontech) can be used to identify the gene of interest, because this human genomic library of Clontech is widely used for the rapid screening of the adenovirus associated gene therapy. 2. Glycogen storage disease type 1 a (GSD 1 a) disease is caused by the deficiency in the synthesis of an enzyme called Glucose 6 phosphatase (G6Pase). G6Pase enzyme is a key enzyme in the glucose homeostasis. Glucose homeostasis is the maintenance of the balance between glyconeogenesis and glycogenolysis. GSD 1a is characterized by hypoglycemia, liver and kidney enlargement, hyperlipidemia, hyperurecemia and retardation of growth. The lack of the Glucose 6 phosphatase is an autosomal recessive disorder. G6Pase catalyses the terminal steps in glyconeogenesis and glycogenolysis. The glusoce 6-Phosphate is concerted into Glucose molecule and phosphate. (Zingone, 2000). G6Pase is present in the endoplasmic reticulum as a membrane spanning protein. The active site of this protein is present facing the lumen. G6Pase requires a transporter molecule to transport the Glucose-6- phosphate from the cytoplasm to the endoplasmic reticulum. The organ transplantation and its effect on the GSD 1a were studied separately for kidneys and liver. It was found that the kidney replacement was not able to eradicate or reduce neither the hypoglycemia nor the hyperurecemia. When the liver was transplanted to the GSD 1a patients they found that they were able to reduce the hypoglycemia and its abnormalities and were able to have an improvement. Thus the gene therapy target was chosen as the liver. After the gene therapy target was selected, the selection of the vector was considered into account. The use of the adenovirus –mediated gene targeting was chosen. The efficiency of the helper-dependent mediated (HDAd) hepatic delivery of the G6Pase was tested and found to be the suitable model for the prolonged survival and the sustained correction of the metabolic abnormalities.(Koeberl, 2007). When the HDAd vector was administered along with the G6Pase into a 2 week old G6Pase knock out mice and tested, it was observed that the survival rate of the test animal was an average of 7 months as compared to the control that died after 3 weeks of age. The reduction in the accumulation of Glycogen , the increase in the growth as that of the wild type, minimal or no symptoms of hypoglycemia and hypercholesterolemia are the results of the gene therapy. As the success rate is very high for the mice, the same amount of success can be expected for the humans also. Here the conclusion is yes, we can clone the mouse gene into the human gene as they both resemble each other. 3. 4. In the exon 3, the change in the code started at the 127th position of the sequence. The change at the 127th position was TAT instead of TAC. Fortunately these two codons have the same code for the amino acid Thymine. Whereas the 128th position amino acid Thymine was replaced by the threonine amino acid in the Von Gierke diseased patient. Similarly the 129th position amino acid Valine was replaced by the Threonine in the patient’s code analysis. The occurrence of the stop codon at the 130th position amino acid must be the main reason for the von gierke disease. The stop codon terminates the translation process. The mutation is the main cause for the variation of the sequence. 5. Even a small variation in the sequence will play a major role in the inactivation of the protein. This is very well understood from the small change in the sequence producing no G6Pase activity in the patient. The occurrence of the Cysteine instead of Proline in the 83rd position of the exon 2 has resulted in no G6Pase activity. The studies have predicted that the active site of the Glucose 6 phosphatase enzyme lies at Arg- 83, His-117, His -176 positions of the enzyme. Arg- 83, His-117, His -176 amino acid residues reside on the same side of the endoplasmic reticulum. The five active site mutations K76N, R83C, R83H, H119l and R170Q are identified in the G6Pase gene of the GSD 1a. The mutations at the K76N, H119L and R170Q have completely inactivated the enzyme. They constitute the ninth transmembrane helical structure. These residues are present in the endoplasmic reticulum membrane and it is highly supported by the nine-transmembrane helical model. R 83C and R83H mutants were devoid of the enzyme activity. Arg -83 is found to have involved in the positioning of the phosphate molecule during the catalysis reaction. So if the Arginine at the position 83 is mutated the energy required for the catalysis is not received by the enzyme and hence the rate of conversion of the Glucose -6- phosphate molecule into glucose and phosphate becomes a question mark. (Pan, 1998). 6. The DNA is a double stranded molecule. For a child, one strand of this double stranded molecule comes from the father and the other comes from the mother. When these two strands join together they contribute the entire genome of the child. The dominant strands of the parents are expressed and thus it results in the similarities from the parents onto the child. Von Gierke disease is caused due to an autosomal recessive condition. The two genes of the DNA must be mutated for a person to receive the autosomal recessive condition. 7. When both the parents have the gene in the recessive condition at the heterozygous state, the chance of occurrence of the disease to their children is 25%. The parents always have a chance of 25% reoccurrence to their children. The prenatal diagnosis can help them to prevent the second child from having the disease. The fetal cells obtained from the amniocentesis at weeks 14- 16 or the chorionic villus sampling at the weeks 9 or 10 will help them to identify whether the fetus is having the disease or not and to abort the child if diagnosed. This is the only method to prevent the occurrence of this disease to their second child. (Meisenberg, 2006). 8. The primer search for the given sequence was done using the Web primer, an inline primer search engine. The url is http://www.yeastgenome.org/cgi-bin/web-primer . It was found that there are 5 primers totally based on the GC content among them are one forward primer and four reverse primers. 9. According to the recent findings, the mutation at the 327th position amino acid, a guanine to adenine substitution has lead to the inactivation of the enzyme Glucose 6 phosphatase. Hence this small variation can be used as a diagnostic feature to detect the G6Pase mutant patients. Thus the short sequence that is cut by the restriction enzyme XyzI will help us to determine the Von gierke disease. Prenatal testing is available for the parents and families for whom the mutation has been identified.The direct sequencing of the 5 exons will prove a better proof for this disease. To analyse the whether The mutation at the 327th position affects the G6Pase enzyme activity, the characterization of the N-terminal deleted and the tagged mutated G6Pase was done. It was found that the deletions of the residues 1-13 abolished G6Pase activity and the mutation of the 5-347 residues abolished the G6Pase activity to an appreciable level.(Lei, 1993), (Pan.1998).When the phosphohydralase activity was checked for these mutants it was found that the deletions and mutations at the 14/357 residues showed very less amount of enzyme activity. The results are given in the picture below. The analysis of the G6Pase mRNA expression and phosphohydrolase activity after transient expression of WT and mutant G6Pase cDNA constructs in COS-1 cells ( Pan , 1998). 10. The Glycogen storage disease is caused by the deficiency of the G6Pase enzyme. When a molecular study was conducted with 350 patients of different origins of the world, it was found that the missense mutations were more among the 60 type of mutations studied. Of all the mutations only a few mutations had a significant frequency. It was found that the R83C and Q347X were fond in common among the 55- 65 % of all the alleles of GSD 1a. A mutation at the 459 insTA appeared frequently among the Hispanic American population. The R83C mutation alone was found among the Chinese and the percentage of occurrence was 40%. No history about the Q347X was found in among the Chinese. Whereas for the Japanese no Q347X and R83C was found, instead they had the occurrence of a silent nucleotide change at the 727G T. This silent mutation led to the deletion of the 91 nucleotides in the 5th exon region. This mutation was found among the 92% of all the GSD1a alleles. This missense mutation affects the active site of the transmembrane helices. i.e. at the R83 active site. PCR and nucleotide sequence analysis are required to be used to identify the locations and the level of the gene expression in the patients and the test people. (Lam, 2008).The enzyme analysis on the liver biopsy can be the clinical diagnosis for this disease. Reference: Koeberl, DD, Sun B, Bird A, Chen YT, Oka K, Chan L 2007, Efficacy of helper- dependent adenovirus vector-mediated gene therapy in murine glycogen storage disease type Ia, Molecular Therapy, vol.15, no. 7:pp.1253-58. Lam CW, But WM, Shek, CC, Tong, SF, Chan, YS, Choy, KW, Tse, WY, Pang, CP, and Hjelm, NM 2008, Glucose-6-phosphatase gene (727G→T) splicing mutation is prevalent in Hong Kong Chinese patients with glycogen storage disease type la, Clinical Genetics, vol.53, no.3, pp.184 – 190. Lei KJ, Shelly LL, Pan CJ, Sidbury JB, Chou JY 1993, Mutations in the glucose-6- phosphatase gene that cause glycogen storage disease type 1a, Science, vol. 262, no. 5133, pp. 580 – 583. Meisenberg, G 2006, Principles of medical biochemistry, 2nd edition, Elsevier publications. Pan CJ, Lei KJ, Annabi B, Hemrika W and Chou JY 1998, Transmembrane Topology of Glucose-6-Phosphatase, Journal of Biological Chemistry, vol. 273, no. 11, pp. 6144–6148. Tenover FC, 1989, DNA probes for infectious diseases, CRC press. Zingone A, Hiraiwa H and Pan, CJ 2000, Correction of Glycogen Storage Disease Type 1a in a Mouse Model by Gene Therapy, The journal of biological chemistry, vol. 275, no. 2, pp. 828–832 Read More