How Would the Mutation Affect the Coding Sequence of the Gene - Essay Example

- How would the mutation affect the coding sequence of the gene Mutations create variation within the gene pool. Less favorable (or deleterious) mutations can be reduced in frequency in the gene pool by natural selection, while more favorable (beneficial or advantageous) mutations may accumulate and result in adaptive evolutionary changes. Mutation is generally accepted by the scientific community as the mechanism upon which natural selection acts, providing the advantageous new traits that survive and multiply in offspring or disadvantageous traits that die out with weaker organisms. Changes in DNA caused by mutation can cause errors in protein sequence, creating partially or completely non-functional proteins. To function correctly, each cell depends on thousands of proteins to function in the right places at the right times. When a mutation alters a protein that plays a critical role in the body, a medical condition can result. A condition caused by mutations in one or more genes is called a genetic disorder. Some mutations alter a gene's DNA base sequence but do not change the function of the protein made by the gene. Studies in the fly Drosophila melanogaster suggest that if a mutation does change a protein, this will probably be harmful, with about 70 percent of these mutations having damaging effects, and the remainder being either neutral or weakly beneficial (Sawyer , et al 2007).If a mutation is present in a germ cell, it can give rise to offspring that carries the mutation in all of its cells. This is the case in hereditary diseases. On the other hand, a mutation can occur in a somatic cell of an organism. Such mutations will be present in all descendants of this cell, and certain mutations can cause the cell to become malignant, and thus cause cancer (Ionov , et al 1993). Although many mutations are deleterious, mutations may have a positive effect given certain selective pressures in a population. For example, a specific 32 base pair deletion in human CCR5 (CCR5-32) confers HIV resistance to homozygotes and delays AIDS onset in heterozygotes(Sawyer , et al 2007). The CCR5 mutation is more common in those of European descent. One theory for the etiology of the relatively high frequency of CCR5-32 in the European population is that it conferred resistance to the bubonic plague in mid-14th century Europe. People who had this mutation were able to survive infection; thus, its frequency in the population increased(Ionov , et al 1993). It could also explain why this mutation is not found in Africa where the bubonic plague never reached. Newer theory says the selective pressure on the CCR5 Delta 32 mutation has been caused by smallpox instead of the bubonic plague(Galvani and Slatkin, 2003). - Render the alignment into a box-shaded diagram. Identify the position of the mutation on the multiple sequence alignment. Can you deduce anything from these data Check that your sequences are appropriately gapped . 3'-AA/860 bp insert : 5'-TTTCATGA----- //----- TCATGAAA-3' 3'-AAAGTACT----- //----- AGTACTTT-5' 3'-TT/860 bp insert : 5'-AATCATGA----- //----- TCATGATT-3' 3'-TTAGTACT----- //----- AGTACTAA-5' 3'-CC/860 bp insert : 5'-GGTCATGA----- //----- TCATGACC-3' 3'-CCAGTACT----- //----- AGTACTGG-5' 3'-GG/860 bp insert : 5'-CCTCATGA----- //----- TCATGAGG-3' 3'-GGAGTACT----- //----- AGTACTCC-5' Note that for each respective PCR product, the last eight bases at each 3'-end of the DNA are identical. Also note that only the first two and the last two base pair positions vary between the four PCR products. The PCR products were designed in this way to directly measure the effect of 3'-base composition on blunt vector and T-vector efficiency (Novy, Yaeger, and Kolb, 2008). From the human protein sequence, present any Prosite motif and conserved sequence domains in a sequence diagram. Identify the position of the mutation on the diagram. Can you deduce anything from these data Representative Sequence Length Mass (Da) A2QKA5 Checksum: FF7C4CB42EEB5629 385 41,846 10 20 30 40 50 60 MPGLVLVFPM LPMIVRLLGH IPSADRIDAI YPFGLAVKMS TVSDSTQDRS GVSGLHFRAV 70 80 90 100 110 120 SAPRPSNAAS TAPGIARQQT HSPVRLYINF CTLIPAAVFI RVRQPPAVTV HHSKLVYSSS 130 140 150 160 170 180 SIRKMARILI TGSVDGLGLE AARQLVHRGH TVYLHARNQQ RAMDAKSACP AAGVLTADL 190 200 210 220 230 240 SSVAETQKLC HDVNAIGNFD AVILNAGLMQ GGFRKTPDTG APALVSVNLI SPYVMSCLLR 250 260 270 280 290 300 PPKRLIFISS NLHRQANMAS MDDMFWFKRG ETRYQDYPAY CDSKLHVMLL ANAVARRFKN 310 320 330 340 350 360 TSVTSVHPGW VATKLGGQGA PDKLEDGVET YVMLAEGDYD EQNLSGAYFE PKRKIGQPIP 370 380 AARDEDLQEK VVQACEEVTG LRLSA PS50237. HECT. 1 hit. PS50030. UBA. 1 hit. PS50330. UIM. False negative. PS50918. WWE. 1 hit. E3 ubiquitin-protein ligase which mediates ubiquitination and subsequent proteasomal degradation of target proteins. Regulates apoptosis by catalyzing the polyubiquitination and degradation of MCL1. Also ubiquitinates the p53 tumor suppressor and core histones including H1, H2A, H2B, H3 and H4. Binds to an upstream initiator-like sequence in the preprodynorphin gene. Regulates neural differentiation and proliferation by catalyzing the polyubiquitination and degradation of MYCN. May regulate abundance of CDC6 after DNA damage by polyubiquitinating and targeting CDC6 to degradation. - On the basis of the expected cDNA sequence, design a pair of primers that would allow you to amplify up the entire coding sequence of the rat cDNA (not genomic DNA). Q06609 B2R8T6 O77507 P70099 UPI000155DA5C Q8MKI8 P37383 UPI0000ECBA5B Q5U0A5 +317 - Show the two primers. Q9H4B7 UPI0000DA24BC UPI000036C574 A2AQ07 UPI00006D397A Q4P235 UPI0001791A7D Q8H6M9 Q94586 +6473 - Show the expected PCR product. Cluster: Tubulin beta-1 chain - Show an alignment between the rat and the human proteins. UPI0000DA24BC - Human UPI000036C574 - Rat A genome position can be specified by the accession number of a sequenced genomic clone, an mRNA or EST or STS marker, a chromosomal coordinate range, or keywords from the GenBank description of an mRNA. The following list shows examples of valid position queries for the rat genome. chr16 Displays all of chromosome 16 chr16:1-5000000 Displays first 5 million bases of chr 16 D20RAT64;D20RAT27 Displays region between STS markers D20RAT64 and D20RAT27 AI501130 Displays region of EST with GenBank accession AI501130 AF199335 Displays region of mRNA with GenBank accession AF199335 apoe Displays region of genome with gene identifier apoE NM_145881 Displays region of genome with RefSeq identifier NM_145881 25728 Displays region of genome with Entrez Gene identifier 25728 pseudogene mRNA Lists transcribed pseudogenes but not cDNAs zinc finger Lists many zinc finger mRNAs kruppel zinc finger Lists only kruppel-like zinc fingers huntington Lists candidate genes associated with Huntington's disease Jones,R Lists mRNAs deposited by co-author R. Jones This sequence was assembled using a hybrid approach that combines the clone-by-clone and whole genome shotgun methods. The assembly is a minor update to version 3.3 that spliced in 54.6 Mb finished BAC sequences; the overall statistics are unchanged from releases 3.0 to 3.4. The 3.x assemblies reflect several sequence additions and software improvements over the previous 2.x assemblies, including the sequencing of over 1100 new BACs to cover gaps, an improved marker set from the Medical College of Wisconsin, a new FPC map from the BC Cancer Agency Genome Sciences Centre, and improved linking of bactigs. - Then design a pair of primers that can be used to re-amplify the previous cDNA product but which will (a) attach a restriction site at the 5' end that can be used for cloning the sequence into the E coli expression vector, (b) delete any signal sequence while preserving (or adding back) the ATG start codon in frame, (c) add an in-frame six-histidine residue tag to the C-terminus of the protein finishing with an in-frame stop codon, (d) add a restriction site at the 3' end that can be used for cloning into the E coli expression vector pET3a. Q7Z6Z7-1 Q7Z6Z7-2 Q7Z6Z7-3 - Show the primers. CDKN2A Q8N726 2 EBI-625934,EBI-625922 TP53 P04637 2 EBI-625934,EBI-366083 - Show the expected PCR product and show how it will be fitted into the vector at the level of the sequence. AY772009 mRNA. Translation: AAV90838.1. DQ097177 mRNA. Translation: AAY98258.1. AY929612 mRNA. Translation: AAX24125.1. - Show a diagram of the vector and the position of its important functional features AL592046, Z94044, Z97054 Genomic DNA. Translation: CAI39580.1. Z94044, AL592046, Z97054 Genomic DNA. Translation: CAI42354.1. Z97054, AL592046, Z94044 Genomic DNA. Translation: CAI42654.1. - Translate the open reading frame of the construct to show the sequence of the protein that you intend to prepare as an antigen. AB071605 mRNA. Translation: BAC06833.1. Frameshift. AB002310 mRNA. Translation: BAA20771.2. AB046798 mRNA. Translation: BAB13404.1. Sequence problems. AF161390 mRNA. Translation: AAF28950.1. Frameshift. BC002602 mRNA. Translation: AAH02602.2. BC063505 mRNA. Translation: AAH63505.1. AF057569 mRNA. Translation: AAC62492.1. Different initiation. CR456813 mRNA. Translation: CAG33094.1. AL162050 mRNA. Translation: CAB82393.1. First strand cDNA synthesis can be primed with a gene-specific primer (not included; supplied by user) or with either the Random Hexamer Primers or Oligo(dT) primer provided in the kit. Oligo(dT) interacts with the 3' poly(A) tail present in most eukaryotic mRNAs and can be used with total DNA or with poly(A)+ RNA (Krug and Berger, 1987). Because of the specificity for mRNA, less optimization is required and results are generally more consistent than with random primers. The recommended amount of Oligo(dT) Primer (0.5 g) is usually sufficient for priming first strand cDNA synthesis of 0.5-5 g total RNA or 50-500 ng poly(A)+ RNA. Oligo(dT) priming is convenient when a single first strand synthesis is to be used as template for PCR amplification of multiple gene-specific targets. The first strand cDNA synthesis reaction can be used directly as template to PCR-amplify a gene-specific sequence. The example protocols which follow use NovaTaq Hot Start DNA Polymerase (Cat. No. 71091) or KOD Hot Start DNA Polymerase (Cat. No. 71086). For downstream applications where high accuracy of the product is needed, we recommend KOD Hot Start DNA Polymearase. Some adjustment of these conditions may be needed depending on factors such as target RNA abundance, primer sequences, and choice of thermostable polymerase. Add 1-5 ul of the cDNA synthesis reaction to a mixture containing PCR components and the user supplied gene-specific primers. PCR reagents are not included in the First Strand cDNA Synthesis Kit. The Positive Control RNA and corresponding primers are useful for verifying system performance and identifying sample-related problems. The expected amplification product from the 3 kb Positive Control RNA is 1000 bp. References Primary Sources Berger, S.L. and Kimmel, A.R. (eds.) (1987) "Guide to Molecular Cloning Techniques", Meth. Enzymol. 152, Academic Press, Orlando. Chomczynski, P. and Sacchi, N. (1987) "Current Protocols in Molecular Biology" Anal. Biochem. 162, 156-159 John Wiley & Sons, New York. Freese, Ernst (1959). "The Difference between Spontaneous and Base-Analogue Induced Mutations of Phage T4". Proc of NAS 45 (4): 622-633. http://www.pubmedcentral.nih.gov/articlerender.fcgiartid=222607. Galvani A, Slatkin M (2003). "Evaluating plague and smallpox as historical selective pressures for the CCR5-32 HIV-resistance allele". Proc Natl Acad Sci U S A 100 (25): 15276-9. doi:10.1073/pnas.2435085100. PMID 14645720. http://www.pubmedcentral.nih.gov/articlerender.fcgiartid=299980. Ionov Y, Peinado MA, Malkhosyan S, Shibata D, Perucho M (1993). "Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis". Nature 363 (6429): 558-61. doi:10.1038/363558a0. PMID 8505985. McCormick, M. and Hammer, B. (1994) InNovations 2, 8-10. Novy, Robert E., Keith W. Yaeger, and Kristin M. Kolb (2008) "Perfectly Blunt Cloning: A superior methodfor cloning PCR products or any DNA." Novagen, Inc. retrieved at http://www.emdbiosciences.com/docs/docs/LIT/inno06-001.pdf Sawyer SA, Parsch J, Zhang Z, Hartl DL (2007). "Prevalence of positive selection among nearly neutral amino acid replacements in Drosophila". Proc. Natl. Acad. Sci. U.S.A. 104 (16): 6504-10. doi:10.1073/pnas.0701572104. PMID 17409186. Secondary Sources Leroi A. (2003). Mutants: On the form, varieties & errors of the human body. 1:16-17. Harper Collins 2003 Maki H. (2002). Origins of spontaneous mutations: specificity and directionality of base-substitution, frameshift, and sequence-substitution mutageneses. Annual Review of Genetics 36:279-303. Taggart R. Starr C. (2006) Biology The Unity and Diversity of Life: Mutated Genes and Their Protein Products. 14.4:227. Thompson Brooks/Cole. Read More