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2/19/10 MUTATION JARGON • Will a base pair substitution, addition or deletion cause a phenotype? • Will a larger scale change such as a gene duplication cause a phenotype? • What information do you need to address this question? Science Dec. 2007 http://www.sciencemag.org/cgi/content/full/318/5858/1842 1 Woe to that child which when kissed on the forehead tastes salty. He is bewitched and soon must die. This adage, from northern European folklore, is an early reference to the common genetic disease recognized today as cystic fibrosis. As the saying implies, the disorder once routinely killed children in infancy and is often identifiable by excessive salt in sweat.. (Scientific American Dec. 1995) cystic fibrosis: most common severe recessive monogenic disorder affecting people of European descent Info about cystic fibrosis http://www.nlm.nih.gov/medlineplus/cysticfibrosis.html http://ghr.nlm.nih.gov/condition=cysticfibrosis http://www.ygyh.org/ 2 the “cystic fibrosis” gene codes for a transmembrane protein involved in chloride transport (note gene is named for its mutant phenotype and not for the protein that it specifies) CFTR= cystic fibrosis transmembrane conductance regulator 3 • All individuals with sickle cell anemia have the same missense mutation in the B globin gene. • In contrast, over 1600 different mutant alleles of the CF gene have been discovered world-wide Cystic Fibrosis Gene: CFTR http://www.genet.sickkids.on.ca/cftr/StatisticsPage.html see statistics, gene, consortium data search database: pull up gene map 4 CF mutations are distributed throughout the gene http://www.genet.sickkids.on.ca/cftr/PicturePage.html Sequence variation = non-disease-causing. It is sometimes designated as “polymorphism”, According to the definition on this web site, a “sequence variation” has to reach an allele frequency of 1% to be a polymorphism. When a sequence variation is found in only one single individual, it is not possible to determine if it is a “non-disease-causing” variation. Retrieval of Genetic Information: Central to any information storage system is the ability to access and retrieve the information and to convert it to a usable form. In addition to the sequence information that will be translated into protein via the triplet code, a gene also contains sequence information that specifies 1. where transcription starts and stops on a given stretch of DNA and which strand of DNA is transcribed 2. where splicing occurs (exon/intron boundaries) 3. where, when and at what level the transcript will be produced 5 find most common mutation: http://www.genet.sickkids.on.ca/cftr/ConsortiumDataPage1.html http://www.genet.sickkids.on.ca/cftr/resource/Table1.html 6 M UTATION J ARGON POINT MUTATION or GENE MUTATION Scale of mutation is small Alteration of a single base pair or a small number of adjacent base pairs ↓ AT THE DNA LEVEL single base pair substitutions: transitions & transversions indels: insertion or deletion of a few base pairs other: transposon insertion AT THE LEVEL OF GENE EXPRESSION AT THE PROTEIN LEVEL promoter mutations splicing mutations regulatory mutations nonsense missense [neutral] silent frameshift AT THE LEVEL OF GENE FUNCTION loss-of-function gain-of-function [neutral] ↑ CHROMOSOME MUTATION involves segments of chromosomes or whole chromosomes CNVs = copy number variations alterations in chromosome structure and number (deletions, duplications, translocations and inversions) 7 DNA TCA 5' 3' AGT transcription TCA 5' 3' 5' 3' 3' UCA 5' AGT splicing and processing in eukaryotes serine codon on mRNA mRNA serine anticodon on tRNA UCA AGU 3' 5' 5' serine attached to tRNA ser at 3' end serine Chemical conversion of TCA into serine. Accuracy of translation depends on precise matching: (1) of an amino acid with its cognate tRNA (2) of the anitcodon of a charged tRNA with its corresponding codon on the mRNA 8 NOTE: code is always in RNAspeak 9 What is a missense mutation? 10 Missense mutation: a mutation that alters a codon so that a different amino acid is specified How will any given missense mutation affect the functioning of a protein? 11 Hard to say a priori without additional information on: • • the nature of the amino acid substitution the site of the mutation in the protein A missense mutation may 1. have virtually no affect on protein function – especially if a chemically similar amino acid is substituted 2. partially or completely inactivate the protein • if the amino acid substitution is in the active site or another site critical for function • if the mutation affects the folding or stability of the protein • if the mutation affects the processing of the protein or interferes with its transit to the appropriate cellular compartment. See interesting example: In Sex Reversal, Protein Deterred by Nuclear Barrier http://fire.biol.wwu.edu/trent/trent/sexreversal.pdf 12 Recent study on a protein called human factor VIII which has a critical role in blood clotting (Nature November 25, 1999) • Factor VIII is a glycoprotein that has a critical role in blood coagulation • This protein circulates as a complex with other proteins • Gene coding for clotting factor VIII is mutated in the Xlinked disease state hemophila A 21 different amino acid residues in factor VIII are known to be sites of deleterious mutations in patients with hemophila • A number of these are in the hydrophobic protein core • other mutated amino acids are involved in hydrogen bonding networks that clearly stabilize protein folding • still others are on the exposed surface of the protein and presumably are important for the interaction of factor VIII with other proteins 13 The enzyme lactate dehydrogenase catalyses the following reaction: pyruvate + NADH lactate the NAD+ What would the effect be of substituting a different amino acid for arginine? 14 Neutral Mutation: • a mutation that has no effect on the Darwinian fitness of its carrier: an allele that has a negligible effect on the ability of the organism to survive and reproduce Neutral Missense Mutation: • a subset of missense mutations in which the effect of the amino acid change on protein function is negligible or is not deleterious to the organism for example: AGA arg AAA lys both are basic amino acids: substitution of arg for lys may not affect protein function arg = arginine lys = lysine 15 BUT: don’t assume that a chemically equivalent substitution will always be neutral Example: Protein: Triose-P-isomerase Glu Asp change in active site decreases catalytic activity 1000X glu= glutamic acid asp = aspartic acid 16 Silent (same sense) Mutation: a mutation that doesn’t change the meaning of the codon (the same amino acid is specified) CUU (leu) CUC (leu) Degenerate code: many amino acids are specified by more than one codon 17 Nonsense mutation: ? 18 Nonsense mutation: a mutation that generates an abnormal stop codon It results in premature termination of translation and a truncated polypeptide 19 Reading Frame: the codon sequence that is determined by reading nucleotides in groups of 3 from some specific start codon (AUG) How does the translation machinery know where to start? How does a eukaryotic ribosome identify the correct reading frame? 20 Frameshift mutatio ? 21 Frameshift mutation: addition or deletion of one or a few base pairs causing a change in the translational reading frame 22 BRCA1 database BRCA1 gene plays a role in the repair of double-stranded breaks in DNA About 5% of breast cancer cases are caused by an inherited susceptibility allele. A mutation in a gene called BRCA1 is thought to account for approximately 80% of families with a high incidence of both early-onset breast and ovarian cancer. Table 1 summarizes some of the data that has been collected on BCRA1 mutations in such families. Table 2 shows neutral polymorphisms found in control families (not showning an increased frequency of breast and ovarian cancer). Table 1. Predisposing mutations in BRCA1. Science 266: 66 1994 NA indicates not applicable. ND = not determined 1901 24 Mutation Nucleotide change -11 bp 2082 1910 2099 2035 1313 1756 1775 NA Extra C T-->G ND Kindred Codon # Coding effect frameshift Gln --> Stop frameshift Met --> Arg Loss of transcript Table 2. Frequency in control chromosomes 0/180 0/170 0/162 0/120 ND Neutral polymorphisms in BRCA1. For the frequency in control chromosomes, the number of chromosomes with a particular base at the indicated polymorphic site is shown (A,C,G, or T). Frequency in control chromosomes Codon Base Name location in codon* A C G T PM1 317 2 152 0 10 0 PM6 878 2 0 55 0 100 PM7 1190 2 109 0 53 0 PM2 1443 3 0 115 0 58 PM3 1619 1 116 0 52 0 *That is, position 1,2 or 3 of the codon. 23 How do point mutations affect the functioning of a gene? DNA RNA PROTEIN Information Contained in the Sequence of a Gene Proper functioning of a gene requires: 1. Coding Region 1. An intact gene product (protein or RNA) specifies RNA & amino acid sequence 2. Other Sequence Information 2. Proper expression of the gene: (signals for generating RNA) a. promoter (RNA polymerase binding site) transcription termination site a. transcript generated from the correct stretch of DNA b. regulatory elements (operators in prok's; enhancers in euk's) b. transcript generated in the appropriate amount at the appropriate time in the appropriate cells c. transcript spliced correctly c. splice site signals 24