12BIS1012012MutatLect12

12BIS1012012MutatLect12 - BIS101-001: Genes and Gene...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: BIS101-001: Genes and Gene Expression Mutations and Mutagenesis Chapters 12 Lecture #12 March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 1 Previous Lecture: Translations Genetic Code: The genetic code is a degenerate, commaless, non-overlapping triplet code. Translation: Synthesizing proteins from mRNAs. Three stages: initiation, elongation, and termination. The SD sequence, AUG codon, tRNAfmet, and 30S ribosome subunit are need for the pre-initiation complex in prokaryotes. Translation needs two methionine tRNAs. Adapter Molecules: The lack of affinity between nucleotides and amino acids makes it necessary for the cell to enlist the help of "adapter molecules" - transfer RNAs and aminoacyl synthetase (AAS). Wobble hypothesis explains isoaccepting tRNA (one tRNA can recognize multiple synonymous codons. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 2 Mutations: Introduction With an understanding of how proteins are synthesized in the cell (translation), the central dogma is now complete. It should now be clear how genetic information is stored, duplicated, and expressed into products essential for the growth and survival of the organism. Because many of these processes require fidelity down to the nucleotide level, slight changes in the gene sequence (e.g., single base substitutions, insertions or deletions) can affect gene function and phenotype. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 3 In this lecture: Molecular Basis of Mutation. Single base pair substitutions, insertions, or deletions can change the way the triple code of the correct open reading frame (zero reading frame) is read. These forward mutations can lead to loss of function or a gene or create a new function. Suppressor Mutations. Mutations can be reversed by a back mutation or by a suppressor. A suppressor is a second mutation within the gene (intragenic) or in another gene (extragenic) Spontaneous Mutations. These mutations are rare and occur as a result of errors in DNA replication, tautomeric shifts in certain bases, and permanent chemical changes in nucleotides. Mutations can be caused by slippage in DNA replication. Induced Mutations. Mutations can be caused or induced by chemicals or physical agents such as ionizing radiation (e.g, X-rays and ultraviolet light). These mutagens can be used to increase the rate of mutations by several orders of magnitude. Ames Test. Bacterial assays that allows us to quickly screen thousands of chemicals for their potential genotoxicity and carcinogenicity. The basis for this test is the reversion rate for a series of well-characterized mutations in the Salmonella bacteria. Complementation analysis: Using mutations to determining the number of genes involved in producing a particular phenotype. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 4 Molecular Basis of Mutation (Cause and Effect) Molecular Basis of Mutation Effects on gene expression How mutations occur in DNA March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 5 The alkaptonuria gene (HGO) and its mutations In 1996, researchers cloned a human gene encoding the enzyme homogentisate 1,2 dioxygenase (HGO). They showed this this gene located on Chr. 3 (3q21-23), corresponded to the AKU gene and that individuals with alkaptonuria had mutations in the HGO gene . exons March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 6 Mutations: Introduction Although DNA must be stable under a wide range of physiological and environmental conditions, it must still be capable of undergoing rare and permanent (i.e., heritable) changes. These changes, together with the processes of independent assortment, recombination and gene conversion, provide a pool of genetic variation from which natural selection will choose the most fit organism for a particular niche. Mutations can arise in several ways: Errors in DNA replication Temporary alterations in H-bonding between bases ( tautomeric shifts) Physical and chemical agents called mutagens that alter DNA structure BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 7 March 19, 2012 Effects of Various Point Mutations Wild Type Gene Sequence Position DNA 1 2 3 5' T T T 5' 4 5 6 7 8 9 T G T T A C A C A U G U Cys A T G U A C Tyr 10 11 12 G T T C G A A U U Val 13 14 15 A G G T A C C G G Glu 3' A A A RNA 5' U U U 5' Amino acid Phe Silent Mutation Position DNA 1 2 3 5' T T C 5' 4 5 6 7 8 9 10 11 12 T G T T A C G T T A C A U G U Cys A T G U A C Tyr C G A A U U Val 13 14 15 A G G T A C C G G Glu 3' A A G RNA 5' U U C 5' Amino acid Phe March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 8 Effects of Various Point Mutations Wild Type Gene Sequence Position DNA 1 2 3 5' T T T 5' 4 5 6 7 8 9 T G T T A C A C A U G U Cys A T G U A C Tyr 10 11 12 G T T C G A A U U Val 13 14 15 G A G C G T C A G Glu 3' A A A RNA 5' U U U 5' Amino acid Phe Nonsense Mutation #1 Position DNA 1 2 3 5' T T T 5' 4 5 6 7 8 9 10 11 12 T G A T A C G T T A C T U G A Non A T G U A C -- C G A U -- A U 13 14 15 G A G C G T A -- C G 3' A A A RNA 5' U U U 5' Amino acid Phe March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 9 Effects of Various Point Mutations Wild Type Gene Sequence Position DNA 1 2 3 5' T T T 5' 4 5 6 7 8 9 T G T T A C A C A U G U Cys A T G U A C Tyr 10 11 12 G T T C G A A U U Val 13 14 15 G A G C G T C A G Glu 3' A A A RNA 5' U U U 5' Amino acid Phe Nonsense Mutation #2 Position DNA 1 2 3 5' T T T 5' 4 5 6 7 8 9 T G T T A A A C A U G U Cys A T T U A A Non 10 11 12 G T T C G A U -- A U 13 14 15 G A G C G T A -- C G 3' A A A RNA 5' U U U 5' Amino acid Phe March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 10 Effects of Various Point Mutations Wild Type Gene Sequence Position DNA 1 2 3 5' T T T 5' 4 5 6 7 8 9 T G T T A C A C A U G U Cys A T G U A C Tyr 10 11 12 G T T C G A A U U Val 13 14 15 G A G C G T C A G Glu 3' A A A RNA 5' U U U 5' Amino acid Phe Missense Mutation Position DNA 1 2 3 5' T T T 5' 4 5 6 7 8 9 10 11 12 T G T C A C G T T A C A U G U Cys G T G C A C His C G A A U U Val 13 14 15 G A G C G T C A G Glu 3' A A A RNA 5' U U U 5' Amino acid Phe March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 11 Effects of Various Point Mutations Wild Type Gene Sequence Position DNA 1 2 3 5' T T T 5' 4 5 6 7 8 9 T G T T A C A C A U G U Cys A T G U A C Tyr 10 11 12 G T T C G A A U U Val 13 14 15 G A G C G T C A G Glu 3' A A A RNA 5' U U U 5' Amino acid Phe Frameshift Mutation (deletion) Position DNA 1 2 3 5' T T 5' 4 5 6 7 8 9 10 11 12 T G T T A C G T T A C A A T G C U G U U A C G Val Thr A U 13 14 15 G A G T A C G 3' A A RNA 5' U U 5' Amino acid Phe A C U G Leu March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 12 Effects of Various Point Mutations Wild Type Gene Sequence Position DNA 1 2 3 5' T T T 5' 4 5 6 7 8 9 T G T T A C A C A U G U Cys A T G U A C Tyr 10 11 12 G T T C G A A U U Val 13 14 15 G A G C G T C A G Glu 3' A A A RNA 5' U U U 5' Amino acid Phe Frameshift Mutation (insertion) Position DNA 1 2 3 4 5 6 5' T T T T T G T 5' 7 8 9 T A C 10 11 12 G T T A U 13 14 15 G A G C T G A Non C G 3' A A A A A C A A T G C A RNA 5' U U U U U G U U A C G U 5' Amino acid Phe Leu Leu Arg March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 13 Intragenic Suppression of Point Mutations Wild Type Gene Sequence Position DNA 1 2 3 5' T T T 5' 4 5 6 7 8 9 T G T T A C A C A U G U Cys A T G U A C Tyr 10 11 12 G T T C G A A U U Val 13 14 15 G A G C G T C A G Glu 3' A A A RNA 5' U U U 5' Amino acid Phe Frameshift Mutation (deletion) Position DNA 1 2 3 5' T T 5' 4 5 6 7 8 9 10 11 12 - - T T A C G T T - - A - - U A T G U A C Tyr C G A A U U Val 13 14 15 G A G C G T C A G Glu 3' A A RNA 5' U U 5' Amino acid Phe March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 14 Intragenic Suppression of Point Mutations Wild Type Gene Sequence Position DNA 1 2 3 5' T T T 5' 4 5 6 7 8 9 T G T T A C A C A U G U Cys A T G U A C Tyr 10 11 12 G T T C G A A U U Val 13 14 15 G A G C G T C A G Glu 3' A A A RNA 5' U U U 5' Amino acid Phe Frameshift Mutation (insertion) Position DNA 1 2 3 5' T T 5' 4 5 6 7 8 9 T G T T T A C A C A A A T G U G U U U A C Val Tyr 10 11 12 G T T C G A A U U Val 13 14 15 G A G C G T C A G Glu 3' A A RNA 5' U U 5' Amino acid Phe March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 15 Extragenic Suppression of Mutation As shown in the previous example, the affect of a mutation can be covered up or "suppressed" by a second mutation. This second mutation can occur in the mutated gene (intragenic suppression) or outside the gene or in another gene (extragenic suppression). The most common type of extragenic suppressor is a mutant tRNA in which a base substitution in the anticodon loop enables the tRNA to recognized one of the stop codons (UAA, UGA, and UAG). UAG amber Gene A X tRNAtyr2 mutation tRNAtyr1 X Anticodon mutation 16 March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 Wild type and Tyrosine Amber Suppressor tRNA Courtesy Stanley Maloy, SDSU Center for Applied and Experimental Genomics March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 17 Molecular Basis of Mutation (Cause and Effect) Molecular Basis of Mutation (How mutations occur) March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 18 Spontaneous Mutations Point Mutations affect only one or two bases in a DNA sequence. A single base change, addition, or deletions are examples of point mutations. Shown below is the effects of point mutations at each of the three positions of the tyrosine codon. GAU asp CAU his UAG non UAA non UAC tyr UUU phe 19 AAU asn UGU cys March 19, 2012 UAU tyr UCU ser BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 Spontaneous Point Mutations Transition: Mutations that change purines and pyrimidines to their alternate forms (e.g., A G; C T, etc.). Transversion: Mutations that change purines to pyrimidines and vice versa (e.g., A C, T G, etc.). Transition A Transversion G Transversion T Transition March 19, 2012 C 20 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 Normal Hydrogen Bonding between Bases March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 21 Rare Pyrimidine Tautomeric Shifts Mispairing of C* and T* to A and G based due to tautomeric shifts. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 22 Rare Purine Tautomeric Shifts Mispairing of A* and G* to C and T based due to tautomeric shifts. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 23 "Fixing Mutations by 2 Rounds of DNA Replication Two rounds of DNA replications are needed to "fix" a mutation in the genome. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 24 Nitrous Acid (HNO2) Nitrous Acid (HNO2) deaminates cytosine and adenine to produce uracil and hypoxanthin (Hx), respectively. Uracil can bond to A while hypoxanthin can bond to C. Nitrous acid produces a GC to AT transitions and TA to CG transitions. Note that two rounds of DNA replication are required to fix the mutation into the gene. G C HNO2 G 1st DNA U replication U 2nd DNA A replication A T March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 25 Nitrous Acid (HNO2) Nitrous Acid (HNO2) deaminates cytosine and adenine to produce uracil and hypoxanthin (Hx), respectively. Uracil can bond to A while hypoxanthin can bond to C. Nitrous acid produces a GC to AT transitions and TA to CG transitions. Note that two rounds of DNA replication are required to fix the mutation into the gene. T A HNO2 T 1st DNA Hxreplication Hx 2nd DNA C replication C G March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 26 Hydroxylamine (NH2OH) Hydroxylamine (NH2OH) adds an OH group in place of the NH2 group on cytosine. This produces hydroxylaminocytosine (or N-4-Hydroycytosine), which can bind to adenine. NH2OH generates GC to AT transitions. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 27 Hydroxylamine (NH2OH) Hydroxylamine (NH2OH) adds an OH group in place of the NH2 group on cytosine. This produces hydroxylaminocytosine (or N-4-Hydroycytosine), which can bind to adenine. NH2OH generates GC to AT transitions. G C NH2OH G 1st DNA C*replication C* 2nd DNA A replication A T March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 28 Chemical Mutagens Alkylating Agents: Ethyl methanesulfonate (EMS), ethyl ethanesulfonate (EES) and methyl methane-sulfonate (MMS) all add alkyl groups such a ethyl or methyl groups to the oxygen on purines and pyrimidines. O-6 ethylguanine is a good example of how EMS works. This produces a GC to AT transition. Alkylating agents can also produce the following transversions: GC to CG, AT to TA, GC to TA and AT to CG. Acridine compounds. Examples, proflavin and acridine orange. These compounds insert themselves between stacked base pairs in double stranded DNA and are therefore called intercalating agents. During the process of DNA replication, single base insertions or deletions are produced. This causes frame-shift mutations in the gene. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 29 Molecular Mechanism of Mutation Physical Mutagens March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 30 Physical Mutagens Herman Muller discovered the first mutagen in the form of Xrays. In 1927, Muller demonstrated that up to a point, the frequency of mutations in certain genes of Drosophila was directly proportional to the X-ray dose. Because the high energy of X-rays, most mutations involved chromosome breakage or gross mutations. Since gross mutations can involve thousands or millions of base pairs, these kinds of mutations are not has helpful for the fine genetic analysis of a genome. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 31 Physical Mutagens One physical mutagen that is capable of creating point mutations is ultraviolet light (UVA, 320400nm; UVB, 290-320nm). Although UV is much less energetic than X-rays, it is still a very potent mutagen because the bases of DNA absorb energy in the UV wavelength range (260nm). Strong UV irradiation can produce chromosomal breaks, however, lower doses can produce permenant photoproducts called pyrimidine dimers. Dimerization can occur between adjacent C and C, U and U, C and U, but T and T (thymine dimers) are the most common photoproduct in UV irradiated DNA. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 32 UV Induced Pyrimidine Dimers Pyrimidine dimers become mutagenic when they go uncorrected. DNA polymerase does not recognized pyrimidine dimers as good templates and will either stall at the dimer or delete or add a nucleotide. Therefore, UV irradiation is useful in producing point mutations of the single nucleotide insertion or deletion. The lac UV5 promoter mutation was created with UV irradiation. BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 33 March 19, 2012 DNA Repair Maintaining the sequence integrity of DNA is critical for the survival and fitness of an organism. For this reason, nature has evolved a number of mechanism for repairing various types of DNA damage. Direct reversal of damage (photolase, alkyltransferase Excision-repair pathways (uvrA, uvrB and uvrC) Specific excision repair (DNA glycosylases) GO system (mutM, mutY and mutT) Post-replication or mismatch repair (adenine methylase) Recombination repair (recA) BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 34 March 19, 2012 Excision Repair of DNA Damage http://www.youtube.com/watch?NR=1&feature=endscreen&v=kodYv-XKhgc March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 35 E. coli Excision Repair uvrA detects the distortion in the DNA and guides uvrB to the damaged site. uvrA dissociates and uvrC binds uvrB at the damaged site. uvrB and uvrC makes cuts about 12 bases apart in the damaged DNA strand. Helicase unwinds the 12mer from the helix. DNA polymerase I synthesizes new DNA in the gap. BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 36 March 19, 2012 Base Excision Repair of DNA Damage Base modifications are sometime too subtle to be recognized by the uvrA protein. In these instances, DNA glycosylases are use to remove damage bases. apurinic site (AP) (AP March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 37 Deficiencies in DNA (excision) Repair Xeroderma pigmentosum is a rare heritable form of skin cancer in humans. Only about 1000 affected individuals world wide. Inheritance is autosomal recessive. The defect is in one of 8 complementation groups (genes) involved in excision repair of UV induced DNA damage (XPA, XPB, XPC, XPD, XPE, XPF, XPG, and POLH) Complementation group XPC is the most common and XPG is associated with neurological disorders. BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 38 March 19, 2012 Molecular Mechanism of Mutation Mutation by "Slippage" in DNA Replication March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 39 Errors in DNA Replication: Slippage 13 b 12 b 13 b 15 b March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 40 Errors in DNA Replication: Strand Slippage During DNA replication, the nascent or template strand may slip and add a base on one strand (nascent) or delete a base on the template strand. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 41 Fragile X Syndrome Fragile X (Xq27.3 ) is the most common inherited form of mental retardation, occurring in 1 of every 1200 males and 1 of every 2500 females. In 1991, the fragile X gene (FMR1) was characterized and found to contain a tandemly repeated trinucleotide sequence (CGG) near its 5' end. The mutation responsible for fragile X syndrome involves expansion of this repeat segment. The number of CGG repeats in the FMR1 genes of the general population varies from six to approximately 50. Courtesy, Dr. David Ussery March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 42 Fragile X Syndrome The defect is due to base slippage during replication of multiple CGG repeats. Repeats in the 6 to 200 range (premutations) appear normal. Males with repeats in this range are called "normal transmitting males" or NTMs. Repeats of 200 and greater show the disease phenotype. Increase in CpG or the CGG repeat is a target for DNA methylation (5 ' methyl cytocine) which seems to reduce the expression of the FMR-1 gene. One strand of the repeated region has been shown to form a hairpin structure that blocks DNA synthesis. The best test for fragile X mental retardation is a PCR-based test using primers flanking the CGG repeats. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 43 Summary: Molecular Basis of Mutation. Single base pair substitutions, insertions, or deletions can change the way the triple code of the correct open reading frame (zero reading frame) is read. These forward mutations can lead to loss of function or a gene or create a new function. Suppressor Mutations. Mutations can be reversed by a back mutation or by a suppressor. A suppressor is a second mutation within the gene (intragenic) or in another gene (extragenic) Spontaneous Mutations. These mutations are rare and occur as a result of errors in DNA replication, tautomeric shifts in certain bases, and permanent chemical changes in nucleotides. These changes can be caused by chemicals or by physical agents such as ionizing radiation (e.g, X-rays and ultraviolet light). Induced Mutations. Chemical and physical agents can be used to induce (i.e., increase the rate of) mutations by several orders of magnitude. Mutations can be caused by slippage in DNA replication. Ames Test. Using our knowledge of mutations to monitor mutagens in the environment. Complementation analysis: Determining the number of genes involved in producing a particular phenotype. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression R.L. Rodriguez 2012 44 ...
View Full Document

Ask a homework question - tutors are online