Class Note for ECOL 320 at UA
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Date Created: 02/06/15
mph ummm at mama s AN ORIGINAL AUDIO DRAMA STARRING PETER DAVISON h SARAH SUTTON Introduction 0 How do we detect genes 1 Detecting phenotypic changes in the organism due to changes in the gene 2 Screening DNA sequences for ORFs with genelike features or similarities to genes already known 0 A mutation is a change in base sequence of a gene or in the arrangement of genes on a chromosome chromosome mutation discussed more later 0 A mutation produces a new allele of the gene 0 The most common allele in a laboratory stock or wild population of an organism is called the wild type allele then a mutation produces a new mutant allele 0 A mutant is an individual carrying a mutant allele Wild type Drosophila 0n the left each of the three mutants is in a different gene Some Kinds of Mutations wild type ATGACCATGA base pair substitution AGGACCATGA AAGACCATGA ACGACCATGA insertion ATGGACCATGA ATGTTACCATGA ATGTTCACCATGA Deletion 6T AGACCATGA 6TG AACCATGA 6TGA ACCATGA 0 Changes of 1 bp or a few contiguous bp are called point mutations 0 Also possible but much less frequent are substitutions of z 2 hp at one tune 0 Transposable elements cause insertions of 102 104 bp Mechanisms of Point Mutation partial list 1 Most are due to errors in DNA replication replication repair GATC gt GATC gt GATC or GGTC CTAG CCAG CTAG CCAG Repair occurs during proofreading or later Slip strand mispairing causes short repeats NNNNNNAGCAGCAGC NNN e g Huntington s disease AGCn an in frame repeat encoding polyGlu The resulting polypeptide causes cell death in parts of the brain and dominant neurological problems N stands for nucleotide ie any base 2 Some are due to spontaneous changes in bases 6 g C gt deamination gt U Mechanisms of Point Mutation continued H J Muller 3 Some mutations are caused by mutagens Chemical mutagens modify bases so they cause mispairing When replicated or are repaired incorrectly UV radiation links adjacent pyrimidines to form dimers Which may be repaired incorrectly Ionizing radiation induces single or double stranded breaks chemically modifies bases or cross links bases may be repaired incorrectly When and Where Mutations Happen Mutations are stochastic events unpredictable random we can never predict exactly when a mutation will occur or what kind of mutation will occur but we can assign a probability frequency or mutation mte to it Most muiau39ons happen during cell division so we usually measure the iaie in muiau39ons per cell division 4 99 9999 6 5 Measured mutation rate would be 7 mutations31 cell divisions or o 225 mutationscell division unrealistically hi gh examplel When and Where Mutations Happen In eukaryotes mutations that occur in the somatic cells somatic mutations are not inherited mutations that occur any time in the germ line are inherited We usually measure the rate in mutations per gamete somatic mutation 44 4 42 4 L a 004 30 0101 germ line mutation Mutation Rates Mutations are stochastic events unpredictable random we can never predict exactly when a mutation will occur or what kind of mutation will occur We can measure the rate at which a given kind of mutation will occur The rate is the probability that it will occur in a unit of time Orders of magnitude animals and plants 10396 10394 muts per gene per gamete 103910 10398 muts per bp per year from evolution rates 10399 mutsbp X year X 3 X 10 9 bp gt 1 new mutation in each gamete we are all mutants Drosophila u 84 X 10399 mutsbp X year U 12 detrimental mutations2N genome bacteria 103910 10396 muts per gene per cell division How to write rates so they can be used in dimensional analysis 10399 muts per bp per year 10399 mutsbp X year NOT 10399 mutsbpyearbecause that is 10399 muts X year bp Mutagenesis increases the rate probability but the mutations are still stochastic Mutation Rates A given mutagen increases the rate of some kinds of mutations but not others In other words it biases the probabilities like weighting a coin 6g 0 Ionizing radiation induces increases the probability of chromosome breaks and hence of large scale chromosome mutations or rearrangements such as deletions or translocations 0 Acridines induce deletion or insertion frameshifts 0 Ethyl methan sulfonate EMS induces chemical changes in bases mainly changing guanine to O6 ethyl guanine Which mispairs resulting in changing GC pairs to AT 0 5 bromouracil Bu is an analogue of thymine and is most often incorporated into DNA in place of thymine It then pairs With G at the next replication so that an AT pair is replaced by a GC pair Less commonly it causes a GC pair to be replaced by AT Modern genetic engineering can reduce or eliminate the stochasticity of mutation allowing us to change a specific base in a specific gene in vilro then put it back in the organism Knockout mutations can be made in specific genes in vivo Phenotypic Effects of Mutations in Exons Review code properties 0 Degenerate 0 Triplet 0 Commaless 0 Start codon 0 Stop codons First Third position Second position position 639 end 339 end U C A G lJlJlJ PheF UCU Ser UAU TyrY UEU Cysc U U UUCPhe UL L Ser S UAC Tyr UBCCVS C UIJA LeuL UL A Ser A UUG Leu UDG Ser UGBTrpW G CUULeu CCU Pro CAU Hi5H CGUArg U C CUCLeu L CCCF rO P CAC His CGCArg R C CUA Leu CCA Pro CAA GinQ CGA Arg A CUG Leu ECG Pro CAB Gln CGG Arg G AUU lie ACU Thr AAU ASHN AGU SerS U A AUClle 1 ACCThr T AAC Asn AGC Ser C AUA lle ADA Thr AAA LysK AGA ArgR A MM ACG Thr AAG Lys AGG Arg G GUU Val GCU Ala GAU AspD GGU Gly U G GUC Val V GCCAla A GAC Asp GGC Gly G C GUA Val GCA Ala GAA GluE GGA Gly A BUG Val 636 Ala GAB Glu GGG Gly G Note Each amino add is given its Conventional abbreviation in both the singlelettar and the threerletter format The codon AUG Wl39llCl39l codes for methionine boxed is generally used for Initiation The codoris are conventionally written With the 539 base on the left and he 3 base on the right Phenotypic Effects of Mutations Useful terms for phenotypes of mutations Amorph nullimorph null knockout mutations mutant allele is completely inactive not transcribed or translated or encodes inactive protein or RNA e g white eye mutant in Drosophila melanogaster Hypomorph mutant allele has reduce activity reduced rate of transcription or translation or encodes protein or RNA with reduced activity e g apricot eye allele of white gene in D melanogaster Neomorph gain of function mutation mutant allele has new activity eg encodes protein or RNA with new enzymatic activity or turns one gene off and another one on or make a gene active in wrong tissue eg Antennapedia in Drosophila The morph terminology was devised by H J Muller who used it mainly to refer to mutation effects inferred from phenotypic effects Consequences of Code Properties First Third position Second position position Degenerate tsenm W U C A G UUU PheF UCU Ser UAU TyrY UGU Cvs U U UUCPhe UCESer S UAC Tvr UGCCvs C UUA LeuL UCA Ser A UUB Leu UCGSer UGGTrpW G CUULeu CCU Pro CAU HisH CGUArg U C CUCLeu L ICC Pro P CAC His CGCArgR C CUA Leu CCA Pro BAA GlnQ CGA Arg A CUGLeu ECG Pro BAG Gln CGGArg G AUU lie ACU Thr AAU AsnN AGU Sergt3 U A AUClle 1 ACCThr T AAC Asn AGCSer C AUA lie ACA Thr AAA Lyslt AGA ArgR A MM AEGThr AAG Lys AGG Arg 3 GUU Val GCU Ala GAU ASpD GGUGIy U G GUCVEI V GCCAIa A GAC Asp GGCGIy C GUAVaI GCA Ala GAA Glu GGA Gly A GUG Val GCG Ala GAG Glu GGG Gly G Nute Each ammo acid is given its conventional abbreviation in both the Singleletter and the threerlettev format The odon AUG which codes for methionine boxed is generally used For initiation The codons are conventionally written with the 539 base on the left and the 3 base on the right Consequences of Code Properties Degenerate therefore in an exon of a protein coding gene there are two kinds of point mutations Synonymous mutations change a codon to a synonymous codon and do not change an amino acid Nonsynonymous missense mutations change an amino acid First Third position Second position position 539 end 339 and U C A G UUU PheF UCU Ser UAU TyrY UGU CysC U U UUCPhe UCCSer S UACTyr UGCCys C UUA LEUL UCA Ser A UUG Leu UCGSer UGGTrpW G CUULeu EEUPro CAU Hi5H CGUArg U C CUC Leu L EEBPro P CAC Hi5 CGCArg R C CUA Leu lJlJA Pro LAA GlnQ CBAArg A CUE Leu ECG Pro CAG Gln C66 Arg G AUU lie AEU Thr AAU AsnN ABU SerS U A AUCHe 1 ACCThr T AAC Asn ABCSer C AUA Jie ADA Thr AAA LysK ABA ArgR A MM ACGThr AAB Lys ABBArg G GUU Val Gnu Ala GAU AspD GGUGIy U G GUC Val V GEE Ala A GAC Asp GGCGIy G C GUA Val GCA Ala GAA GIUE GGA Gly A BUG Val GCG Ala GAG Glu GGG Gly G Nnte Each amino acid is given its Conventionai abbreviation in both the singieietter and the threerietter format The codon AUG which codes for methionine boxed is generain used For il liliatlo The codons are conventionaliy written with the 539 base on the left and lhc 3 base on the right Consequences of Amino Acid Changes Synonymous mutations do not change an amino acid Therefore they usually have no effect on phenotype Nonsynonymous missense mutations change an amino acid The phenotypic effect depends on the nature of the change and the location in the protein Some changes have no effect on protein function therefore no effect on phenotype Changes that are more likely to have an effect include Changing to amino acid With very different side chain charge hydrophobicity etc Change in active site or ligand binding site or site required for folding or joining With other polypeptides Changes on outside of molecule that affect hydrophobicity or charge of protein Hemoglobin Heme in red two oz and two 3 chains in different colors Phenotypic Effects of Mutations First Third position Second position position 539 and 339 endl U C A G I I UUU PheF ucu Ser UAU TvrY usu Cvsc U U UUCPhe UCCSer 5 UAC Tyr UGCCvs C UUA LeuL UCA Ser m A UUG Leu UCGSer UGG TrpW G II CUULeu CCUPro CAU HisH CGUArg U C CUCLeu L CCCPro P CAC His CGCArg R C CUA Leu CCA Pro CAA GlnQ CGA Arg A CUG Leu ECG Pro CAB Gln CGGArg G AUU lle ADU Thr AAU AsnN ABU Sers U A AUCllB 1 ACCTI Ir T AAC Asn ABCSer C AUA lle ADA Thr AAA LysK ABA ArgR A AUGMst ADGTI Ir AAG Lys ABG Arg G BUU Val GCU Ala GAU ASDD GEUGly U G BUB Val V GEE Ala A GAC Asp GGCGly G C BUA Val GCA Ala GAA GIUE GEA Gly A BUG Val GCG Ala GAG GlLl GEE Gly G Nate Each amino acid is glth its convenhonal abbreviation in both the singleletter and the threerletter Format The codon AUG which codes For methlonine boxed ls genevally used For initiation 39l he Codons ave conventlonally wnlten wrth the 539 base on lhe left and lhe3 base on the right First Third position Second position position Phenotyplc Effects u c A e Of uuu PheF ucu Seri UAU TyrY usu Cysc u U UUCPhe UCESer S UAC Tyr usccvs c UUA LeuL UCA Serf UAA St Em A UUGLeu UCGSer UAGSt UBBTrpW G Trlplet cuu Leu CCU Pro CAU HisH csu Arg u C CUCLSL L EEC Pro P CAC His CGCAFQ R C CUA Leu BEA Pro CAA Gln CGA Arg A o CUG Leu EEG Pro CAG GlniQ CGG Arg G AUU lie ACU Thr AAU AsnN AGU Sers u A AUCIIE1 ACCThr T AAC Asn AGCSer c AUA lie ACA Thr AAA Lys AGA Arg A Deletions of 1 or 2 bp are frameshift mutations MM m mi W m K AGE M R G GUUVBI ECU Ala GAU ASpD GGUGIy U G GUCVBI V EEC Ala A GAC Asp GGCGIy G C Consequences GUA Val EDA AlaJ GAA GIuE GSA Gly A GUGVBI ECG Ala GAE Glu GGG Glyi G Note Each ammo en its Conventionai abbreviation in both the singieie ter and the threeriette e codon AUG which Codes for methionine boxed is generain used For muanen The codons are conveniionaliy written with the 539 base on the left and lhe 3 base on the ngm Met Val His Leu Thr Pro Glu His Term CACCAIGGIGCACQIGAQTCQTGAGmQACUAAGCU Met Val Thr Term CACCAISGI39QAE TQACTCCTGAGCACUAAGCU C Met Val Pro Pro Asp Ser Term CACCAIGGIGCQAQQTGACTQCTQAGmCACUAAGCU Met Val His Leu Thr Arg Leu Ser CACCEGCEGCACCAGETQAMAQAAEU 39 First Third Phenotyplc Eff 60tS 223quot 722quot 0 U C A G Of UUU PheF UEU Ser UAU TyrV UGU CysC U U 332 E251 EEK Siiis AW 6 5 5i UUGLeuJL UEGSer m UGGTrpW G Deletlons of 1 or 2 bp 1n exons are frameshlft CUULZE UPI CAUHEEiH 5332 E mutatlons 0 33A he L SEA 22 332 AAAAAS R A CUBLeu CCG Pro CAB Gln CGG Arg G Consequences 33 1 1 22331 223 23 2E3 3238 E 0 Change of one or more amlno a01ds phenotype A AAA AA AAA m T AAA WK AAA Argh A I I I I M AEGThr AAG Lys AGG Arg G depends on the ammo a01ds and the1rlocat10n 233 3 25 A 223 20 223 Sly E Premature termlnatlon G AAAVA V AAA AA A AAAAIEE AAA A G A GUGVSI GCG Ala GAE Glu GGG Gly G 0 Late termination 0 Usually nullimorphs or hypomorphs Met Val His Leu Thr Pro Glu Met Val Thr Term termination or stop Note Each amino acid is iv 39t g en 1 s orwentionai abbreviation in both the Singieietter and the threerietter format The codon AUG which gemeraily used For initiation The codons are the left and the 3 base on the right His codes for methionine boxed is convehhohaliy WHHEH With the 539 base on Term CACCAISGESQACQTQAQTCELTGAGQACUAAGCU CACCAIEGEQAEE TQACTCCTGAGCACUAAGCU C Met Val Pro Pro Asp Ser Term CACCATGGTGCCACCTGACTCCTGAGCACUAAGCU C Met Val His Leu Thr Arg CT Leu CACCEGgTGgAchGACRECLCAQAAEU Ser Phenotypic Effects of Mutations in Exons 0 Start codon 0 Stop codon First Third position Second position position 1539 end 339 end U c A G UUU PheF UIZU Ser UAU TyrY UBU Cysc U U UUCPhe UEESer S UAC Tyr UBCCys c UUA LeuL UIZA Ser MA 3 A UUGLeu UEGSer UAG St UBBTrpW G CUULeu CCU Pro CALI HisH CGUArg U C cuc LieuL cm ProP CAC His CGC ArgR C CUA Leu DEA Pro CAA Gln CGA Arg A BUG Leu ECG Pro CAG Gln Q 66 Arg G AUU Ile ADU Thr AAU AsnN AGU Sers U A AUCIIe1 ACCThrT AACAsn ABCSer c AUA lie ADA Thr AAA LysK ABA ArgR A MM Ace Thr AAG Lys ABE Arg G GUU Val ecu Ala GAU AspD GGU Gly U G GUC Val V GEE Ala A GAC Asp GGCGIin c GUA Val SEA Ala GAA Glu GGA Gly A GUG Val EEG Ala GAB Glu E 566 Glyi G Note Each amino acid is given its conventional abbreviation in both the singieietter and the threeriettev format The codon AUG Wi39iiCi i Codes for methionine boxed is genevaily Used For in ation The codons are conventionaliy written with the 539 base on the left and the 3 base on the right Phenotypic Effects of Mutations in Exons Any change in start codon 0 Late start first part of polypeptide missing Out of frame start Likely result is polypeptide With no function nullimorph or abnormal function hypomorph First Third position Second position position 539 end 339 end U C A G UUU PheF UCU Ser UAU TyrY UBU CysC U U UUCPhe UCESer S UAC Tyr UBCCVs C UUA LeuL UIZA Ser UAA St A UUGLeu UCGSer quotAG 8 UBBTrpW G CUULeu CCU Pro CAU HisH CGUArg U C CUCLeu L EEC Pro P CAC His CGCArg R C CUA Leii CEA Pro CAA GinQ CGA Arg A BUG Leii ECG Pro CAG Gln CGG Arg G AUU lie ACU Thr AAU ASI IN AGU SerS U A AUC lie 1 ADC Thr T AAC Asii AGE Ser C AUA lie ACA ThrJ AAA LysK AGA ArgR A MM AUG Thr AAG Lys AGE Arg G GUUVaI ECU Ala GAU AspD GGUGIy U G GUCVBI V GEE Ala A GAC Asp GGCGIy G C GUA Val EBA AlaJ GAA GIHE GGA GIyJ A GUGVBI EEG Ala GAG GILi GGG Gly G Nara Each amino acid is given its Conventional abbreviation in both the singleletter and the threeriettei format The odori AUG Wi iiCi i Codes for methionine boxed is Change of stop codon to a sense codon Polypeptide has extra segment at the end Possible result is polypeptide With no function reduced function or abnormal function amorph hypomorph or neomorph Nonsense mutations change a sense codon for an amino acid to a nonsense 2 stop codon Shortened polypeptide Which usually but not always has reduced or no function generain used For initiation The codons are conventionally written with the 539 base on the ieii and the 3 base on the right Mutations Due to Transposable Elements g I r l x l t39 i l Transposable elements TEs are segments of DNA that can move from one location to another in the genome or have a copy made and moved to a new location Insertion of a TE in a gene can cause frameshifts or cause additional amino acids to be added to a protein product This usually results in a null mutation Insertion of TEs in controlling elements or between a gene and its controlling elements can cause major changes in transcription no transcription or transcription at inappropriate times andor places Transposable elements are more common in some organisms eg Drosophila melanogaster than in others and can cause a major proportion of all visible mutations Genes Encoding Enzymes and Auxotrophic Mutants Some genes code for enzymes Many enzymes catalyze steps in biosynthetic pathways Steps are sequential enzymes act sequentially Usually l enzyme catalyzes 1 reaction or step in a pathway Genes G 1 G2 G3 l l l Enzymes E1 E2 E3 A i gt B i gt C i gt D e g tryptophan biosynthesis in E coli Text says 5 steps but don t show the pathway Notice that names of genes are italicized or underliuned Chorismic acid ASase 4 trpE Anthranilic aci gtlt PRTase trpD PEA CDRP InGPSase 4 trpC lnGP TSaseB 4 trpB lndole TSaseA 4trpA L Tryptophan Genes Encoding Enzymes and Auxotrophie Mutants Special classes of mutants especially useful with microorganisms Antibioticresistant Auxotrophic can t make essential nutrient must be supplied in culture medium Test for growth on minimal medium MM which has things wild type needs energy C N sources salts etc only wild type prototroph grows because it can make everything it needs from these simple ingredients 0Control is complete medium CM which has yeast extract proteose peptone etc that supply everything all genotypes grow One Gene One Enzyme Hypothesis not quite right but focused on idea that genes have their phenotypic effects by encoding proteins Tryptophan Auxotrophs Chorismic acid ASase 4 trpE AnthraniW PRTase 4 trpD P EA CDRP InGPSase 4 trpC InGP TSaseB lt trpB Indole gtltTSaSCA 4 mm L Tryptophan Grows On CM MM MMTrp MMindoIe MMInGP MMCDRP trp trpA trpB trpC trpD trpE Visible Phenotypes 0f Pathway Mutants lry pvlophzn Irwxophan pyrmlzse yHoirmIkynurenm i formamidase kwurenine J kwumnvneanydrwase lt7 cinnabar 3hydmxykynul enme phenoxazinone symmase xamhomrrmin Drosophi la melanogaster wild type has red eyes which require a number of pigments Makes eye pigment xanthommatin from tryptophan Cinnabar ant gene encodes kynurenineShydroxylase Cinnabar cn mutant has Cinnabarcolored eyes Phenotypic Effects of Mutations in Introns Usually no effect unless mutation in splicing signal sequence gt failure to remove intron 0r splicing at incorrect site ATG GTG CAC m GCAthtggtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcatgtggag AcagagaagactcttgggtttctgataggcactgactctctctgcctattggtctattttcccacccttagGCTGCTG GTGATGGTGCAC m AG ATG caa ata CTG GTG tag ggc CTG GCAGGCTGCTGG No splicing wrong amino acids up to a stop codon CAC m GCA th ggt atc aag gtt aca aga cag gtt taa gga gac aaa ctg ggc atg tggagacagagaagactcttggg ttt ctg act gac tct ctc tgc cta ttg gtc tat ttt ccc acc ctt agG GTG Phenotypic Effects of Mutations in Genes Encoding Functional RNAs rRNA genes Complicated because rRNA genes present in hundreds or thousands of copies tandem repeats mutation affects only one in which case has little or no effect but can spread to all copies after many generations The mechanism of spreading is unequal crossing over and gene conversion which we will discuss later in the course If it spreads can have no effect nonfunctional or subfunctional RNAs and ribosomes no or reduced synthesis of all proteins encoded same genome Often lethal or very detrimental ni IW i us 55 255 ms 525 ms ms 555 255 tES 555 255 ssvaA 525mm mumx Phenotypic Effects of Mutations in Genes Encoding Functional RNAs tRNA genes Mutations in the anticodon or in the region that is recognized by the aminoacyl tRNA synthetase can result in wrong amino acids being inserted at many sites in many proteins Mutations and Gene Interactions mRNA protein i quoti Gene A I Gene B Control of gene expression involves interaction with the products of other genes More detail in next section of lectures 0 Sometimes Whether a gene is transcribed or not depends on Whether or not a protein produce of another gene binds to an upstream controlling region 0 Mutations in controlling gene can modify presence absence or abundance of product of controlled gene 0 There are many other ways in Which products of different genes interact 0 Consequence change in expression of a gene may be due to mutation in gene itself or in its control sequences or in other genes that interact With it 0 What appears to be a mutation in gene B could be a mutation in gene A if A encodes a protein that is required for transcription of B Dominance An important aspect of genetics is being able to relate the genotype with respect to a particular gene to the phenotype Dominance allele A1 is dominant to allele A2 if the heterozygote A1A2 has the same phenotype as the homozygote A1A1 Dominance of a mutant allele depends on the effects of the mutation at the molecular level eg a null alllele Will be recessive to the Wild type in a gene that codes for a product that is detected fairly directly You are invited to try to predict the phenotypes of heterozygotes for various kinds of mutations Mutation Summary Mutations can result from 0 errors in DNA replication or repair 0 damage by chemical or radiation mutagens 0 movement of transposable elements The effects of mutations depend on 0 the kind of gene in which the occur 0 the function or lack thereof of the site Within the gene 0 the nature of the mutation 0 interactions of the mutant gene or sequence with other genes
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