Genetics: From genes to genomes - Chapter 9: Analysis of genetic information

*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or displayPowerPoint to accompanyGenetics: From Genes to GenomesFourth EditionLeland H. Hartwell, Leroy Hood, Michael L. Goldberg, Ann E. Reynolds, and Lee M. SilverPrepared by Mary A. BedellUniversity of GeorgiaCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*CHAPTERAnalysis of Genetic InformationCHAPTERPART IIIDigital Analysis of DNACHAPTER OUTLINE9.1 Sequence-Specific DNA Fragmentation 9.2 Cloning Fragments of DNA 9.3 Hybridization 9.4 The Polymerase Chain Reaction9.5 DNA Sequence Analysis9.6 Bioinformatics: Information Technology and Genomes9.7 The Hemoglobin Genes: A Comprehensive ExampleRestriction enzymes fragment the genome at specific sitesEach restriction enzyme recognizes a specific sequence of bases anywhere within the genomeCuts sugar-phosphate backbones of both strandsRestriction fragments are generated by digestion of DNA with restriction enzymesHundreds of restriction enzymes now availableRecognition sites for restriction enzymes are usually 4 – 8 bp of double-strand DNA (see Table 9.1)Often palindromic – base sequences of each strand are identical when read 5'-to-3'Each enzyme cuts at same place relative to its specific recognition sequence (Figure 9.2) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Ten commonly used restriction enzymesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Table. 9.1Restriction enzymes produce restriction fragments with either blunt or sticky endsBlunt ends – cuts are straight through both DNA strands at the line of symmetrySticky ends – cuts are displaced equally on either side of line of symmetryEnds have either 5' overhangs or 3' overhangsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.2Different restriction enzymes produce fragments of different lengthAverage fragment length is 4n, where n is the number of bases in the recognition site4-base recognition site occurs every 44 bp, average restriction fragment size is 256 bp3 billion bp genome/256 = 12 million fragments6-base recognition site occurs every 46 bp, average restriction fragment size is 4100 bp (4.1 kb)3 billion bp genome/4100 = 700,000 fragments8-base recognition site occurs every 48 bp, average restriction fragment size is 65,500 bp (65.5 kb)3 billion bp genome/65,500 = 46,000 fragmentsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Sites for three restriction enzymes in a 200 kb region of human chromosome 11Names and location of genes in this region are shown below the restriction sitesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.3bGel electrophoresis distinguishes DNA fragments according to sizePreparing an agarose gel for electrophoresisCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.4aGel electrophoresis distinguishes DNA fragments according to size (cont)Load DNA samples into wells in gel, place gel in buffered aqueous solution, and apply electric current Electrophoresis (movement of charged particles in an electric field)DNA has negative charge, so moves toward positive chargeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.4aWith linear DNA fragments, migration distance through gel depends on sizeAfter electrophoresis, visualize DNA fragments by staining gel with fluorescent dye, and photograph gel under uv lightCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Gel electrophoresis distinguishes DNA fragments according to size (cont)Fig. 9.4aDetermine size of unknown fragments by comparison to migration of DNA markers of known sizeDifferent types of gels separate different-sized DNA moleculesPolyacrylamide gels (left) separate small fragmentsAgarose gels (right) separate larger fragments Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.4bRestriction maps provide sequence-specific landmarks in the DNA terrainRestriction maps show the relative orders and distances between multiple restriction sitesConstruction of restriction mapDigest DNA sample with different restriction enzymes, single digests vs double digestsRun gel and determine fragment sizes for each digestDeduce restriction arrangement of sites by process of elimination (see Fig 9.5)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Deducing a restriction mapCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.5(b) Load each digest into gel along with size markers(a) Do single and double digests with two restriction enzymes(c) Use process of elimination to derive the only possible arrangement that accounts for all the observed fragmentsCloning fragments of DNAGenomes of animals, plants, and microorganisms are too large to analyze using simple techniques such as gel electrophoresis and restriction mappingCloning is a means to purify a specific DNA fragment away from all other fragments, and make many identical copies of the fragmentThe cloned fragment can then be analyzed by restriction mapping and DNA sequencingCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Two strategies to purify and amplify individual fragments of DNAMolecular cloningPurification and amplification of previously uncharacterized DNACut DNA and insert fragments of specific sizes into vectorsTransport vector-insert molecules into living cells that make many copies of the recombinant vectorClones have amplified sets of purified DNA moleculesPolymerase chain reactionPurification and amplification of previously sequenced genomic regionsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Molecular cloning step 1: Splicing inserts to vectors produce recombinant DNACopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.6aIn this example, EcoRI fragments of human DNA are cloned into the single EcoRI site in a plasmid vectorLigase is used to seal the phosphodiester backbones at the ends of vector and insert fragmentComplementary sticky ends increase the efficiency of ligation between vector and insert DNAChoice of cloning vectors Simplest vectors are plasmids that replicate in bacteriaMultiple restriction sites for cloning insert DNAOrigin of replication so that it replicates independently of bacterial chromosomeSelectable marker (e.g. gene for ampicillin resistance)Easily purified from chromosomal DNALargest capacity cloning vectors are artificial chromosomesBacterial artificial chromosomes (BACs) can carry inserts of up to 300 kb Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Molecular cloning step 2: Host cells take up and amplify recombinant DNACopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.7b, cTransformation – the process by which a cell or organism takes up foreign DNAIn E. coli, only 0.1% of cells will be transformed with plasmidSelection (e.g. antibiotic resistance) for cells that take up plasmid is neededOnly the cells with plasmid will grow on on media with ampicillinEach cell multiplies to produce a cellular clone containing millions of genetically identical cellsA plasmid that allows a screen for insert-containing DNAPlasmid carries lacZ gene, which encodes b-galactosidase (b-gal)Insertional inactivation - restriction site for cloning insert DNA is located in the middle of lacZCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.7aScreen for b-gal activity:Plasmid that doesn't carry insert will have intact lacZPlasmid that carries insert will have disrupted lacZMolecular cloning step 2: Host cells take up and amplify recombinant DNA (cont)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.7dUsing a screen to distinguish cells carrying recombinant molecules from cells carrying non-recombinant moleculesX-gal is substrate for b-gal and is converted to blue pigmentIntact lacZ  blue colonyDisrupted lacZ  white colony Libraries are collections of cloned fragmentsGenomic libraryLong-lived collection of cellular clones that contains copies of every sequence in the whole genome inserted into a suitable vectorcDNA libraryLong-lived collection of cellular clones that contains copies of every mRNA expressed in a particular tissue or condition inserted into a suitable vectorSeries of in vitro reactions used to make cDNA copies of mRNA Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Genomic librariesComplete genomic libraryCollection of clones that contain one copy of every sequence in the entire genomeGenomic equivalent – number of clones in a perfect libraryTo determine number of clones needed, divide the length of the genome by the average size of insert fragments Impossible to obtain a perfect libraryUsually libraries are made that have four to five genomic equivalentsGives an average of four or five clones for each locus (95% probability that each locus is present at least onceCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Converting RNA transcripts to cDNA: Obtaining mRNA from red blood cell precursorsEukaryotic mRNAs have poly A tails at 3’ endmRNAs purified by affinity to oligo(dT) – single strand DNA fragments of 20 nucleotides made of dT only Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.8aConverting RNA transcripts to cDNA (cont): Synthesis of hybrid cDNA-mRNA molecule In vitro synthesis using reverse transcriptase (a DNA-dependent RNA polymerase) + dATP + dGTP + dTTP + cCTPPrime DNA synthesis using oligo(dT) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.8bCreating the second DNA strand complementary to the first cDNA strand mRNA digested with RNAse3’ end of cDNA folds back and acts as a primer for 2nd strand synthesisIn the presence of dNTPs and DNA polymerase, the first cDNA strand acts as a template for synthesis of the second cDNA strandDouble-stranded cDNA can be cloned into a plasmidCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.8c, dA comparison of genomic and cDNA librariesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.9Clones from a genomic library with 20 kb inserts that are homologous to this regionRandom 100 kb genomic fragmentClones from cDNA librariesHybridization is used to identify similar DNA sequencesComplementary single-stranded DNA or RNA will base pair and form stable double helicesHybridization probes can be from cloned fragments of DNA, PCR products, or chemically synthesizedProbes are labeled with radioactive or fluorescent tagComplementary region must be sufficiently long and accurate to produce a large enough number of H bondsCohesive force formed by large numbers of H bonds counteracts thermal forces that disrupt the double helixHybridization can be DNA/DNA, DNA/RNA, or RNA/RNACopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*How to make oligonucleotide probes for screening a libraryAutomated DNA synthesizer is used to synthesize specified oligonucleotides of defined length and sequenceCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.10bReverse translation – generating a degenerate DNA sequence that contains all possible codons for a specific amino acid sequenceSouthern blots allow visualization of rare DNA fragments in complex samplesCut genomic DNA with restriction enzyme (s) and separate DNA fragments by electrophoresis on agarose gelCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.11DNA is transferred from gel to nitrocellulose membrane by blottingDNA fragments on the membrane (blot) are in the same migration pattern as in the gelCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.11Southern blots allow visualization of rare DNA fragments in complex samples (cont)After electrophoresis, gel is treated with NaOH to denature the transferred DNA and the blot is treated with uv and high temperature to attach single-stranded DNACopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.11Southern blots allow visualization of rare DNA fragments in complex samples (cont)After hybridization of probe to the blot, autoradiography reveals fragments in restriction digests that have sequences complementary to the probe Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.11Southern blots allow visualization of rare DNA fragments in complex samples (cont)PCR generates copies of target DNAPolymerase chain reaction (PCR) first developed in 1985Faster, less expensive, and more flexible way to amplify specific fragments of DNA than molecular cloningExtremely efficient – can amplify DNA from a single cell or from some archaeological samplesOligonucleotides are designed from previously known DNA sequence and serve as primers for DNA synthesisTarget sequence located between primer sequences are exponentially amplified by 25-30 cycles of DNA synthesis Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Two oligonucleotide primers (16 – 26 nt) are needed for PCR reactionsRegion between the two primers will be synthesizedOne primer is complementary to one strand of DNA at one end of the target regionThe other primer is complementary to the other strand of DNA at the other end of the target regionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.12PCR consists of repeated cycles of DNA synthesis, with three steps in each cycleCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.12The three steps in each cycle of PCRCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.12(1) Denature strands(2) Base pairing of primers(3) Polymerization from primers along templatesExponential increase in the amount of target DNA during PCR Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.12Some of the uses of PCRPCR fragments can be labeled to produce hybridization probes and can be sequenced (next section of this chapter)Genotype detection and gene mapping (Chapters 10 and 11)Determine evolutionary relationships of living and extinct species (molecular evolution, Chapter 22)Study genetic variation and changes in nucleotide sequence in groups of individuals over time (population genetics, Chapter 19)Detection of infectious diseases (e.g. HIV) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*DNA sequence analysisTwo methods originally developedMaxam-Gilbert methodChemical cleavage of DNA at specific nucleotidesSanger methodEnzymatic extension of DNA strands to a defined terminating baseBoth methods can determine sequence of 500-700 bp per reaction and have 99.9% accuracySanger method is much more amenable to automationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Sanger sequencing generates sets of nested fragments separated by sizeTwo steps to the Sanger method:1. From a portion of a template DNA, generate a complete series of complementary single-stranded subfragmentsEach subfragment differs in length by a single nucleotide from preceeding and succeeding fragments (nested array)Each subfragment is defined by its terminal nucleotide2. Polyacrylamide gel electrophoresisSeparates DNA molecules that differ in length by one nucleotideCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Sanger sequencingTemplate DNA is denatured and mixed with radio-labeled oligonucleotide primer, dNTPs, and DNA polymeraseCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.13Split sample into four aliquots, each aliquot receives a different dideoxyribonucleotide (ddNTP)During DNA synthesis, ddNTPs are incorporated into DNA like dNTPs, but lack 3’OH group so cannot be extendedEach ddTTP reaction produces a series of different-sized fragments that terminate with insertion of T opposite an A on the template strandCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.13Fragments produced by a ddTTP reaction in the Sanger sequencing methodPolyacrylamide gel electrophoresis to separate fragments generated by Sanger sequencingThe appearance of a DNA fragment of particular length demonstrates the presence of the particular ddNTP5’-to-3’ sequence of synthesized strand is read from the bottom of the gelSequence of template strand is complementary to the synthesized strandCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.13Automated DNA sequencingEach ddNTP is labeled with a different color fluorescent dye and all four are used in a single synthesis reactionAll four ddNTP reactions are run together in a single lane on a gelAfter electrophoresis, fragments flow through a fluorescence detector and the color of the fragment is digitally recordedCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.14aFluorescent bands in an automated sequencing gelEach lane displays the sequence obtained from a separate DNA sample and primerEach fragment has terminated with a specific ddNTP labeled with a specific fluorescenceCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.14bChromatogram and inferred DNA sequence from automated Sanger sequencingComputer reads of sequence complementary to the template strandSequence is read from left to right (5'-to-3' synthesis from primer)Ambiguity in sequence is recorded as "N"Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.14cAccumulation of genome sequence dataParallel revolutions in acquisition of genome sequence and information technologyGenBank – first official open-access, online repository for DNA sequences (1982, National Institutes of Health) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.15aUltrahigh-throughput DNA sequencing2008 - New generation of nanotechnology-based DNA sequencers100 billion base pairs of sequence can be determine in a single experimentMillions of DNA clones can now be sequenced simultaneouslyCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.15bBioinformatics provides tools for visualizing functional features of genomesBioinformatics is the science of using computational tools to decipher biological information1988 – National Center for Biotechnology Information (NCBI) establishedOversees GenBankCreated additional public databases of biological informationDeveloped bioinformatic tools for analyzing, systemizing, and disseminating the dataRefSeq – species reference genome sequence, a single, complete, annotated version of the species genomeIs not from one individual, but is a composite from several individualsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Visualizing genes of the human RefSeq genome with the UCSC Genome BrowserCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.16aA 3 Mb region of human chromosome 7From human RefSeq on NCBI Sequence ViewerBetween sequence positions 116,000,001 and 119,000,000Shows locations of nine genes, including the CFTR gene Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.16bA gene desertVisualization of a 540 kb region of human chromosome 7 containing the CFTR geneFrom human RefSeq on NCBI Sequence ViewerFor each gene, Exon/intron structure; blue boxes and connected linesSpliced RNA products; red boxesProtein coding sequences; black boxes Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.16cWhole-genome comparisons distinguish genomic elements conserved by natural selectionCharles Darwin proposed "descent with modification"Genome sequencing of many species has shown that the DNA sequence undergoes descent with modificationTwo perfectly matched 50 bp DNA sequences found in different species are almost certainly derived from an ancestral speciesProbability of occurrence = (0.25)50 = 8 x 10-31DNA sequence conservationHomologous sequences in two species that show evidence of being derived from a common ancestorCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.17aSpecies relatedness and genome conservation between H. sapiens and other vertebratesBranch points represent a series of nested common ancestorsNumber at each branchpoint is millions of years before the presentProportions of human protein-coding sequences found in each vertebrate genomeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.17bHomology map for a 100 kb region of the human genomeDNA sequence conservation between species can be visualized directly on the genome sequenceDark lines represent conservation of sequence homology in each of five species compared to human RefSeq genomeNote that the coding regions (exons) of the genes are most highly conserved Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.18The hemoglobin genes: A comprehensive exampleHemoglobin disorders are the most common genetic diseases in the worldHemoglobin consists of four polypeptides that switch to adapt its oxygen carrying capacity to different stages of development (see Fig. 9.1c) Adult form consists of two a and two b polypeptidesEmbryonic form consists of two a-like ξ chains and two b-like e chainsFetal form consists of two α chains and two b-like g chainsGenes for a- and b-globins are clustered and are transcribed in order at different stages of developmentCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*The genes for human hemoglobin are located in clusters on two different chromosomesThe a-globin (HBA) locus – 28 kb on chromosome 16 contains five functional genes (HBZ, HBM, HBA2, HBA1, HBQ1Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*The b-globin (HBB) locus - 45 kb on chromosome 11 contains five functional genes (HBE, HBG2, HBG1, HBD, HBB)Fig. 9.19a, bCorrelation of globin gene order with timing of expressionLinear organization of a- and b-globin genes reflects the order that they're expressed during developmenta-like chains – HBZ is expressed during first 5 weeks in embryogenesis, HBA2 and HBA1 is expressed during fetal and adult lifeb-like chains – HBE is expressed during first 5 weeks in embryogenesis, HBG2 and HBG1 are expressed during fetal life, and HBB is expressed with a few months of birthLocus control region (LCR) – DNA sequence located at 5' end of each locusSpecialized DNA binding proteins bind to LCR and control transcription of each gene in the locus (discussed more in Chapter 18) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Globin-related diseases result from a variety of mutationsTwo general classes of human disorders are caused by mutations in hemoglobin genesHemolytic anemias (e.g. sickle-cell anemia)Mutations affect amino acid sequence and the three dimensional structure of a- or b-globin chainResults in destruction of red blood cellsThallasemiasMutations result in reduction or elimination of one of the two globin polypeptidesDeletion of entire HBA or HBB gene; mutations in noncoding, regulatory sequence; nonsense mutationsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Mutations in the DNA for hemoglobin produce two classes of diseaseMajor types of structural variants causing hemolytic anemias Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.20a.1Basis of sickle-cell anemiaCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.20a.3Fig. 9.20a.2Mutations in the DNA for hemoglobin produce two classes of disease (cont)Clinical results of various a-thallasemia genotypesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.20b.1A b-thallasemia patient makes only a globin, not b globinCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.20b.2Fig. 9.20b.3Regulatory regions affecting globin gene expressionMutations in the TATA box associated with HBB gene eliminate transcription and cause b-thallasemiaCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.21aMutations in the the LCR can prevent expression of all HBA genes, resulting in severe a-thallasemiaFig. 9.21bEvolution of the globin gene familyCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 9*Fig. 9.22The a- and b-globin lineages were created by duplication of an ancestral gene followed by divergenceWithin the separate lineages, further rounds of duplication and divergence occurred