GENETIC ANALYSIS Genetic Analysis: Principles, Scope and Objectives John R. S. Fincham © 1994 Blackwell Science Ltd. ISBN: 978-0-632-03659-2 GENETIC ......................................... ANALYSIS Principles, Scope and Objectives JOHN R. S. FINCHAM PhD, SeD, FRS. 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Nopart ofthis publication may be reproduced, sloTed in a retrieval system,ortransmitted, in Acataloguerecord for thistitle any form or byany means,electronic,mechanical, isavailable from theBritish Ubrary photocopying.. r~ordingorotherwise,exceptaspermitted bythe UK Copyright, Designsand PatentsAct 1988, ISBN0-632-0365~-1 without thepriorpermission ofthecopyrightowner. LibraryofCongress firsT published 1994 Cataloging-in-Publication Dat'3 Setby ExcdTypesetters Co., Hong Kong Fincham,j.R.S. Prillled and bound in Great Britain Geneticanalysis/John R.S. Fincham. atthe Alden Press,Oxfotd and Northamplon p. em. Includesbibliographical references and index. ISBN0-632-0365~-1 1. Genetics-Technique. 2. Nucleotidesequence_ 3. Genomemapping. I. Title. [DNLM: I. Genetics. 2. Genome. 3. SequenceAnalysis. DNA. QH 430 F492ga 1~~4] QH44LF56 1~~3 574.87'322-dc20 Preface, viii Introduction: the expanding scope of genetics, ix CONTENTS , Dissecting the genome using natural genetic systems. , The eukaryoricsystem, 1 Eukaryoresdistinguished from probryO[es. Mitosisand chromosomestructure,4 Alternarion ofhaploid and diploid phases in the sexual cycle, 7 Meiosisand the rules ofclassicalgenetics, 9 Meiosis, 9 2 From mutations to genes, 47 Single-facrotgenetic ratios and their Definingthegene by mutation and explanation, 12 complementation,47 Independent assortmentofdifferentallele- What is it mat the markers mark? 47 pairs, 14 The collection ofmutants,47 Tn:ringdara for fit to theoretical raetas, 17 Sortingofmutants intocomplementation Lmkagegroupsand the natureofcrossovers, 18 groups, 48 Map unirs and map distanlX, 24 An interim definicion ofthegene,55 Placing linkedgenes in sequence. 24 Mapping within thegene, 55 Distinguishing between independentassortment Thedetection ofrecombination within genes,55 and distantlinkage, 25 Mapping by reference flanking markers, 58 (0 Mappingcemromeres usingtetrads, 25 Recombination frequency, 58 Sex linkage, 27 Deletion mapping, 58 Segregation and linkage in human genetics, 28 Thedeterminationbygenesofproteinstructure,60 Assigninglinkage groups to chromosomes, 32 Colinearity ofthegeneand itsencoded protein Genetic analysisofbacteria and bacteriophage, 34 product, 60 Three modesofgene rranster in bacteria, 34 Biochemistryofpolypeptidesynthesis, 61 Time-of-entry mappingin E. coli, 37 NotaU genesencode proteins, 64 Analogy with sexual recombination and Conclusions, 64 segregation, 38 References, 66 Stable partial diploids- F' plasmids, 40 Bacteriophage lambda- a virus in the 3 The gene as DNA sequence. 67 chromosome, 41 Characterization ofDNA fragments, 67 BacteriophageT4 - a model recombinational Restriction endonudeases, 67 system, 42 Separatingand sizing DNA fragments, 67 Eukaryoticorganellegenetics. 42 Probing for specific sequences, 68 Exceptions to the rule ofequal results from Making resrrictioll"site maps, 72 reciprocal crosses, 42 Cloningand cloningvectors, 72 Chloroplast variants, 42 Making recombinant DNA molecules, 72 Mitochondrial variants, 43 F.scherichia coliplasmid vtttors, 72 Summary and perspective,44 Yeast 2"I-.Iln plasmid and shuttle vectors, 76 Further reading, 44 Lambda (1.) bacteriophage and cosmid References,44 vectors, 76 Yeast artificial chromosomes (YACs), 79 Screening DNA Hbraries for functional genes, 82 Genomic libraries and cDNA libraries. 82 v VI Contents Gene identification by complementation of Locus~specifictrans-actingcontrol- mutants. 84 transvection, 125 Screeninggene libraries with DNA probes. 85 Restructured genes and edited messages, 126 Designing gene-specificprobes, 86 Conclusion - whatis thegene? 127 Use ofcDNA probes. 87 References. 127 Screening for geneexpression. 87 Probingacrossspeciesor group boundaries. 88 5 Analysis of the whole genome, 129 PositionaJ doning. 88 Introduction. 129 Confirming the identiryofcloned sequences. 88 Total DNA- quantitities and patterns of DNA sequence andopen reading frames, 88 complexiry, 129 Using theclone to disruptthecorresponding C~values,129 gene, 88 Degreesof repetition amonggenomic Hybridization back to chromosomes, 90 sequences, 129 Simultaneous mutagenesis and gene tagging, 91 The narureofrepetitive DNA, 132 The principle, 91 Satellite DNA, 132 Transposon tags. 91 Functional genes in repetitive arrays, 132 Conclusion, 92 Dispersed repeats, 133 References, 92 Chromosomecharacterization by microscopy, 138 Features visible in unspecifically stained 4 The evolving concept of the gene. 95 chromosomes, 138 Thegeneas acis-acting unit, 95 Chromosome banding, 140 Thegene as a unitoftranslation, 95 Chromosome sorting- chromosome-specific Singlegenes- single enzymic functions, 95 probes and libraries, 142 Singlegenes- multipleenzymic functions, 98 Pulsed-field gel electrophoresis (PFGE1, 142 Singlegenes- polyproteins. 100 Fluorescence-activated chromosomesorting Single transcripts- multiple proteins - polar (FACS), 142 effectSon translation, 101 Useofhuman-rodent hybrid cells, 143 Thegene as a unitoftranscription, 102 Physical dissection ofthe chromosomes, 144 GenomicDNA compared tocDNA- the Gettingchromosome-specificDNAmarkers- the detection ofintrons, 102 use ofPCR, 144 Comparison ofgenomic and cDNA restriction Comprehensive genetic mappingwith molecular site maps, 103 markers. 147 Introitsdefined by sequenceanalysis, 103 Molecular markers presenrin populations Definingtheend-points oftranscription. 106 RFLPs, 147 Processingofthe primary transcript. 106 Standard genorypes for geneticmapping. 149 RNApolymerases- promotersandprocessingof Physical mapping, 151 transcripts. 106 From physical map to complete DNA Different products from thesame primary sequence. 153 transcript, 110 Differenrgene densities in different Genes nested within othergenes' introns, 113 organisms, 153 Retroviral genome expression - unitsofall DNAmethylation- undermcthylated<jslands' as kinds! 115 markers for activegenes, 156 Thecomplex functions ofpromoters, 116 How manygenes? 156 The E. coliparadigm, 116 DNA oftheorganelles, 157 Analysis ofeukaryotic promoter regions by Mitochondrial genomes- total analysis. 157 mutation. 117 The mitochondrion as an aliensystem, 159 Enhancers- theexpandingfrontiersofthegene,122 Chloroplasr DNA, 159 The ADH genesof Drosophila. 122 Conclusion: total analysisofgenomes- what Howdogenesinteractwiththeirenhancers?The benefits to understanding? 160 caseofthe B-globin locuscontrol region. 123 References, 160 Contents vii 6 Accounting for heritable variation, Epistasisand its imc:rprelation, 184 163 &lllllpks from the: YC3SlS. 185 IlltrU<.hx:tion, 163 Control oftnc cdl cy,k- an exampleofa B.ll;kground, 16] protein cascade, 185 Kindsofgttl~icvariarion, 163 Galactose mnabotism in buddingyeast Molecular.dentinc:uion ofmutlilionsofStrong ulilllscriprional regulation and protein effea, 164 protein interaction, 186 Usingthe mutation to lind the gene, 164 General control ofaminoacid biosynthesis Ways ofscreening known genes for ;l prutein kinasecascade that regulale~ sequence: variation, 166 rranslMion, 189 Thediversity ofmutant alleles in Another levelofcontrol - RNA splicing in populations, 168 Drosophila sexual development, 192 Screening for known mutations, 169 The emergenceofrnorphologkal pattern, 194 DNA sequence variation wilhom phenorypic The maternal contribution - analysis in corncquences, 169 Drosophila, 194 The scope for neutral muration in prOlein Thetstablishmemofcompanmc:nts in encodingge:nes, 169 Drosophila developmenl, 196 Sequence variation outsidecoding Selc:aorgenes in Drosophilaand other sc:qurnces:, 172 organisms, 201 QuantilOlti,'c variation and ils hc:rilOlbiliry, 172 Drosophila,20I Mcans and variances, 172 Mouse, 203 Heritability ofquantitnive variation, 173 Flowetingplants, 204 Heritability assessed by correlations Timingofgeneexpression - a function for between relatives, 173 introns? 205 Estimatesofheritability in humans, 175 Stabilizinggene acrivity - epigenetice:ffe,ts, 207 How many genes? 176 Transcriptional aCliviry/inacliviry - mechani~ms What kmds01 8"'nt:S are invoh"cd in ofmainl~ancc,207 cominuoos varialion? In X-chromosome inactiv,uion in mammals, 209 Attributingherilable quantitative "ariation to Dosage:compensation in Drosophila,209 chromosomal loci, 178 Drosophilaposition-effect variegation and the Quantitative trait loci distinguishinginbred maintemlllQofstates ofgeneactiviryduring Slrain~ 178 development, 210 Looking for QTLs in oolbred populations, 180 Imprintingofmammalianchromosomes, 213 Conclusions and perspectives, 181 The natureand significance:ofchromatin References, 182 $tcuclUre,214 Towardsthe compleredescription ofthe 7 Gene interactions and the genetic organism, 215 programme. 183 Further reading, 2I6 lmroduction, 183 References, 216 duesto gene function, 183 Index. 219 Molecular m«hanismsofgene intet:lClion, 184 Geneticsincreasinglydominatesbiologicalscience. What began as a tather esoteric field, concerned with the mode of inheritance of minor variations and quirks, has become the PREFACE main road to understanding how living organisms function. The great expansion of the scope of genetics is due to the reinforcement of classical genetic methodology with the newer molecular analysis. The purpose of this book is to explain how moderngeneticsisactuallydone,toreviewwhatit tells us about the structure and functions of the genetic material, and to consider the extent to which all this newknowledgehassolved, or looks portingthesespeci6cexamples,though I havealso like solving, the classical biological problems of listed some more general SOurces of information. variation and development. I have tried to show This is not intended as a comprehensive gene both the great power of genetic analysis and its tics text-book. The emphasis is on 'mainline' present incompleteness - the enormous com eukaryotic organisms, with bacteria and viruses plexity of living systems will not yield to toral treated rather summarily, mainly in connection analysis in the forseeable future. with their relevance to genetic manipulation. I Inotderto keepthebooktoa teasooablelength have also concentrated on the problems that can it has been necessary to be very selective in the be attacked through controlled investigations in choice of material. I have thought it best to con laboratories, and have left aside questions of sidera limited numberofexamples in somedetail popuJational genetk change and evolution. These so as to show the kinds ofanalysis that are poss latter areas require different kinds ofanalysis and ible, even at the expense of marginalizing other even diffetent styles ofthought. importanttopics. Agooddealofthemoredetailed J.R.S.F. information is presented in the figures and their March 1994 legends. The bibliogtaphyis mainly aimed atsup- viii Genetics can be said to have started with the rediscovery in 1900 of Mendel's rules, published in 1866, for the transmission of clearly defined distinguishing traits from one generation to the INTRODUCTION: next. Originally formulared for peas, they were soon found to hold true for plants and animals THE EXPANDING SCOPE generally. Clear-cut inherited differences could be attributed tohereditary units thatsooncameto be OF GENETICS called genes. Initially, me genes were justsymhols in a set of algebraic formulae, set up to describe the patterns of transmission of the differences. But, mainly as the result of the work of T.H. Morgan's school of fruit fly (Drosophila melano gaster) geneticists, the genes acquired a physical menrs. It became apparent that no feature of the location. Each gene could be shown to reside at a organism was immune to the effeers of mutation. specific posirion (locus) on one of the chromo All parts of the living sysrem appeared to be somes ofthe cell nucleus. dependenton the integrityofwhatever itwas that This phase of genetic analysis is described in resided at the chromosome loci. Chapter 1. It was based entirely on the narural Nevertheless, it remained true for a long time breeding systems of the organisms concerned that genes were detectable only in so far as they notonly me sexual cycleofplant and animals but mutated. As late as the 1950s, at least one distin also the very different modes of gene transfer guished geneticist, Richard Goldschmidr, argued found in bacteria. These natural systems have thatthe'gene' wascreated by the mutation- that tended to beovershadowed in recent years by the the <mutant gene' was just a scar on the chromo 'generic engineering' made possible by molecular some which, in its unscarred state, was an inte technology, but mey still provide the geneticist gratedwhole,notdividedintofunctionallydistinct with an essential set of analytical tools. More components. over,theyarewhatgoeson all the time in thereal What, more than anythingelse, gave solidity to world outside the laboratory, which in itself is thegeneconcept,wasthedetailedstudy,especially a more than sufficient reason for knowing about in Drosophila, ofrecurrent mutations at thesame memo chromosome locus. The effeers of such allelic Until meadventofmolecularbiology, thegenes mutations tended to be variations on a common remained intangible. The chromosome locus was theme. By the criterion of non-complementation merelythesiteofthedeterminantofa difference (discussed in Chapter 2) they seemed to represent usually a difference between the normal form of different degrees ofdefect in the same function of the organism and an aberrantvariant, or mutant. theorganism. Whatever it was that resided at the Forseveral decadesafterthe formal establishment chromosomelocus. itclearly had a high degreeof of the chromosome theory of Mendelian inherit functional specificity. ance,thereremainedconsiderablescepticismabout As genetic mapping was pursued to a higher its general importance. The apparently trivial or level ofresolution, first in Drosophila and then in freakish character ofmany ofthe inherited differ even more detail in micro-organisms, it was dis ences used in classicaIgenetics encouraged some covered that the functional units, or genes. were to argue mat, even if genes existed, mey were not indivisible, as had previously been assumed, responsible only for superficial quirks superim but consisted of linear arrays of individually posed on an essentially invariant species-specific mutablesites.Andastheeffectsofgenemutatinns substructure. This view became increasingly im were analysed to the biochemical level, it became plausibleasthenumberand rangeofknownMen clear in certain casesthat mutations within a gene delian variants was increased, especially through causedchangesinthesequenceofaminoaddsina the use of radiation and other mutagenic treat- protein, and that the amino acid sequence of the ix x J"troduction protein was encoded in the linear structure ofthe objectives,andtheextentstowhich theyarelikely gene. to be realized. The final three chapters of this This phase of genetic analysis, culminating in bookaddresswhat maybeconsidered asthe three the concept ofthe gene as a repository oflinearly grand objectives ofgenetics. encoded information for thesynthesisofaspecific Thefirst, deahwith i~ Chapter5, isthegenome macromolecule, will be described in Chapter 2. It proiect, ar presentarrraetingagreardeal ofatten proceeded againS! the background of the proof rion as applied to humans, but being pursued in (by transformation experiments) of the generic several other species as well. The project is to function ofDNA and concurrently with theeluci make a complete molecular map and ultimately dation of the biochemical mechanisms whereby obtain complete DNA sequences of all the chro DNA is transcribed into RNA, which is then mosomes, at least for one representative individ translated intO protein strueture~ ual.Formorecomplexorganismsitisaformidable Until the 19705, however, it stiLi remained task, hut feasible gi,-en sufficient resources. Once true that genes were recognized only through the a complete DNA sequence has been obtained, it effectSoftheirmutations.Thegeneas amolecular should be possible to recognize the genes, or po structure in itself remained elusive. The great tential genes, by computer-based scanning. Find change came with the development of new mol ing out what all the genes do will be much more ecular techniques, first for detection (probing) of difficult. Itwillbepossibletodeducemuch,though specific DNA sequences and then for amplifying not everything, about me structures of the pro and purifying genes or gene fragments as mol teinsthatthey encode, buttheonlywayoffinding ecular clones. These revolutionary developments outwhat a given protein does for theorganism is are described in Chapter 3. They gave physico· to see what difference it makes when it is lost or chemical reality to the genes and showed them modified, either by random mutation or by DNA to be !lacts of DNA. Genes at the DNA level engineering. This brings us back tothe analysis of conform ro theexpectationsofclassicalgeneticsin genetic variants with which genetics started, but rhatthey residewithinspecificand relativelyshort wotkingfrom thegenetotheeffeerratherthanthe chromosome segments hut, as we shall see in other way round. This approach is feasible with Chapter 4, they turn our to display a complexity yeast, far more difficult with the mouse, and and variety of structure and function that could possible with humans only at one remove - by not previously have been imagined. using the mouse as a proxy or model. As a consequence of the molecular revolution, A second grand objective of genetics, which is theagendaofgeneticanalysishasbecomeradically considered in Chapter 6, is to account for natural changed. Formerlyitstarredwithgeneticvariation inherited variation in termsofdefined genediffer and attempted to define and map the genes re ences at the molecular level. A comprehensive sponsible. The challenge was [0 account for in account is clearly out of the question for any herited variation in terms of gene differences. species. It would involve the completion of a Now genes of all kinds can be detected and whole genome project for every individual. There mapped, whatever their functions and whether is, however, some prospectofbeingable to define they mutate or not. Moreover, once genes have some of the gene diiferences that make relatively been isolated as DNA, alterarions of any desired large contributions to populational variation, kind can be made in them to order outside the even if their individual effeers are not completely cell, and the altered genes reintroduced into the clear-cut. Gene differences of small effect - and Hvingsystem for observation oftheconsequences. the effecrs grade aU the way down to zero - are Analysis ofgene function is no longer necessarily generally not wonb pursuing. Nevenheless, if a dependent on random mutation. particular variant form of a gene, whatever the With thepowetful combinationofclassical and magnitudeofitseffect,hasalreadybeendefined in molecular methods of analysis, the scope and oneindividual, ircan berelativelystraightforward ambition of genetics is greatly increased. It be to screen for it in other individuals. It is aU a comespossible[0posequestionsabout maximum question of knowing what to look for. Introduction xi The final ambition ofgenetics,dealt with in the complete description will ever be possible for any final chapter, is to accounr for the entire develop but the simplest organisms. But, as Chapter 7 menr ofthe organism in rerrns ofthe information attempts to show, we do begin to seesomeofthe encoded in the genome. The problem here lies in general principles of inreraction that, with in thesheercomplexityoftheoperation. Itwould be numerablesubtle variations, may account in prin quite unrealistic to think in terms of a number, ciple for the development of living systems. The however large, of parallel and separate connec most fruitful approach is again through the study tions between genes and traus of the developed of heritable variation, bur now tracing cause organism. In reality, thesystem isa network, with effect relationships in both directions - from a very large number of primary elements (the observableinheriteddifferencesbacktothegenes, 10000 or 100000 genes and the proteins that and ('genetics in reverse') from defined changes in they directly encode) forming innumerable cross the genes out to the phenotype. connections and loops. We cannot be sure that a