bio in for matics

7/16/2019 Bio in for Matics

1/54

Updated:February2011

TheGeneGatewayWorkbookAcollectionofactivitiesintroducingnewusersto

thewebresourcesthatscientistsaccesstolearn

aboutgeneticdisorders,genes,andproteins.

Using hereditary hemochromatosis as a model,access a variety of websites and databases to

Learnaboutageneticdisorderanditsassociatedgene.

Identifymutationsthatcausethedisorder.

Findthegeneonachromosomemap.

Examinethegenessequenceandstructure.

Accesstheaminoacidsequenceofagenesproteinproduct.

Explorethe3-Dstructureofthegenesproteinproduct.

To view the chromosomes of the Human Genome

Landmarks poster online, order your free copy of

the poster, or download additional copies of this

workbook, go to the Gene Gateway website:

genomics.energy.gov/genegateway/
http://genomics.energy.gov/genegateway/http://genomics.energy.gov/genegateway/


2/54

U.S.DepartmentofEnergyOfficeofBiologicalandEnvironmentalResearch

TheGeneGatewayWorkbook genomics.energy.gov/genegateway/ Updated:Feb2011

2

Acknowledgements

ThisworkbookwaslastupdatedFebruary2011.Withmuchappreciation,wewouldliketo

acknowledgeCarrieMorjan,AuroraUniversity,whofacilitatedthecreationofthislatestversion

bysharingherupdatestoTheGeneGatewayWorkbookforuseinhergeneticsclasses.

ThisworkbookwasfirstproducedbytheBiologicalandEnvironmentalResearchInformation

SystematOakRidgeNationalLaboratory,OakRidge,Tennessee,July2003,withsupportfrom

theU.S.DepartmentofEnergyOfficeofBiologicalandEnvironmentalResearch.

OfficeofBiologicalandEnvironmentalResearch

OfficeofScience

U.S.DepartmentofEnergy(DOE)

ForMoreInformation

ThisworkbookisfreelydownloadablefromtheGeneGatewaywebsite(seelinkbelow).For

questionsorcommentsconcerningthisdocument,contactJenniferBownasbyemailat

[email protected].

GeneGateway

genomics.energy.gov/genegateway/

HumanGenomeProjectInformation

www.ornl.gov/hgmis/home.shtml

DOEGenomicScienceProgram

genomicscience.energy.gov

DOEOfficeofBiologicalandEnvironmentalResearch

science.energy.gov/ber/
mailto:[email protected]://genomics.energy.gov/genegateway/http://www.ornl.gov/hgmis/home.shtmlhttp://genomicscience.energy.gov/http://science.energy.gov/ber/http://science.energy.gov/ber/http://genomicscience.energy.gov/http://www.ornl.gov/hgmis/home.shtmlmailto:[email protected]://genomics.energy.gov/genegateway/


3/54



3

TableofContents

Introduction...................................................................................................................4 WhyUseHereditaryHemochromatosisasaModel?..............................................................5SomeBasicConceptstoUnderstandBeforeStarting..............................................................5

Activity1........................................................................................................................6OnlineMendelianInheritanceinMan(OMIM).......................................................................6GeneTests............................................................................................................................11

Activity2......................................................................................................................14NCBIMapViewer.................................................................................................................14

Activity3......................................................................................................................22NCBIEntrezGeneandGenBank...........................................................................................22

Activity4......................................................................................................................32UniProtProteinKnowledgebaseandBLASTSearching.........................................................32

Activity5......................................................................................................................38ProteinDataBank................................................................................................................38

TableofStandardGeneticCodeforTranslatingDNASequenceRecords.......................50

HereditaryHemochromatosisWorksheet....................................................................51


4/54



4

Introduction

TheGeneGatewayWorkbookisacollectionofactivitieswithscreenshotsandstep-by-step

instructionsdesignedtointroducenewuserstogenetic-disorderandbioinformaticsresources

freelyavailableontheWeb.Itshouldtakeabout3hourstocompleteallfiveactivities.

Theworkbookactivitieswerederivedfrommoredetailedguidesandtutorialsavailableatthe

GeneGatewaywebsite( genomics.energy.gov/genegateway/ ).Thiswebsitewascreatedasa

resourceforlearningmoreaboutthegenes,traits,anddisorderslistedontheHumanGenome

Landmarks(HGL)poster,butitcanbeusedtoinvestigateanygeneorgeneticdisorderofinterest.

Manyguidestousingbioinformaticresourcesaredesignedforbioscienceresearchersandaretoo

technicalfornonexperts.ThisworkbookandotherGeneGatewayresourcestargetamore

generalaudience:teachers,highschoolandcollegestudents,patientswithdisordersandtheir

families,andanyoneelsewhowantstolearnmoreabouthowlifeworksatamolecularlevel.

Thisworkbookshowsyouhowtogetstartedusingbioinformaticsresourcesthatoftenintimidate

andoverwhelmnewusers.Italsodemonstrateshowinformationfromoneresource,suchasannotatedproteinsequencedatafromtheUniProtProteinKnowledgebase,canbeusedto

reinforceandclarifyinformationavailablefromanotherresource,suchasthree-dimensional(3-D)

structuresfromProteinDataBank(PDB).GeneGatewayprovidesuserswithasystematic

approachtousingmultiplebioinformaticsdatabasestogainabetterunderstandingofhowgenes

andproteinscancontributetothedevelopmentofaparticulargeneticcondition.

Usingthegeneticdisorderhereditaryhemochromatosisasamodel,thisworkbookshowsyou

howtoaccess:

OnlineMendelianInheritanceinMan(OMIM)andGeneReviewstolearnaboutagenetic

disorder,itsassociatedgeneorgenes,andcommondisease-causingmutations.

NCBIMapViewertofindagenelocusonachromosomemap.

NCBIEntrezGeneandGenBanktoexaminethesequenceandstructureofagene.

UniProtProteinKnowledgebasetofindtheannotatedaminoacidsequenceofagenes

proteinproduct.

ProteinDataBanktoviewandmodifythe3-Dstructureofthegenesproteinproduct.

Skillsgainedbyworkingthroughtheactivitiesinthisworkbookcanbeappliedtolearningabout

othergeneticdisorders,genes,andproteins.

Thisworkbookandothergenomescienceresourcesareavailablefromthewebsiteforthe

genomeprogramsoftheOfficeofBiologicalandEnvironmentalResearch,U.S.DepartmentofEnergyOfficeofScience( genomics.energy.gov/ ).
http://genomics.energy.gov/genegateway/http://genomics.energy.gov/http://genomics.energy.gov/http://genomics.energy.gov/genegateway/


5/54



5

Why Use Hereditary Hemochromatosis as a Model?

Hereditaryhemochromatosis,adisorderinwhichtoomuchironaccumulatesincertain

tissuesandorgans,iscausedbychangesintheDNAsequenceofasinglegene,sothegenetic

basisofthisconditioniseasiertounderstandthanmorecomplexdisorderscausedby

alterationsinmultiplegenes.

Thegeneanditsproteinproductarerelativelywellstudied.Three-dimensionalstructuresof

theproteinproductareavailableinPDB,theinternationalrepositoryformacromolecular

structuredata.

Hereditaryhemochromatosisisthemostcommonautosomalrecessivedisorderaffecting

individualsofNorthernEuropeandescent(about1in200Caucasiansdevelophereditary

hemochromatosis).

Effectivemethodsfortreatmentareavailablewithearlydiagnosis.

Some Basic Concepts to Understand Before Starting

Genesarethebasicphysicalandfunctionalunitsofheredity.Eachgeneislocatedona

particularregionofachromosomeandhasaspecificorderedsequenceofnucleotides(the

buildingblocksofDNA).

Centraldogmaofmolecularbiology:DNARNAProtein

- GeneticinformationisstoredinDNA.

- SegmentsofDNAthatencodeproteinsorotherfunctionalproductsarecalledgenes.

- GenesequencesaretranscribedintomessengerRNAintermediates(mRNA).

- mRNAintermediatesaretranslatedintoproteinsthatperformmostlifefunctions.

Eukaryoticgeneshaveintronsandexons.Exonscontainnucleotidesthataretranslatedinto

aminoacidsofproteins.Exonsareseparatedfromeachotherbyinterveningsegmentsof

DNAcalledintrons.Intronsdonotcodeforprotein,andtheyareremovedwheneukaryotic

mRNAisprocessed.Exonsaresplicedbacktogethertoformtheintron-freemRNAstrandthat

isusedasatemplatetomakeproteins.

Specialcellularcomponents(ribosomes)usethetripletgeneticcodetotranslatethe

nucleotidesofanmRNAsequenceintotheaminoacidsequenceofaprotein.ATableof

StandardGeneticCodeisprovidedonpage50ofthisworkbook.

Thereare20differentaminoacids.Proteinsarecreatedbylinkingaminoacidstogetherina

linearfashiontoformpolypeptidechains.SeetheTableofStandardGeneticCodeonpage50

forsingle-letterandthree-letterabbreviationsforthe20differentaminoacids.

Polypeptidechainsfoldinto3-Dstructuresthatcanassociatewithothermolecularstructures

toperformspecificfunctions.


6/54



6

Activity 1Online Resources: OMIM and GeneTests

Learn about the genetic disorder and its associated gene.

Identify mutations that cause the disorder.

Online Mendelian Inheritance in Man (OMIM)

OMIMisacomprehensivedatabaseofhumangenes,genetictraits,anddisorderscreatedby

researchersatJohnsHopkinsUniversity.TheOMIMdatabase,whichisupdateddaily,isaccessible

throughtheNationalCenterforBiotechnologyInformation(NCBI)suiteofonlineresources.Each

recordinOMIMsummarizesthebodyofresearchrelevanttoaparticulargene,trait,ordisorder.

ToaccessOMIM,letsgototheNCBIhomepage( www.ncbi.nlm.nih.gov)shownbelow,andthen

clickonOMIMintheboxontheupperright.

AscreenshotoftheOMIMhomepageisshownonthefollowingpage.Theeasiestwaytobegina

searchistosimplytypeadisordernameinthesearchboxatthetopoftheOMIMpageand

submityoursearch.However,NCBIalsosupportsavarietyoffeaturesfornarrowingasearchand

browsingdisordersalphabetically(usingOMIMMorbidMap)orbychromosomallocation(using

OMIMGeneMap).

Tonarrowasearch,NCBIhasoptionsfortypingsearchfieldqualifiersintothesearchbox[see

OMIMHelp(www.ncbi.nlm.nih.gov/Omim/omimhelp.html)formoreinformation]orselecting

searchfieldsusingthe Limitstab.Thisexercisewilldemonstratesearchesusingthe Limitstab.
http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/Omim/omimhelp.htmlhttp://www.ncbi.nlm.nih.gov/Omim/omimhelp.htmlhttp://www.ncbi.nlm.nih.gov/


7/54



7

1. SelecttheLimitstabatthetopoftheOMIMpageshowninthescreenshotbelow.

URLforOMIMhomepage:www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM

Mostgenes,disordersandtraitslistedontheHumanGenomeLandmarks(HGL)posterwere

takenfromthetitlefieldsofOMIMrecords,sowecannarrowoursearchtolookonlyforthose

recordsthathavehemochromatosisinthetitlefield.Byselectinghemochromatosisfromthe

HGLposter,wealsoknowthatthegeneforthisdisorderisfoundonchromosome6.

2. FromtheLimitspage,enterhemochromatosisintothesearchboxandselectthe Titlebox

andchromosome6asshowninthescreenshotbelow.ClickGotosubmityoursearch.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIMhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM


8/54



8

NOTE:SearchingforOMIMrecordsassociatedwithmulti-genedisorders,such

asbreastcancerordiabetes,whicharecausedbyalterationsingeneson

differentchromosomes,mayprovidemultipleOMIMrecordsinthesearch

results.Limitingyoursearchtojustonechromosomeforamulti-genedisorder

mayonlyretrieveasubsetofalltherecordsassociatedwiththatdisorder.

3. Thesearchshouldreturnoneresult: MIMID#235200.AscreenshotofthefullOMIMrecord

forthehemochromatosisdisorderisshownbelow.

4. Letsexaminesomeofthefeaturesofthisrecord:

EachOMIMrecordisassignedauniquesix-digit MIMIDnumberlocatedatthetopof

eachentry.Forhemochromatosis,theMIMIDis235200.Asauniqueidentifierfora

disorder,theMIMIDcanbeusedtosearchotherdatabasesforinformationabouta

particulardisorder.

Thenumbersign(#)prefixinfrontoftheMIMIDmeansthatthisentryreferstothe

descriptionofaphenotype,andthemolecularbasisforthisphenotypeisknown.For

moreinformationaboutotherMIMnumberprefixes,seeOMIMHelp

(www.ncbi.nlm.nih.gov/Omim/omimhelp.html#MIMnumberPrefix).

BelowtheMIMID,youwillfindthedisordernameandtheofficialgenesymbol(shownin

theimageonthenextpage).Theofficialgenesymbol,whichis HFEforhemochromatosis,

servesasauniqueidentifierforagene.Tobe"official,"agenesymbolmusthavebeen

approvedbytheHUGOGeneNomenclatureCommittee( www.genenames.org).Thegene
http://www.ncbi.nlm.nih.gov/Omim/omimhelp.html#MIMnumberPrefixhttp://www.ncbi.nlm.nih.gov/Omim/omimhelp.html#MIMnumberPrefixhttp://www.genenames.org/http://www.genenames.org/http://www.ncbi.nlm.nih.gov/Omim/omimhelp.html#MIMnumberPrefix


9/54



9

symbolisespeciallyusefulwhensearchingotherdatabases(suchassequence,genome-

mapping,andstructuredatabases)forgene-specificinformation.

NOTE:Foradisorderlikehemochromatosis,which

isprimarilycausedbymutationsinasinglegene,

theofficialgenesymbolmaybeincludedinthe

recordtitle.Forcomplexdisorderslikebreast

cancer,officialsymbolsforassociatedgeneswillbe

describedinthefirstparagraphoftext.

TheGenemaplocusdescribeswhereagenecanbefoundonachromosome.Forthe

genelocus6p21.3,6isthechromosomenumber,pindicatestheshortarmofthe

chromosome,and21.3isanumberassignedtoaparticularregionofthechromosome.

ClickingonagenemaplocusopenstheOMIMGeneMap,atableofgenesorganizedby

chromosomallocation.

TheamountoftextwithinanOMIMrecordvariesaccordingtowhatisknownabouta

particulargene,disorder,ortrait.Sincehemochromatosisiswellstudied,alotof

informationisknownaboutthisdisorderanditsgene.Somedifferenttypesof

informationthatmaybeincludedinanOMIMrecordaredisorderdescription,inheritance,moleculargenetics,genotypeandphenotypecorrelations,diagnosis,

populationgenetics,andanimalmodels.

EachrecordincludesaTableofContentsboxontherightwithquicklinkstodifferent

sectionswithintherecord.

5. Tolearnmoreaboutthemolecularbasisofhemochromatosis,selecttheMolecularGenetics

linkintheTableofContentsbox(seescreenshotonpreviouspage).TheMolecularGenetics

sectionoftheOMIMrecordforhemochromatosisisshownbelow.

Onestudyshowedthatabout83%ofhemochromatosiscasesarerelatedtotheC282Y

mutation.TheC282YnotationmeansthatamutationoccursintheDNAsequencethat

changestheaminoacidatposition282intheproteinproductfromacysteine(C)toa

tyrosine(T).

6. ClickonthefirstlinkfortheC282Ymutation, 613609.0001.Thislinkwilltakeyoutothe

OMIMrecordfortheHFEgene(MIMID*613609;theasteriskprefixindicatestherecord

representsageneofknownsequence).OMIMoftenmaintainsseparaterecordsfor


10/54



10

phenotypes(suchasthedisorderhemochromatosis)andthegenesassociatedwiththose

phenotypes.

7. TheAllelicVariants sectionoftheOMIMrecordfortheHFEgeneisshowninthescreenshot

below.Thissectiontypicallydescribessomeofthemostnotablegenemutations(alsocalled

allelicvariants)thatproducediseasephenotypes.NotethattheC282Ymutationisalsoknown

astheCYS282TYRmutation,anditisthefirstofseveralmutationsthathavebeenidentifiedfortheHFEgene.ToseealistingofthedifferentmutationsfortheHFEgene,clickonthe See

allelicvariantsintabulardisplay link.

8. NowyouarereadytoanswerQuestions12forActivity1intheworksheetonpage51.

9. ScrolltothetopofthisOMIMrecord,andclickonthe Limitstab.Letsuseoptionsonthe

Limitspagetodeterminehowmanygenesinthehumangenomehavebeendescribedin

OMIM.

UnchecktheboxesforTitleandchromosome6.

ChecktheboxesbesidetheMIMNumberPrefixoptionsfor *genewithknownsequence

and+genewithknownsequenceandphenotypeasshowninthescreenshotonthenext

page.

ThenclicktheGobuttonbesidethesearchboxatthetopofthepage.


11/54



11

10.Youshouldretrieveover13,500searchresults.Oftheestimated20,000to25,000genesin

thehumangenome,about13,500geneshaverecordsinOMIM.Youmaywanttotestyour

newsearchskillsbyusingOMIMtosearchforothergenesorgeneticconditions.Inaddition

toOMIM,anothergoodresourceforlearningaboutgeneticdisordersandassociatedgenesis

theGeneTestswebsite,whichisdescribedinthenextpartofthisactivity.

GeneTests

TheGeneTestswebsiteisamedicalgeneticsinformationresourcedevelopedbyresearchersand

healthcareprofessionalsandfundedbytheNationalInstitutesofHealth.Inadditiontoproviding

up-to-date,authoritativereports(GeneReviews)ongeneticdisorders,thesitealsoincludes

educationalmaterials(e.g.,factsheetsongenetictestingandcounseling,PowerPointslides,and

anillustratedglossary)andonlinedirectoriesofgeneticlaboratoriesandclinics.

Thisactivityfocusesonaccessingandusinggeneticdisorderinformationavailablefrom

GeneReviews.Allentriesarewrittenandreviewedbyphysicians,sothelanguageissimilartothat

ofmedicaltext.Whiletheamountandkindofcontentcanvarygreatlyfromrecordtorecordin

OMIM,allreportsinGeneReviewswillprovidesimilarkindsofinformationandsharethesame

organizationalstructure.

LetsgototheGeneTestswebsite( www.genetests.org)tofindaGeneReviewforhereditary

hemochromatosis.ThescreenshotoftheGeneTestshomepageisshownonthenextpage.
http://www.genetests.org/http://www.genetests.org/


12/54



12

1. Clickon inthenavigationbaratthetop.

2. AttheGeneReviewssearchpage(shownbelow),usethe GeneSymbolsearchoption,select

exactlymatchesfromthedrop-downmenu,andenter HFEintothesearchbox.ClickGoto

submityoursearch.


13/54



13

3. BesidethesearchresultHFE-AssociatedHereditaryHemochromatosis,selectthe

linktoaccessthehereditaryhemochromatosisreviewshownbelow.

4. Ontherightsideofthescreenisanavigationcolumnwithlinkstodifferentsectionsofthe

HFE-AssociatedHereditaryHemochromatosisGeneReview.

5. AccesstheSummarysectiontolearnaboutdiseasecharacteristicsandtreatmentfor

hemochromatosis.ThissectioncanhelpanswerQuestion3forActivity1intheworksheeton

page51.

6. AccesstheMolecularGeneticssectionforabriefoverviewofthisdisordersmolecularbasis.

Withinthissectionyoucanfindinformationabout:

officialsymbolforthegeneassociatedwiththisdisorder.

chromosomallocusofthegene.

genesizeandthenumberofexonsinthegene.

nameofthegenesproteinproduct.

descriptionoftheproteinsfunction. mutationsinnucleotideandaminoacidsequencesthatcauseabnormalproteinproducts

anddiseasephenotypes.

linkstoscientificliteratureandotherdatabasesformoreinformation.


14/54



14

Activity 2Online Resource: NCBI Map Viewer

Find the hereditary hemochromatosis gene on a chromosome map.

NCBI Map ViewerNCBIMapViewerisaWeb-basedtoolforviewingandsearchinganorganism'scompletegenome.

Usersalsocanviewmapsofindividualchromosomesandzoomintospecificregionswithin

chromosomestoexplorethegenomeatthesequencelevel.

MapViewerprovidesaccesstoseveraldifferenttypesofmapsfordifferentorganisms.Manyof

thesemapsaremeaningfulonlytoscientificresearchers.Adiscussionofallthedifferenttypesof

mapsandgenomicdataisbeyondthescopeofthisactivity,whichwillfocusonlyonhowtolocate

aspecificgenelocusonachromosomemap.

1. GototheNCBIMapViewerwebsite( www.ncbi.nlm.nih.gov/mapview/).Inthelistof

Primates,clickontheBuild37.2linkforHomosapiens(human).
http://www.ncbi.nlm.nih.gov/mapview/http://www.ncbi.nlm.nih.gov/mapview/


15/54



15

2. TheMapViewerpagefortheentirehumangenomeisshowninthescreenshotbelow.

Homosapiensgenomeview:www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9606

3. InActivity1,welearnedthattheofficialsymbolforthehereditaryhemochromatosisgeneis

HFE,anditslocusis6p21.3.LetsfindtheHFEgeneonchromosome6.

Whatisalocus?

Thelocusforaparticulargenedescribestheregionofa

chromosomewherethatgenecanbefound.Forthe6p21.3

locus:6isthechromosomenumber,pindicatestheshort

armofthechromosome,and21.3isthenumberassignedto

aparticularbandorregiononachromosome.When

chromosomesarestainedinthelab,lightanddarkbands

appear,andeachbandisnumbered.Thehigherthenumber,

thefartherawaythebandisfromthecentromere.Alocus

containingqisfoundonthelongarmofachromosome.

4. InthesearchboxatthetopoftheMapViewerpage,enter HFE[sym]andthenclicktheFind

buttontosubmityoursearch.Addingthe[sym]searchfieldqualifiertotheendofyoursearch

termspecifiesyourquerysothatonlythoseresultscontainingtheHFEgeneareretrieved.
http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9606http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9606


16/54



16

5. Redtickmarksshouldbedisplayedonchromosome6,indicatingtheapproximatelocationof

theHFEgeneinthemiddleoftheshortarmofchromosome6(seescreenshotbelow).The

rednumber(61)labelingchromosome6indicatesthenumberofobjectsmappedto

differentassembliesofthehumangenomethatincludetheHFEgene.

6. Clickonthenumber6linkbelowthechromosome.Thiswillopenaviewofchromosome6

thatshouldlooklikethescreenshotbelow.Inthenextstepwewillmodifythisviewsowecan

seeanideogramshowingtheregionofchromosome6wheretheHFEgenecanbefound.


17/54



17

7. Tomodifythedisplayoptions,clickontheMaps&Optionsbuttonintheupperrightcorner.

Thiswillopenawindowforcustomizingmapoptions.Makethefollowingadjustments.

RemoveallmapslistedunderMapsDisplayed(lefttoright) excepttheGenemap.To

removeamap,selectitwithyourmouseandthenclicktheREMOVEbutton.

UnderAvailableMapsselectideogr(youwillneedtoscrollthroughmorethanhalfofthe

availablemaps)andthenclicktheADDbutton.Theideogrammapisagraphicshowing

thebandingpatternofachromosome.

TheMapsDisplayedlistshouldlooklikethescreenshotbelow.TheGenemapshouldbe

designatedasyourmastermap.Tomakeamapthemaster,selectitwithyourmouseand

thenclicktheMakeMaster/MovetoBottombutton.Inthechromosomeview,amaster

mapisshownattherightsideofthescreenalongwithitsdetailsanddescriptivetext.The

Genemapincludeslinksforlearningmoreaboutthegenesmappedtoaparticularregion

ofgenomicsequenceonachromosome.

UnderMoreOptions nearthebottomofthewindow,changePageLengthfrom30to10.

ThePageLengthoptionishighlightedinthescreenshotbelow.Thiswilladjusttheheight

ofthedisplayedmap.

BeforeyouclicktheOKbuttontosubmityourchanges,theoptionswindowshould

resemblethescreenshotbelow.

8. Thenewmapofchromosome6shouldresemblethescreenshotonthenextpage.


18/54



18

9. CheckoutsomeofMapViewersfeaturesdisplayedinthescreenshotabove.

Theportionofchromosome6displayedinMapViewerishighlightedontheideogramin

thebluenavigationcolumnontheleft.Noticethattheredmarkindicatingthepositionof

theHFEgenelinesupwiththeideogramatthe6p22chromosomeband,not6p21.3.

Roundedtothenearestthousandth,theregionofsequencedisplayedbeginsataboutthe

26,086,000th

nucleotideandendsataboutthe26,100,000th

nucleotideoftheDNA

sequenceofchromosome6.ThetotalDNAsequenceforchromosome6isabout171

millionbasepairslong,butthisviewonlyshowsabout14,000basepairs.

ClickingontheIdeogramorGenes_seqmaps(notthelabels)willopenapop-upwindow

withoptionsforzoominginoroutonthedisplayedmaps.MapViewerhaszoomedinso

muchtoshowtheHFEgene,thereisntmuchoftheideogrammapdisplayed.Youcan

alsozoominandoutusingthezoomoptioninthebluenavigationcolumn.

TheGenes_seqmapprovideslinkstogene-specificentriesinotherNCBIdatabases.

o HFELinkstotheHFEentryintheEntrezGenedatabase,acompendiumofgenes

andmappedphenotypes.

o OMIMLinkstothehemochromatosisentryintheOnlineMendelianInheritancein

Man(OMIM)databasecoveredinActivity1.


19/54



19

o HGNCLinkstothegenesymbolreportmaintainedbytheHUGOGene

NomenclatureCommittee.

o svLinkstoSequenceViewer,agraphicalinterfaceforinvestigatingthegenes

sequenceaswellasgenomicsequenceupstreamanddownstreamofthegene.

o prLinkstosequencerecordsforthegenesproteinproductmaintainedinNCBIs

Proteindatabase.

o dlLinkstoapagefordownloadingtherangeofsequencedatadisplayedinMap

Viewer.

o evLinkstoEvidenceViewer,atoolforfindingbiologicalevidencethatsupportsa

particulargenemodelandforexploringthedifferenttypesofexpressedsequences

thataligntoaparticularareawithinagenome.

o mmLinkstoModelMaker,atoolforbuildingyourownversionofagenemodelby

addingorremovingexons.

o hmLinkstoHomologene,aresourceforcomparinggenesinhomologoussegments

ofDNAfromdifferentorganisms.

o stsLinkstoUniSTS,acomprehensivedatabasethatintegratesgeneticmarkerand

mappinginformation.Asequencetaggedsite(STS)isashort(200to500basepairs)

DNAsequencethathasasingleoccurrenceinthehumangenome.Detectableby

polymerasechainreaction(PCR),STSsareusefulforlocalizingandorientingthe

sequencedatareportedfrommanydifferentlaboratories.

o CCDSLinkstotheCCDSproject,anefforttoensurethatcodingregionswithinthe

humangenomeareconsistentlyannotated.

o SNPLinkstorecordsforsinglenucleotidepolymorphisms(SNPs)andotherareasof

sequencevariationthathavebeenidentifiedintheselectedgene.

10.Letszoomouttoviewtheentirechromosomeusingthe Maps&Options window.

ClickonMaps&Options againtoopentheoptionswindow.

Deletethenumbersdefiningthe RegionShownatthetopoftheoptionswindow.

Thiswillmodifythedisplaysoitshowstheentirechromosome.

UnderMoreOptionsnearthebottomofthewindow,changePageLengthfrom10to

20.ThePageLengthoptionishighlightedinthescreenshotonthenextpage.Thiswill

display20labeledgenesinthemastermapandshouldprovideenoughspaceonthe

screentoviewtheentirechromosomewithreadablelabelsforthechromosome

bands.

OncetheMaps&Optionswindowresemblesthescreenshotonthefollowingpage,

clicktheOKbuttontosubmityourchanges.


20/54



20

11.Yourviewofchromosome6shouldresemblethescreenshotonthenextpage.

Toseeamorecomprehensivelistingofgenesonchromosome6,selectthe DataAsTable

Viewlinkinthebluenavigationcolumnontheleft.The DataAsTableViewdisplays

1,000ofthegenesonchromosome6andshowswheregenesstartandstopinthe

chromosomesDNAsequence.

Scrolldowntothebottomofthemaptoexaminethe SummaryofMapssection.Usethis

informationandwhatyouhavelearnedaboutMapViewertoanswertheQuestionsforActivity2onpage51.


21/54



21


22/54



22

Activity 3Online Resources: NCBI Entrez Gene and GenBank

Examine gene sequence and structure.

NCBI Entrez Gene and GenBank

EntrezGeneisanNCBIresourcethatservesasasingle-queryinterfaceforaccessingsequence

andotherbiologicalinformationforspecificgenesfromavarietyofsequencedorganisms.

GenBankisNCBIscomprehensiverepositoryofannotatedDNAsequences.

ThisactivitycovershowtouseEntrezGenetoaccessthegenomicDNAsequenceofthe

hereditaryhemochromatosis(HFE)gene.Thenbyexaminingsomedifferentfeaturesofa

GenBankrecordfortheHFEgene,wewilllearnaboutthegenesstructure(e.g.,intronandexon

composition,codingsequence).

1. Tobegin,letsgototheEntrezGenehomepage( www.ncbi.nlm.nih.gov/gene).Inthesearch

boxatthetop,enterHFE[sym]ANDhuman[orgn]asshowninthescreenshotbelow.Besure

tocapitalizeanyBooleanoperator(AND,OR,andNOT)includedinyoursearchstatements.

Thensubmityoursearch.
http://www.ncbi.nlm.nih.gov/genehttp://www.ncbi.nlm.nih.gov/gene


23/54



23

SearchTip:Adding[sym]totheendofyourquerytermtellsEntrezGenethatyouare

searchingbygenesymbolonly.Ifyoudonotspecifythatyouwanttosearchthegene

symbolfield,thesearchwillreturnmultiplerecordsthatincludethequeryterm

anywherewithinarecordscontent.Adding[orgn]toasearchtermlimitsthesearch

togenesfromaspecificorganism.Formoreinformationonoptionsforrefiningyour

search,seetheSearchFieldDescriptionsandQualifierssectionoftheEntrezHelp

Document(www.ncbi.nlm.nih.gov/entrez/query/static/help/Summary_Matrices.html).

2. Submittingthissearchshouldretrieveasingleresult.TheHFErecordisshownbelow.

3. IntheSummarysectionyoucanfindinformationaboutthefunctionofthegenesprotein

product.TheHFEproteinisthoughttohavearoleinregulatingirontransportintocells,and

defectsintheHFEgenecancausetheironabsorptiondisorderhereditaryhemochromatosis.

UseinformationprovidedintheSummarysectiontoanswerQuestion1forActivity3inthe

worksheetonpage52.
http://www.ncbi.nlm.nih.gov/entrez/query/static/help/Summary_Matrices.htmlhttp://www.ncbi.nlm.nih.gov/entrez/query/static/help/Summary_Matrices.html


24/54



24

4. BelowthesummarysectionistheGenomicregions,transcripts,andproductssection.

ThesequenceviewerboxshowsagraphicmodeloftheHFEgeneconsistingofathingray

line(representingintronsthatareremovedwhenthemRNAisprocessed)connectedto

thickergreenboxes(representingexons).

Theportionofthechromosome6sequenceincludedinthesequenceviewerboxisnoted

intheupperleftcorner.

ClickontheGenBanklinkintheupperrightcornertoaccesstheGenBankrecordforthe

HFEgenesequencethatispartofthesequencedatageneratedbytheInternational

HumanGenomeProject.AscreenshotofthisGenBankrecordisshownonthenextpage.


25/54



25

5. CheckoutsomeofthefollowingfeaturesintheGenBankrecord.

Atthetopweseethatonlyaverysmallportionofchromosome6(from26,087,448th

base

to26,097,059th

base)isincludedinthisrecord.

ThefirstReferencelistedforthisrecordidentifiestheInternationalHumanGenome

SequencingConsortiumasthesourceforthissequenceinformation.Thusthissequence

isaproductoftheinternationalHumanGenomeProject.

Evenafteragenomesequenceispublishedinajournalandreportedascomplete,the

researchcommunitycontinuestoanalyzethegenomesequencedataandimprovethe

annotationthatdescribesdifferentfeaturesencodedwithinthegenomesequence.Note

thatthisrecordwaslastmodifiedOctober25,2010.

6. ScrolldowntotheFEATURESsectionofthisGenBankrecord(seescreenshotonnextpage).

TheHFEgeneis9,612basepairs(bp)long.

TheinformationinthisGenBankrecordfortheHFEgenewasDerivedbyautomated

computationalanalysisusinggenepredictionmethodasapartoftheHumanGenome

Project.

FromthemultipleentriesformRNAlistedinthisrecord,weseethatmorethanone

mRNAtranscriptcanbegeneratedfromtheHFEgene.Forexample,anexonincludedin

onemRNAtranscriptmightbeleftoutinanothertranscript.Eachofthesedifferent

mRNAtranscriptsfromthesamegeneisknownasavariant.


26/54



26

7. UseyourbrowsersbackbuttontoreturntotheEntrezGenepageforthehumanHFEgene.

8. LetsaccessanotherGenBankrecordfortheHFEgenesequencetoseehowinformationcan

varyinrecordsthatcomefromdifferentsources.Asshowninthescreenshotonthenextpage,selecttheRelatedsequenceslinkintheTableofcontentsboxontherightsideofthe

screen.


27/54



27

9. IntheRelatedSequencesfortheHFEgene(seescreenshotonthenextpage),selectthe

genomicsequencerecord Z92910.1.

Howdidyouknowwhichgenomicsequencetoselect?

TheproblemwitharchivalsequencedatabaseslikeNCBIsGenBankisthatthey

usuallyhavemultiplesequencerecordsforthesamegene.Youmayneedtoopeneachrecordindividuallyandbrowsethroughdefinition,sequence

annotation,andcommentstodeterminehowmuchofthegenesnucleotide

sequenceiscontainedwithineachrecord.

Forexample,theU91328.1recordcontainsthesequenceofagenomicsegment

thatnotonlyincludestheHFEgenesequencebutalsosequencesforother

genes.Y09801.1containsonlysequenceinformationfortheHFEpromoterand

theHFEgene'sfirstexon.Ofthegenomicrecordslisted,Z92910.1hasthemost

completesequenceinformationfortheHFEgene.

InsequencedatabasessuchasGenBank,genomicDNAsequencerecordsfor

eukaryoticorganismscontainbothexonsandintrons,whilemRNAsequences

areintron-freeDNAsequences.AllsequencesinGenBankandsimilarrepositoriesusethesingle-letterabbreviationsfortheDNAbasesadenine(A),

cytosine(C),guanine(G),andthymine(T)torepresenteachnucleotide.Even

mRNAsequencerecordsuseA,C,G,andTwhereTisusedtoreplaceeach

uracil(U)inthemRNAsequence.


28/54



28

RelatedSequencesfortheHFEGeneinEntrezGene

10.AscreenshotoftheGenBankrecord Z92910.1fortheHFEgeneisshownonthenextpage.

TheDNAsequenceincludedinthisrecordis12,146basepairs(bp)long.Inadditionto

containingthegenomicsequenceoftheHFEgene,thisrecordalsocontainsseveralhundredadditionalbasepairsofsequenceupstreamanddownstreamofthegene.

ThisrecordwasoriginallysubmittedbyaresearchertoGenBankin1997,sothesequence

oftheHFEgenewasknownseveralyearsbeforetheHumanGenomeProjectwas

complete.

ScrolldowntotheFEATURESsectionofthisrecordandusethisinformationtoanswer

Questions24forActivity3onpage52.Notethatclickingonthe genelinkinthe

FEATURESsectionshowsthatthelengthoftheHFEgeneisdifferentfromwhatwe

observedintheGenBankrecordexaminedinstep5ofthisactivity.


29/54



29

GenBankRecordZ92910.1forHumanHFEGene

11.SomefeaturesofthesequenceinGenBankrecordZ92910.1include

source:RequiredforeveryGenBankrecord,thesourceprovidestheentiresequence

lengthandthescientificnameofthesourceorganism.Othertypesofsourceinformation

mayincludechromosomenumber,maplocation,andcloneorstrainidentification. gene:Thisfeatureprovidesnucleotidenumbersindicatingwherethegenestopsand

starts.Thislinkopensanewsequencerecordthatshowsonlythegenesequence.

exon:Thisfeatureprovidesnucleotidenumbersindicatingwhereeachexonbeginsand

ends.Youwillseeseveraloftheseentriesasyouscrolldown.Eachexonisasequence

segmentthatcodesforaportionofprocessed(intron-free)mRNA.Thenameofthegene


30/54



30

towhichtheexonbelongsandtheexonnumberareprovided. Anexonlinkopensa

newsequencerecordthatshowsonlytheexonsequence.

CDS:Thecodingsequence(CDS)consistsofnucleotidesthatactuallycodeforaminoacids

oftheproteinproduct.Thisfeatureincludesthecodingsequence'saminoacidtranslation

andmayalsocontaingenename,geneproductfunction,alinktoproteinsequence

record,andcross-referencestootherdatabaseentries. ACDSlinkopensanewsequencerecordthatshowsonlythecodingsequence.

intron:Thisfeatureprovidesthenucleotidenumbersindicatingwhereeachintronbegins

andends.Anintronisasegmentofnoncodingsequencethatistranscribedbutremoved

fromthetranscriptbysplicingtogethertheexonsoneithersideofit. Anintronlink

opensanewsequencerecordthatshowsonlytheintronsequence.

Whatsthedifferencebetweenexons

andcodingsequence?

Exonsoftenaredescribedasshortsegments

ofproteincodingsequence.Thisisabitofan

oversimplification.Exonsaresegmentsof

sequencesplicedtogetherafterintronshave

beenremovedfrompre-mRNA.Exonscarry

thecodingsequenceofagene,butsome

exonsmaycontainnocodingsequence.

Portionsofexonsorevenentireexonsmay

containsequencethatisnottranslatedinto

aminoacids.Thesearetheuntranslated

regions(UTR)ofmRNA.UTRsarefound

upstreamanddownstreamoftheprotein-

codingsequence.Seediagramonright.

12.SequenceinformationinaGenBankrecordcanalsobedisplayedusinggraphicsintheNCBI

SequenceViewer.ToaccessSequenceViewerfromaGenBankrecord,clickonthe Graphics

linkintheupperleftcorner(asshownbelow).


31/54



31

13.TheSequenceVieweroptionforGenBankrecordZ92910.1isshowninthescreenshotbelow.

ThetoppaneldisplaystheentiresequenceincludedintheGenBankrecord,thegreenbar

representstheHFEgenesequence,andtheblueoutlineofaboxwitharrowsindicates

whichportionofthesequenceisshowninthepanelbelow.Clickanddragthearrowson

theblue-boxoutlinetochangehowmuchofthesequenceisdisplayedinthelowerpanel.

Youcanalsousethearrowsontheleftsideofthelowerpaneltomovealongthe

sequenceandseewhereexonsandothergenefeaturesbeginandend.Thesliderbelow

thearrowscanbeusedtozoominandoutonthesequence.


32/54



32

Activity 4Online Resources: UniProt Protein Knowledgebase and BLAST Searching

Access the amino acid sequence of a genes protein product.

Compare the HFE protein sequence with protein sequences of otherorganisms.

UniProt Protein Knowledgebase and BLAST Searching

TheProteinKnowledgebase,whichispartoftheUniversalProteinResource(UniProt),isa

comprehensive,freelyaccessibledatabasethatthescientificcommunityusestoaccesshigh-

qualityproteinsequenceandfunctionalinformation.ThisactivitycovershowtouseUniProtto

learnabouttheaminoacidsequenceandotherfeaturesofthehereditaryhemochromatosis

protein.

1. GototheUniProthomepage( www.uniprot.org),enterHFEintothequeryboxasshownin

thescreenshotbelow,andthensubmityoursearch.

2. Fromthelistofresults(showninthescreenshotonthenextpage),noticethatsomeentries

havegoldstarsandothershavegraystars.Thosewithgoldstarshavedescriptionsofprotein

functionsandcharacteristicsthathavebeenmanuallyreviewedbyexperts.Entrieswithgray

starshavedescriptionsthatwereautomaticallygenerated,andexpertshavenotyetreviewed
http://www.uniprot.org/http://www.uniprot.org/


33/54



33

theserecords.Thusselectingasearchresultwithagoldstarwillprovideyouwithricher,

higherqualityinformationaboutaprotein.

SelectaccessionnumberQ30201fortheHFE_HUMANentryforthehereditary

hemochromatosisprotein.

3. TheUniProtentryfortheHFEproteinisshownonthenextpage.Thebluenavigationbarat

thetopofthescreencontainslinkstodifferentpartsoftheUniProtrecordforthisprotein.

Makeanoteoftheaccessionnumber(Q30201)forthisprotein.Wewillusetheaccession

numbertosearchforproteinstructuralinformationinActivity5.

Scrolldownthroughtherecordandreviewthe Proteinattributes andtheGeneral

annotationsectionstoanswerQuestions13forActivity4intheworksheetonpage52.

4. IntheProteinattributes section,forSequenceprocessing,noteThedisplayedsequenceis

furtherprocessedintoamatureform.ThismeansthatpartoftheHFEproteinchainneedsto

becutoffbyaproteolyticenzymetoformthematurefunctionalprotein.


34/54



34

5. ClickonSequenceannotationinthebluenavigationbarnearthetopoftherecord(markedin

thescreenshotabove).

6. TheSequenceannotationsectionoftheHFEproteinrecordisshowninthescreenshotonthe

nextpage.

UnderMoleculeprocessingintheSequenceannotationsection,noticethatthesignal

peptidecomprisesaminoacids122.Thefirst22aminoacidsarenotassociatedwithany

domains(functionalunitswithinaprotein).Thisportioniscleavedfromthecomplete

proteinsequencetomakethemature,functionalHFEprotein,whichconsistsofaminoacids23348.ClickingonthebluePosition(s)numbersinthesequenceannotationwill

openawindowshowingtheselectedsequencehighlightedwithinthecontextofthe

entireproteinsequence.

InActivity1welearnedthatthecysteineataminoacidposition282ischangedtoa

tyrosineinacommonmutationthatcauseshemochromatosis.ReviewtheRegionsand

AminoacidmodificationspartsoftheSequenceannotationsectiontoanswer

Questions45forActivity4onpage52.


35/54



35

7. ScrolldowntotheSecondarystructurepartofthe Sequenceannotationsection(shownin

imagebelow)andclickonDetailsbelowthecoloredbar.

8. Thesecondarystructuredetailsshowwhichsegmentsofproteinsequencemakeupbeta

strands,alphahelices,ortheturnsthatformbetweenbetastrandsandalphahelices.These

secondaryelementsareimportantindeterminingthethree-dimensionalproteinstructure.

UsethissecondarystructuralinformationtoanswerQuestion6forActivity4onpage52.

9. ReturntothetopoftheHFEproteinrecordbyscrollingorbyclicking Namesintheblue

navigationbar.ClickontheBlasttabatthetopofthepage.


36/54



36

NOTE:BLAST(BasicLocalAlignmentSearchTool)isatoolusedtocalculatehowsimilar

nucleotideorproteinsequencesareamongthesameordifferentkindsoforganisms.Many

resourcesthatmaintainbiologicalsequenceinformationoftensupporttheirownBLAST

searchingcapabilitiestoretrieveandcomparesequencedata.Formoreinformationabout

BLAST,seeTheNCBIHandbook(www.ncbi.nlm.nih.gov/books/NBK21097/).

ProteinsequencesareoftenpreferredovernucleotidesequencesforBLASTsearchingbecauseofthegreatervariabilityinnucleotidesequences.Rememberwiththegenetic

code,differentcodonsofnucleotidescanspecifythesameaminoacid.Thusproteinsthat

havesimilaraminoacidsequencesmayhaveconsiderablydifferentnucleotidesequences

encodingthoseproteins.

10.AscreenshotoftheBLASTsearchfeaturefortheHFEproteinisshownbelow.

TheaminoacidsequenceofthecompleteHFEproteinisautomaticallyenteredintothe

textboxontheleft.Thesingle-letterabbreviationsusedtorepresenteachaminoacidare

explainedintheTableofStandardGeneticCodeonpage50.

ClickontheBlastbuttontocomparetheaminoacidsequenceoftheHFEproteinwithall

thesequencesavailablefromtheUniProtKnowledgebase.Bepatient.ABLASTsearchmaytakeseveralminutesdependingonhowbusytheserveris.

11.Oncetheresultsareretrieved,scrolldowntothe DetailedBLASTresults(seescreenshoton

nextpage).

TheIdentitycolumnontherightprovidesthepercentofeachentrysaminoacid

sequencethatisidenticaltothesequencesubmitted.Tosortallofyourresultsfrom

highesttolowestIdentityvalues,clickonthearrowsatthetopofthe Identitycolumn.

Toseemoreresults,clickNextintheupperrightcorner.

UsetheDetailedBLASTresultstoanswerQuestion7forActivity4onpage53.
http://www.ncbi.nlm.nih.gov/books/NBK21097/http://www.ncbi.nlm.nih.gov/books/NBK21097/


37/54



37

DetailedBLASTResultsfortheHFEProteininUniPRot


38/54



38

Activity 5Online Resources: Protein Data Bank

Explore the sequence and structure of the genes protein product.

Protein Data Bank

Thisactivitydemonstrateshowtofindandviewaproteinstructureusingtoolsandresources

availablefromtheProteinDataBank(PDB).PDBisaninternationalarchiveof3-Dstructural

informationforbiologicalmacromolecules.PDBrecordsprovideaccesstoseveralinteractive

moleculargraphicsprograms.ThisactivityalsousesFirstGlanceinJmol,aresourcethatworksin

mostbrowsersforviewingthemajormolecularfeaturesofastructurewithjustafewmouse-

clicks.

Before You Begin

ManyfeaturesofthePDBwebsiterequirenewerWebbrowserswithJavaScriptandcookies

enabled,andpop-upsshouldnotbeblocked.FormoreinformationonsystemrequirementsseePDBFrequentlyAskedQuestions(www.rcsb.org/pdb/static.do?p=home/faq.html).

Some Protein Structure Basics

Proteinsarecreatedbylinkingaminoacidsinalinearfashiontoformpolypeptidechains.The

aminoacidsequenceofapolypeptidechainistheprimarystructureofaprotein.Seethe

TableofStandardGeneticCodeonpage50forsingle-letterandthree-letterabbreviationsfor

the20differentaminoacids.

Aminoacidshavedifferentchemicalproperties.Forexample,someaminoacidresiduesare

strictlyhydrophobic(waterfearing)andmustbeprotectedfromaqueousenvironments,whileotheraminoacidsarehydrophilic(waterloving).Thesubstitutionofjustoneamino

acidforanotherwithverydifferentchemicalpropertiescanhaveseriousconsequencesfora

proteinsstructureandfunction.

Thefoldingofregionswithinthepolypeptidechainintoalphahelicesandbetasheetsisa

proteinssecondarystructure.

Thepackingoftheentirepolypeptidechainintoathree-dimensionalglobularunitisa

proteinstertiarystructure .

Ifaproteinmoleculeisacomplexofmorethanonepolypeptidechain,thenthecomplete

structureofthismolecularcomplexiscalledaproteins quaternarystructure.

Adomainisadiscreteportionofaproteinwithitsownfunctionandspecificthree-

dimensionalstructure.Thecombinationofdomainsinasingleproteindeterminesitsoverall

function.

Differentpartsofapolypeptidechaincanbelinkedbydisulfidebridgesthatformbetween

twocysteineresidues.Disulfidebridges(ordisulfidebonds)stabilizeaproteinsthree-

dimensionalstructure.Thelossofadisulfidebridgewouldbedetrimentaltoaproteins

overallstructure.
http://www.rcsb.org/pdb/static.do?p=home/faq.htmlhttp://www.rcsb.org/pdb/static.do?p=home/faq.html


39/54



39

Finding a Structure Record in PDB

1. Tobegin,letsgototheProteinDataBank( www.rcsb.org/pdb/).

NOTE:IfyouarenewtoPDB,besuretocheckoutthe Educationlinksinthelight

bluecolumnontheleftofthescreen.Under EducationalResourcesyoucanfind

posters,tutorials,activities,andlessons.MoleculeoftheMonth isacollectionof

vignettes,eachfeaturingadifferentmolecularstructureanditsimportanceto

humanwelfare.

2. BesidethesearchboxatthetopofthePDBhomepage,select AdvancedSearch.
http://www.rcsb.org/pdb/http://www.rcsb.org/pdb/


40/54



40

3. OntheAdvancedSearchpage,fromthe ChooseaQueryTypedropboxselectUniProtKB

AccessionNumber(s).InActivity4weaccessedthehumanhemochromatosisproteinrecord

Q30201intheUniProtProteinKnowledgebase.Enter Q30201inthesearchbox.The

advancedsearchpageshouldlooklikethescreenshotbelow.Selectthe SubmitQuerybutton

tosubmityoursearch.

4. Thesearchshouldreturntwohits.Scrolldownthepagetoseeabriefsummaryofeach

searchresult.Onerecord(1DE4)providesstructuralinformationonthehemochromatosisproteinHFEcomplexedwithareceptor,andtheotherrecord(1A6Z)justprovidesstructural

informationfortheHFEprotein.Clickon 1A6ZHFE(HUMAN)HEMOCHROMATOSISPROTEIN

toopenthisPDBrecord.


41/54



41

5. Thesummarytabofthe1A6Zrecordisshowninthescreenshotabove.

NotetheMolecularDescriptionboxinthecenterofthescreenshot.Thisstructureisa

complexoffourpolymerchains:A,B,C,andD.AandCareidenticalHFEpolypeptide

chains,andBandDareidenticalchainsofanotherproteincalledbeta-2-microglobulin.

NotethePrimaryCitationinthe1A6Zrecord.Thebestwaytolearnaboutstructure

detailsistoaccessthearticlelistedastheprimarycitation.Althoughthefulltextforsome

articlesmaybefreelyavailableonline,manyarticlesareaccessibleonlybysubscription.

Someuniversityresearchlibrariesmayprovidepublicaccesstotheirjournalcollections.

Thearticleforthisstructurehasbeenaccessedtorevealthefollowingdetails:

o OnlythesolubleportionoftheHFEpolypeptidechainisincludedinthe1A6Zstructure.Thetransmembranedomainismissing,sotheHFEproteininthis

structurehasonly275ofthe348aminoacidsinthecompleteHFEprotein

sequence.

o Thefirst22aminoacidsoftheHFEpolypeptidesequencehavebeenexcluded

becausetheyarenotpartofthemature,functionalprotein.Therefore,thefirst

aminoacidinthisstructureisreallythe23rd

,andcysteine260isthecysteine

residueinvolvedintheCYS282TYRmutationthatwelearnedaboutinActivity1.


42/54



42

o EachHFEpolypeptidechainiscomplexedwithanotherpolypeptidechaincalled

beta-2-microglobulin.

o The1A6ZstructureconsistsoftwoHFEbeta-2microglobulincomplexes.

6. SelecttheSequencetabtoexaminethesequenceandsecondarystructuredetailsforthis

structure.

7. TheSequenceandStructureDetailsforrecord1A6Zareshowninthescreenshotbelow.

TheHFEproteinsequence(polypeptidechainA)ispresentedfirst.Eachletterinthe

proteinsequencerepresentsadifferentaminoacid.Cstandsforcysteine.SeetheTable

ofStandardGeneticCodeonpage50todeterminewhichaminoacidisrepresentedby

eachletter.

Secondarystructuredetailsaremappedontosequencedetails.Differentgraphical

symbolsareusedtorepresentextendedbetastrands,alphahelixes,bends,andturns.


43/54



43

8. Selectthedisplayexternal(UniProtKB)sequencelinkhighlightedinthepreviousscreenshot.

9. ThesequencepagewillreloadanddisplaytheaminoacidnumbersfortheUniProtHFE

proteinsequence(thatweexaminedinActivity4)abovethelineofsingle-letteraminoacid

abbreviations(seescreenshotbelow).

Findcysteine282intheUniProtsequence.Cysteine282istheaminoacidthatisreplaced

bytyrosineintheCYS282TYRmutation.

Youwillseethatcysteine282intheUniProtsequenceisatposition260inthePDB

structuresequence.InActivity4,welearnedthatcysteine282formsadisulfidebond

withcysteine225intheUniProtHFEproteinsequence.IntheHFEproteinsequencefor

PDBstructure1A6Z,weseethatcysteine260formsadisulfidebondwithcysteine203

(whichcorrespondstocysteine225intheUniProtsequence).Disulfidebondsarecritical

toformingtheproperstructuralarrangementneededtomakeafunctionalprotein;

therefore,thelossofcysteine260wouldbedetrimentaltoproteinstructure. Answerthe

firsttwoquestionsforActivity5intheworksheetonpage54.

Viewing the Structure

10.SelecttheSummarytabnearthetopofthepagetoreturntothe1A6Zrecordsummary.In

theBiologicalAssembly1boxintheupperrightcornerofthepagethereareseveraloptions

forviewingthemolecularstructure.Clickingonthe MoreImageslinkwillopenapagewith

optionsfordownloadingastillimageoftheHFEmolecularcomplex1A6Z.AlthoughPDBprovidesaccesstoseveraldifferentmolecularviewersforexamininga3-Drepresentationofa

molecularcomplex,manyoftheseoptionsweredesignedforscientistswhospecializein

studyingmolecularstructures.Inthisactivity,wewilluseamolecularviewercalled

FirstGlanceinJmol,whichisoneofthemoreuser-friendlyoptionsfordisplayingthemajor

structuralfeaturesofamolecule.FirstGlanceinJmolwasdevelopedtoworkinallpopular

webbrowserswithouthavingtodownloadandinstallanything.


44/54



44

11.ToaccessFirstGlance,firstclickontheleftarrownexttothe BiologicalAssembly1 label

abovethemolecularimage.ThisshouldchangetheboxlabeltoAsymmetricUnit.

12.ByclickingonthearrownexttoOtherViewers,adrop-downmenuwillappear.Select

FirstGlancefromthedrop-downmenu(seescreenshotbelow).


45/54



45

13.Anewpageshouldopendisplayingstructure1A6Zusing FirstGlanceinJmol (seescreenshot

below).

Tostopthespinningofthemolecule,clickthe Spinboxintheupperleft.

ToremovetheS-labels,unchecktheShowboxbesideLabelsX,S-,?.

14.Thestructureisinitiallydisplayedusingthe Cartoonoption,whichassignsadifferentcolorto

eachmolecularchaininthestructure.ChainsA,B,C,andDshouldbedisplayed.Earlierinthe

activitywelearnedthatchainsAandCareidenticalHFEchainsandchainsBandDare

identicalbeta-2-microglobulinchains.

Clickinganywhereonthemoleculewillgeneratealabelinthelowerleftcornershowing

theaminoacidresidueandtheproteinchainthatyouhaveselected.

ClickoneachcoloredchaintofindChainA,whichisoneofthetwoHFEproteinchains.In

thescreenshotonthenextpage,ChainAisthebluechain.

Ifyouneedtorotatethestructure,simplyclickonthestructureanddragwithyourmouse.

Toundoanyofthechangesyouhavemadeandresetthestructuretoitsoriginal

configuration,clickResetintheupperleftcorner.


46/54



46

Byclickingonthebluechain,thelabelinthelowerleftindicatesthatthebluechainisChainA.

15.LetshideallthechainsexceptChainA.Clickonthe Hide..linkintheupperleftcornerand

thenclickoneachchainexceptChainA.Yourscreenshouldlooklikethescreenshotbelow.

16.ClickontheCenterVisibleChains link(highlightedinscreenshotabove)toplaceChainAin

thecenterofthedisplaypanel.


47/54



47

17.OnceChainAiscentered,usethe ZoomtooltoenlargeChainA.InadditiontousingtheZoom

arrowsintheupperleftcorner,youcanzoominandoutbyclickingonthebackgroundofthe

structureandthenusingthewheelonyourmouse.Alternatively,youcanalsoholddownthe

Shiftkeyanddragthemouseupanddownoverthemoleculetozoominandout.Yourscreen

shouldsomethinglooklikethescreenshotbelow.

18.Letsfindcysteine260andcysteine203(thecysteineresiduesthatformthedisulfidebond

involvedintheCYS282TYRmutation).Clickonthe Find..link(highlightedinscreenshot

above).

19.TheFindoption(showninthescreenshotonthenextpage)allowsyoutosearchfor

particularresidueswithinamolecule.Thelocationsoftheresiduesareindicatedusingyellow

dots.ThebackgroundcolorautomaticallychangestoblackwhenyouselectFind.Ablack

backgroundmakestheyellowdotseasiertosee.Youcantogglebetweenblackandwhite

backgroundcolorsbyclickingontheBackgroundboxintheupperleftcorner.

TypeCYS260,CYS203intothetextbox.

PresstheEnterkeyonyourkeyboardtosubmityoursearch.

Yellowdotsshouldindicatewherethesetworesiduesareintheproteinchain.Youmay

needtorotatethestructurebyclickinganddraggingyourmouseoverthemoleculesothatyoucanobtainagoodviewoftheyellowdots.Notethattheyellowdotssurrounda

thingoldbar.Thisthingoldbarrepresentsadisulfidebond.Youcanseethatabond

betweencysteines203and260wouldcreateastrongconnectionbetweentwodifferent

strandswithintheprotein.


48/54



48

FindingCysteineResiduesintheHFEProtein

20.ToobtainabetterviewofthedisulfidebondsintheHFEprotein,clickonthe MoreViews..

linkintheupperleftcorner,andthenclickonthe DisulfideBonds:ShowAll link.Thepage

shouldchangesothatitlookslikethescreenshotbelow.Thebackboneoftheproteinchainis

modifiedtoathinline(whichisdifficulttoseeinthescreenshot),andthedisulfidebonds

becomethickerandeasiertosee.Thecysteineresiduesarealsolabeled. AnswerQuestions

34forActivity5intheworksheetonpage54.


49/54



49

21.Nowthatyouarefamiliarwithafewoptionsformodifyingamolecularstructureusing

FirstGlance,youmaywanttoResetthestructureandpracticewhatyouhavelearned.In

additiontothedisplayoptionsintheupperleftcornerofthescreen,youcanalsousepop-up

menustomodifythestructurebyclickingon Jmolinthelowerrightcornerofthedisplay

panel(highlightedinthepreviousscreenshot).

22. Ifyouareinterestedincopyingorsavingaparticularviewofastructurethatyouhave

created,checkoutthePresentingMolecularViewsfromFirstGlanceinJmolpage

(molvis.sdsc.edu/fgij/slides.htm).

Protein Structure and Hereditary Hemochromatosis Development

ByexaminingtheHFEproteinssequenceandstructure,wediscoverthatthecysteinelostinthe

CYS282TYRmutationhasanimportantroleinestablishingthecorrectthree-dimensionalHFE

structure.Inthismutation,acysteineresidueisreplacedbyanotheraminoacid,tyrosine,andthe

disulfidebondbetweentwocysteinesinthepolypeptidechainislost.Thisisdetrimentaltothe

protein'sstructure.Asaresult,theHFEproteincannolongerperformitsnormalfunctionofregulatingironuptake,andcellsbecomeoverloadedwithiron.Thisbuildupofironincells,if

untreated,canleadtoorgandamageandothercomplications.
http://molvis.sdsc.edu/fgij/slides.htmhttp://molvis.sdsc.edu/fgij/slides.htm


50/54



50

Table of Standard Genetic Code forTranslating DNA Sequence Records

T C A GT

TTT Phe (F)TTC Phe (F)TTA Leu (L)TTG Leu (L)

TCT Ser (S)TCC Ser (S)TCA Ser (S)TCG Ser (S)

TAT Tyr (Y)TACTAA STOPTAG STOP

TGT Cys (C)TGCTGA STOPTGG Trp (W)

CCTT Leu (L)CTC Leu (L)CTA Leu (L)CTG Leu (L)

CCT Pro (P)CCC Pro (P)CCA Pro (P)CCG Pro (P)

CAT His (H)CAC His (H)CAA Gln (Q)CAG Gln (Q)

CGT Arg (R)CGC Arg (R)CGA Arg (R)CGG Arg (R)

AATT Ile (I)ATC Ile (I)ATA Ile (I)ATG Met (M) START

ACT Thr (T)ACC Thr (T)ACA Thr (T)ACG Thr (T)

AAT Asn (N)AAC Asn (N)AAA Lys (K)AAG Lys (K)

AGT Ser (S)AGC Ser (S)AGA Arg (R)AGG Arg (R)

GGTT Val (V)GTC Val (V)GTA Val (V)GTG Val (V)

GCT Ala (A)GCC Ala (A)GCA Ala (A)GCG Ala (A)

GAT Asp (D)GAC Asp (D)GAA Glu (E)GAG Glu (E)

GGT Gly (G)GGC Gly (G)GGA Gly (G)GGG Gly (G)

Key to the Table of Standard Genetic Code

Alanine ALA A Arginine ARG R

Asparagine ASN N Aspartic acid ASP D

Cysteine CYS C Glutamic acid GLU E

Glutamine GLN Q Glycine GLY G

Histidine HIS H Isoleucine ILE I

Leucine LEU L Lysine LYS K

Methionine MET M Phenylalanine PHE F

Proline PRO P Serine SER S

Threonine THR T Tryptophan TRP W

Tyrosine TYR Y Valine VAL V

START = Initiation Signal (signifies the beginning of apolypeptide chain)

STOP = Termination Signal (signifies the end of a

polypeptide chain)


51/54



51

Hereditary Hemochromatosis WorksheetThisworksheetprovidesquestionstobeansweredasyoucompletetheactivitiesintheGene

GatewayWorkbook.

Questions for Activity 1

1) Whatistheofficialgenesymbolofthehereditaryhemochromatosisgene?

2) Whichallelicvariant(geneticmutation)mostcommonlycauseshereditaryhemochromatosis?

3) Whataresomecharacteristicsofhereditaryhemochromatosis?Howisittreated?


1) Onthediagramtotheright,markthegeneralregionwhere

theHFEgenecanbefoundonchromosome6.

2) Abouthowmanygenesareonchromosome6?

3) HowlongistheDNAsequenceforchromosome6?


52/54



52


1) UsingthesummaryfromtheEntrezGenerecordfortheHFEgene, brieflydescribethe

functionofthegenesproteinproduct.

Use the GenBank sequence record Z92910.1 to answer questions 24.

2) IntheFeaturessectionofrecordZ92910.1,selectthe genelink.Howmanybasepairs(bp)are

inthegenomicsequenceoftheHFEgene?

3) ScrollthroughtheFeaturessectionofthe genesequenceinZ92910.1.Howmanyexonshave

beenidentifiedinthissequence?

4) ReturntothemainrecordZ92910.1.Selectthe CDSlink.Howmanybasepairsareinthecodingsequence?


1) Howmanyaminoacids(AA)areinthecompleteHFEprotein?

2) InwhatpartofthecellistheHFEproteinlocated?

3) WhattypeoftissuedoesnotexpresstheHFEprotein?

4) Iscysteine282foundontheextracellularorcytoplasmicsideoftheHFEprotein?

5) Whatisthenumberofthecysteineresiduethatformsadisulfidebondwithcysteine282?

6) Whatkindofsecondarystructuralelementcontainscysteine282:alphahelix,turn,orbeta

strand?


53/54



53

7) UsingtheBLASTsearchresults,listthefirst10non-humanorganismsthathaveproteins

similartothehumanHFEproteinsequence.Includethepercentidentityscorewitheach

organismyoulist,andorderthelistfromhighesttolowestidentityscore.Skipanyhuman

entries,anddonotlistanyorganismmorethanonce.

OrganismName Identity%

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.


54/54



1. ExaminetheaminoacidsequenceforthehumanHFEproteinfromtheUniProtProtein

Knowledgebase(shownbelow).Findcysteine282,theaminoacidthatisreplacedbytyrosine

intheCYS282TYRmutation.RefertotheTableofStandardGeneticCodeonPage50forhelp

withthesingle-letteraminoacidabbreviations.

10 20 30 40 50 60

| | | | | |

MGPRARPALL LLMLLQTAVL QGRLLRSHSL HYLFMGASEQ DLGLSLFEAL GYVDDQLFVF

70 80 90 100 110 120

| | | | | |

YDHESRRVEP RTPWVSSRIS SQMWLQLSQS LKGWDHMFTV DFWTIMENHN HSKESHTLQV

130 140 150 160 170 180

| | | | | |

ILGCEMQEDN STEGYWKYGY DGQDHLEFCP DTLDWRAAEP RAWPTKLEWE RHKIRARQNR

190 200 210 220 230 240

| | | | | |

AYLERDCPAQ LQQLLELGRG VLDQQVPPLV KVTHHVTSSV TTLRCRALNY YPQNITMKWL

250 260 270 280 290 300

| | | | | |

KDKQPMDAKE FEPKDVLPNG DGTYQGWITL AVPPGEEQRY TCQVEHPGLD QPLIVIWEPS

310 320 330 340

| | | |

PSGTLVIGVI SGIAVFVVIL FIGILFIILR KRQGSRGAMG HYVLAERE

2. ComparetheaminoacidsequenceabovewiththeHFEsequencedetailsprovidedforPDB

structure1A6Z.Inquestion1,underlinetheportionoftheaminoacidsequenceincludedin

thePDBstructure.

3. Howmanydisulfidebondsarepresentinthehereditaryhemochromatosisprotein?

4. WhyisthecysteineresidueaffectedintheCYS282TYRmutationimportant?

bio in for matics

Documents

govge bio

human genomelandmarks

free copy ofthe poster

govb mailto

gene gateway website

variety of websites