medicalmedical genetics...

MedicalMedical GeneticsGenetics

CytogeneticsCytogeneticsCy gCy g

HumanHuman chromosomechromosome

ChromosomalChromosomal aberrationaberrationChromosomalChromosomal aberrationaberration

ChCh didiChromosomeChromosome diseasedisease

Hi tHi t ff C t tiC t tiHistoryHistory ofof CytogeneticsCytogenetics

DarkDark AgesAgesPainterPainter TSTS.. StudiesStudies inin mammalianmammalian spermatogenesisspermatogenesis.. IIII..TheThe spermatogenesisspermatogenesis ofof manman.. JJ ExpExp ZoolZool.. 19231923;; 3737:: 291291--336336

HypotonicHypotonic PeriodPeriodDarkDark AgesAges

HsuHsu TCTC.. MammalianMammalian chromosomeschromosomes inin vitrovitro.. II.. TheThe

HypotonicHypotonic PeriodPeriod

karyotypekaryotype ofof manman.. JJ HeredHered.. 19521952;; 4343:: 167167--172172TjioTjio JHJH,, LevanLevan AA.. TheThe chromosomechromosome numbernumber ofof manman..AmAm JJ ObstetObstet GynecolGynecol.. 19561956;; 130130:: 723723--724724

T. C. Hsu (T. C. Hsu (徐道觉徐道觉,April 17,1917,April 17,1917--July,9,2003)July,9,2003)

TjioTjio && LevanLevan ((19561956))

Sh dSh d thth ll hh hh bb tt bb 4646ShowedShowed thethe normalnormal humanhuman chromosomechromosome numbernumber toto bebe 4646..

HypotonicHypotonic PeriodPeriodDarkDark AgesAges

TrisomyTrisomy PeriodPeriodHypotonicHypotonic PeriodPeriod

yyLejeuneLejeune JJ,, etet alal.. EtudeEtude desdes chromosomeschromosomes somatiquessomatiques dedeneufneuf enfantsenfants mongoliensmongoliens.. GG.. RR.. AcadAcad.. SciencesSciences.. 19591959;;gg ;;248248:: 17211721--17221722FordFord CECE,, etet alal.. AA sexsex chromosomalchromosomal anomalyanomaly inin aa casecase ofofgonadalgonadal dysgenesisdysgenesis (Turner's(Turner's syndrome)syndrome).. LancetLancet.. 19591959;;11:: 711711--713713JACOBJACOB PAPA,, etet alal.. AA casecase ofof humanhuman intersexualityintersexualityhavinghaving aa possiblepossible XXYXXY sexsex determiningdetermining mechanismmechanism..NatureNature 19591959;; 183183:: 302302 303303NatureNature.. 19591959;; 183183:: 302302--303303..

JérômeJérôme LejeuneLejeune ((19591959))

HypotonicHypotonic PeriodPeriodDarkDark AgesAgesHypotonicHypotonic PeriodPeriodTrisomyTrisomy PeriodPeriodBandingBanding EraEra

yy

CasperssonCaspersson TT,, etet alal.. DifferentialDifferential bandingbanding ofof alkylatingalkylatingfluorochromesfluorochromes inin humanhuman chromosomeschromosomes.. ExpExp CellCell ResRes..19701970;; 6060:: 315315--319319

HypotonicHypotonic PeriodPeriodDarkDark AgesAgesHypotonicHypotonic PeriodPeriodTrisomyTrisomy PeriodPeriod

l ll lBandingBanding EraEra

yy

MolecularMolecular EraEraParduePardue MLML,, etet alal.. MolecularMolecular hybridizationhybridization ofof radioactiveradioactiveDNADNA toto thethe DNADNA ofof cytologicalcytological preparationspreparations.. ProcProc.. NatlNatl..AcadAcad.. SciSci.. USAUSA.. 19691969;; 6464:: 600600 -- 604604PinkelPinkel DD,, etet alal.. CytogeneticCytogenetic analysisanalysis usingusing quantitative,quantitative,highhigh--sensitivity,sensitivity, fluorescencefluorescence hybridizationhybridization.. ProcProc.. NatlNatl..highhigh sensitivity,sensitivity, fluorescencefluorescence hybridizationhybridization.. ProcProc.. NatlNatl..AcadAcad.. SciSci.. USAUSA.. 19861986;; 8383:: 29342934--29382938..

HistoryHistory ofof CytogeneticsCytogeneticsyy y gy g

19101910 19261926 MM ThTh ThTh ff thth GG b i ib i i19101910--19261926 Morgan,Morgan, TheThe TheoryTheory ofof thethe Gene,Gene, ----beginningbeginningofof cytogeneticscytogenetics

19231923 P i tP i t 22 4848 XXXX XYXY19231923 Painter,Painter, 22n=n=4848,, XXXX oror XYXY19521952 HsuHsu TT C,C, hypohypo--osmoticosmotic preparingpreparing19561956 TjiTji && LL 22 4646 b i ib i i ff hh19561956 TjioTjio && Levan,Levan, 22n=n=4646,,——beginningbeginning ofof humanhuman

cytogeneticscytogeneticsii19591959 Lejeune,Lejeune, TrisomyTrisomy--2121

19601960 DenverDenver systemsystem19701970 CasperssonCaspersson && QQ--bandingbandingFISH(FISH(19861986),), microdissectionmicrodissection technique(technique(19891989),), connectconnect

toto cytogeneticscytogenetics —— molecularmolecular cytogeneticscytogenetics

2 1 1 H Ch ti2.1.1 Human Chromatin

☺☺DefinitionDefinition

C tC t☺☺ComponentsComponents

☺☺ Structure Structure ☺☺ Structure Structure

☺☺CompactionCompactionpp

2 1 1 Human Chromatin D fi iti2.1.1 Human Chromatin -Definition

Chromatin is the proteinChromatin is the protein--DNA DNA l i hi h ti t i l l i hi h ti t i l complex in which genetic material complex in which genetic material

exist in interphase nucleusexist in interphase nucleusx px p

DDuring metaphase, chromatin uring metaphase, chromatin ‘‘condensescondenses’’ into chromosomesinto chromosomes

2.1.1 Human Chromatin -Definition

EuchromatinEuchromatinLightly stainedLightly stained

Chromatin Loose structureLoose structure

Genetically activeGenetically active

Heterochromatin

Genetically activeGenetically active

Deeply stainedDeeply stained

Compact structureCompact structure

Genetically silentGenetically silent


Constitutive HeterochromatinAlways highly condensedAlways highly condensed

Enriched at centromeres and Enriched at centromeres and Heterochromatin

Enriched at centromeres and Enriched at centromeres and telomerestelomeres

Facultative Heterochromatin

Y chromatinY chromatin

Facultative HeterochromatinSpecifically inactivatedSpecifically inactivated--specific timesspecific timesspecific timesspecific times

Switch between two statesSwitch between two states

X chromatin X chromatin

2.1.1 Human Chromatin -Componentsp

Chromatin is the proteinChromatin is the protein--DNA DNA l i hi h ti t i l l i hi h ti t i l complex in which genetic material complex in which genetic material

exist in interphase nucleusexist in interphase nucleusx px p

ComponentsComponents--DNA , associated DNA , associated proteins, and small amount of RNAproteins, and small amount of RNA

2 1 1 Human Chromatin Components2.1.1 Human Chromatin -Components

DNA: 24 different kinds of moleculesDNA: 24 different kinds of molecules

Two types of proteinsTwo types of proteinsHistonesHistonesHistonesHistones

Small, wellSmall, well--defined, highly conserved basicdefined, highly conserved basicNonNon--histone chromosomal proteinshistone chromosomal proteinsNonNon--histone chromosomal proteinshistone chromosomal proteins

–– Large numberLarge number–– DiverseDiverse–– Mostly not well characterizedMostly not well characterized

2 1 1 Human Chromatin Components2.1.1 Human Chromatin -Components

HistonesSmall proteins:Small proteins:

Histones

Arginine or lysine rich: positively chargedArginine or lysine rich: positively charged

i h i l h d Ai h i l h d AInteract with negatively charged DNAInteract with negatively charged DNA

Can be extensively modified(Can be extensively modified(--make them make them Can be extensively modified(Can be extensively modified( make them make them less positively chargedless positively charged

Five types: H1, H2A, H2B, H3, H4Five types: H1, H2A, H2B, H3, H4

2 1 1 Human Chromatin Structure2.1.1 Human Chromatin -Structure

N f b f hNucleosomes: fundamental subunits of chromatin

Electronic Microscope

Evidences

Nuclease digestion

2 1 1 Human Chromatin Structure2.1.1 Human Chromatin -Structure

EM of chromatin shows presence ofnucleosomes as “beads on a string”

Nucleosome Structure

Contains ~ 200 bp of DNAContains ~ 200 bp of DNA

Nucleosome Structure

Contains ~ 200 bp of DNAContains ~ 200 bp of DNA

DNA wrapped around a core of eight DNA wrapped around a core of eight pp gpp gbasic proteins called histones basic proteins called histones –– histone histone octamer octamer

Linker DNALinker DNA linking nucleosome linking nucleosome Linker DNALinker DNA-- linking nucleosome linking nucleosome togethertogethergg

H1 binds to linker DNAH1 binds to linker DNA

The Histone OctomerThe Histone OctomerFour proteins: H2A, H2B, H3, and H4Four proteins: H2A, H2B, H3, and H4p , , ,p , , ,H3 and H4 are arginine rich and highly H3 and H4 are arginine rich and highly

ddconservedconservedH2A and H2B are slightly enriched in lysineH2A and H2B are slightly enriched in lysineg y yg y yBoth arginine and lysine are basic amino acids Both arginine and lysine are basic amino acids making the histone proteins both basic andmaking the histone proteins both basic andmaking the histone proteins both basic and making the histone proteins both basic and positively chargedpositively chargedThe octomer is made of two copies of each The octomer is made of two copies of each proteinproteinp o ep o e

The Fifth Histone H1The Fifth Histone, H1

A fifth protein, H1, is part of the A fifth protein, H1, is part of the nucleosome, but resides outside the nucleosome, but resides outside the ,,octomeroctomerH1 i b t ti dH1 i b t ti dH1 varies between tissue and H1 varies between tissue and organisms and seems to stick to the organisms and seems to stick to the 19 bases attached to the end of the 19 bases attached to the end of the core sequence core sequence qq

Hi tHi t HH11 ll i t ti t t llHistoneHistone HH11 playsplays anan importantimportant roleroleinin organizingorganizing thethe chromatinchromatin fiberfiber

AA PortionPortion ofof thetheNucleosomeNucleosomeNucleosomeNucleosomeLookingLooking fromfrom thethetoptop andand showingshowingthethe DNADNA rappedrappedthethe DNADNA wrappedwrappedaroundaround thetheHistonesHistones

2 1 1 Human Chromatin Compaction2.1.1 Human Chromatin -Compaction

Human DNAHuman DNA’’s total length is ~2 meters! s total length is ~2 meters! Human DNAHuman DNA s total length is 2 meters! s total length is 2 meters!

This must be packaged into a nucleus that This must be packaged into a nucleus that is about 5 micrometers in diameter is about 5 micrometers in diameter

This represents a compression of more This represents a compression of more This represents a compression of more This represents a compression of more than 100,000 times !than 100,000 times !

It is made possible by wrapping the DNA It is made possible by wrapping the DNA around nucleosomes and then packing around nucleosomes and then packing around nucleosomes and then packing around nucleosomes and then packing these in helical filamentsthese in helical filaments


P ki ti i th l th f th DNA di id d bP ki ti i th l th f th DNA di id d bPacking ratio is the length of the DNA divided by Packing ratio is the length of the DNA divided by the length into which it’s packagedthe length into which it’s packagedSmallest human chromosome (21) has 4x10Smallest human chromosome (21) has 4x1077 bp bp of DNAof DNAof DNAof DNAEquivalent to 14mm of extended DNAEquivalent to 14mm of extended DNAIn most condensed state the chromosome is In most condensed state the chromosome is about 2mm longabout 2mm longggPacking ratio = 14000/2 = 7000Packing ratio = 14000/2 = 7000


There are many levels of DNA compactionThere are many levels of DNA compactionThere are many levels of DNA compactionThere are many levels of DNA compaction

First level of DNA compaction is to form First level of DNA compaction is to form nucleosomenucleosome

Nucleosomes become helically arranged Nucleosomes become helically arranged Nucleosomes become helically arranged Nucleosomes become helically arranged into a 30nM chromatin fiber (solenoid)into a 30nM chromatin fiber (solenoid)

The fiber is super coiled by attachment to a The fiber is super coiled by attachment to a nonnon--histone protein scaffold (not histone protein scaffold (not nonnon histone protein scaffold (not histone protein scaffold (not completely understood)completely understood)

30 nm fiber30 nm fiber

Packaging DNAPackaging DNAPackaging DNAPackaging DNAHistoneHistoneoctomer

Histone proteins

2DNA Helix 2 nm

Packaging DNAPackaging DNAPackaging DNAPackaging DNAHistone11 nm Histoneoctomer

Histone proteins

Nucleosome2DNA Helix 2 nm

Packaging DNAPackaging DNAPackaging DNAPackaging DNA

Histone H1

Packaging DNAPackaging DNAPackaging DNAPackaging DNA11 nm“Beads on

ia string”

30 nm LoopedTight helical

fib

Looped Domains200 nm

fiber

Protein scaffoldProtein scaffold

Packaging DNAPackaging DNAPackaging DNAPackaging DNA11 nm

N lMetaphase

Ch

Nucleosomes

Chromosome700 nm30 nm

200 nm Looped DomainsTight helical fiber

Protein scaffold2 nm

Protein scaffoldDNA Helix

2 nm – DNA double helix

10 nm – Nucleosome (10 nm fiber

30 nm – 30 nm Fiber

300 nm – Loops I

700 nm – Loops II

1400 nm – chromosome1400 nm chromosome

2.1.2 Metaphase Chromosomesp

C ll l

Interphase - replication

Cell cycle Prophase - condensed

Dividing stages

A h t d

Metaphase

Anaphase - seperated

TelophaseTelophase

Metaphase chromosome Metaphase chromosome ppPreparationPreparation

Cultured cells are arrested at metaphase Cultured cells are arrested at metaphase by adding colchicineby adding colchiciney gy g

This is when cells are most condensed This is when cells are most condensed and easiest to identifyand easiest to identifyyy

Metaphase chromosome Metaphase chromosome ppPreparationPreparation

Arrested cells are broken openArrested cells are broken openMetaphase chromosomes are fixed and stainedMetaphase chromosomes are fixed and stainedChromosomes are photographed through Chromosomes are photographed through p g p gp g p gmicroscopemicroscopePhotograph of chromosomes is cut up and Photograph of chromosomes is cut up and Photograph of chromosomes is cut up and Photograph of chromosomes is cut up and arranged to form karyotype diagramarranged to form karyotype diagram

Then you can examine the preparations under microscope.

Chromosome MorphologyChromosome Morphologyp gyp gy

Each chromosome has a primary constriction called a centromere. The centromere can be found almost anywhere along thefound almost anywhere along the chromosome—near the top, near the middle, or in-between, but not at the very ends of the , ychromosome.

The centromere divides the chromosome into two "arms " and unless the centromere is righttwo arms, and unless the centromere is right in the middle, there will be a short arm, labeled p, and a long arm, labeled q.p, g , q

IdeogramIdeogram::IdeogramIdeogram::

1960, Denver System1960, Denver System

Autosomes were named in order of their Autosomes were named in order of their

size (1size (1~22)~22) and centromere position, and and centromere position, and

were divided into 7 groups (A~G).were divided into 7 groups (A~G).

X chromosome belongs to group C, and Y X chromosome belongs to group C, and Y

chromosome belongs to group Gchromosome belongs to group Gchromosome belongs to group G.chromosome belongs to group G.

IdeogramIdeogram::AA：：11～～33..

11,,33 metacentricmetacentric;; 22 submetacentricsubmetacentric;;11qq secondarysecondary constrictionconstriction;;largestlargestlargestlargest

BB：：44,,55..submetacentricsubmetacentric;;

CC：：66～～1212,X,X..,,submetacentricsubmetacentric;;99qq secondarysecondary constrictionconstriction;;

DD：：1313～～1515..acrocentricacrocentric;;acrocentricacrocentric;;satellitessatellites

EE：：1616～～1818..1616 metacentric,metacentric, 1616qq secondarysecondaryqq yy

constrictionconstriction;;1717,,1818 submetacentricsubmetacentric

FF：：1919,,2020metacentricmetacentricmetacentricmetacentric

GG：：2121,,2222,Y,Yacrocentricacrocentric;;2121,,2222 satellitessatellites;; 2222>>2121;;smallestsmallest

IdeogramIdeogram ofof humanhuman chromosomeschromosomes showingshowing thethe GiemsaGiemsabandingbanding patterns,patterns, arrangedarranged andand numberednumbered accordingaccordinggg p ,p , gg ggtoto thethe ParisParis classificationclassification ofof 19711971

2 1 4 Chromosome bandingStaining with a dye called Giemsa chromosomes will

2.1.4 Chromosome banding

Giemsa chromosomes will appear to have between 400 or more alternating light and dark b d Thi i ll d Gibands. This is called Giemsa banding or G-banding.Centromere

These bands, which can vary in intensity from very light to y y galmost solid black, have been numbered by scientists and provide location markers withprovide location markers with which breakpoints and small structural aberrations can be identifiedidentified.

BandingBandingBandingBandingbandband

AA tttt ff li htli ht dd d kd k b db d ll ththAA patternpattern ofof lightlight andand darkdark bandsbands alongalong thethearmsarms ofof chromosomeschromosomes showedshowed byby usingusing specialspecialstainingstaining techniquestechniques

S ifiS ifiSpecificSpecificStableStable

landmarklandmarkAA distinctivedistinctive signsign onon chromosomechromosomecentromerescentromeres 、、 telomerestelomeres 、、 somesome distinctivedistinctive

bandsbands

regionregionggthethe regionregion betweenbetween twotwo neighborhoodneighborhood

landmarkslandmarks

NamingNaming ofof bandsbandsNamingNaming ofof bandsbands

RegionRegion——namednamed fromfromcentromerecentromere toto telomeretelomerecentromerecentromere toto telomeretelomereBandsBands inin thethe samesame regionregion----namednamed fromfrom centromerecentromere toto

lltelomeretelomereLandmarkLandmark——asas thethe firstfirstbandband ofof thethe furtherfurther regionregionbandband ofof thethe furtherfurther regionregion

bandlandmark

regionregion

• The standard nomenclature for banding of human chromosomes

1. the chromosome number;

2. the arm symbol (p or q);

3 the "region" number along that arm;3. the "region" number along that arm;

4. the band number within that region. g

DescriptionDescription ofof bandsbands

ChromosomeChromosome numbernumber armarmb lb l ii bb b db dsymbolsymbol regionregion numbernumber bandband

numbernumber11pp3232 11qq2323

1p32

11pp3232 11qq2323

1p11

1 11

The band divided by centromere were respectively named as p11 and q11

1q23

1q11respectively named as p11 and q11.

1q23

Sometimes, sub-bands, separated by a decimal point from the band designation are also noted

Centromere

designation, are also noted.

For example, the location of a t (ABL1) i t dprotooncogene (ABL1) associated

with chronic myeloid leukemia, sub-band 1 of band 4 in region 3 ofsub-band 1 of band 4 in region 3 of the long arm of chromosome 9.

Though microscopic in size a sub 9 34 1Though microscopic in size, a sub-band can contain tens of individual genes.

9q34.1

gKaryotyping using Giemsa Bandinghttp://www.pathology.washington.edu/Cytogallery/Frame_setup/cytoimages.html

BandingBanding techniquestechniquesBandingBanding techniquestechniques

☺☺QQ bandingbanding☺☺QQ--bandingbanding☺☺GG--bandingbanding☺☺RR--bandingbanding☺☺CC--bandingbandinggg☺☺TT--bandingbanding☺☺NN bandingbanding☺☺NN--bandingbanding☺☺SCESCE stainingstaining☺☺HighHigh resolutionresolution GG bandingbanding☺☺OtherOther stainingstaining andand bandingbanding techniquestechniquesgg gg qq

QuinacrineQuinacrine banding,banding, QQ--BandingBanding：：

AA techniquetechnique thatthat stainsstainschromosomechromosome preparationspreparations withwithQuinacrineQuinacrine mustardmustard oror similarsimilarfluorescentfluorescent dyedye ..ThisThis resultsresults inin aa patternpattern ofof lightlight andandThisThis resultsresults inin aa patternpattern ofof lightlight andanddarkdark bandsbands thatthat isis uniqueunique forfor eacheachhumanhuman chromosomechromosome andand allowsallows thetheunequivocalunequivocal definitiondefinition ofof eacheachchromosomechromosome..EarliestEarliest usedusedEarliestEarliest usedused..traitstraits

11.. veryvery clearclear22.. can’tcan’t bebe keptkept forfor aa longlong timetime33.. needsfluorescentneedsfluorescent microscopemicroscope

2012年4月6日星期五 Medical Genetics

GiemsaGiemsa--stainedstained banding,banding, GG--BandingBandingGG g,g, GG gg

AA techniquetechnique involvesinvolves gentlegentle trypsintrypsint t tt t t ff hhtreatmenttreatment ofof chromosomechromosomepreparationspreparations followedfollowed byby stainingstainingwithwith GiemsaGiemsa

traits:

withwith GiemsaGiemsa

traits:•1. Very clear, too•2. Can be kept for a long time. Ca be ept o a o g t e•3. Needs light microscope only

Most widely used


G banded ChromosomeG-banded Chromosome

telomere

centromereG-pale bands

G d k b d GC-richmany genes

G-dark bandsAT-rich

few genes

telomere

f g

light band in Q-banding

dark band in G-banding

dark band in Q-banding

Staining with Giemsa chromosomes will appear to have 350~550

light band in G-banding

w appea to ave 350 550alternating light and dark bands.

ReversedReversed GG--band,band, RR--BandingBanding

PretreatedPretreated withwith saltsalt solutionsolution thenthen stainedstained byby GiemsaGiemsa..ThiThi ltlt ii tttt ff dd b db d tt GG b db dThisThis resultsresults inin aa patternpattern ofof reversedreversed bandsbands toto GG--bandsbands..

light band in R-banding

dark band in G-banding

dark band in R-banding

light band in G-banding


For examining the light bands in G-banding, also terminal of chromosomes.

G-banding R-banding

CConstitutiveonstitutive heterochromatinheterochromatin bandingbanding,,C t iC t i b dib di CC BB didioror CentromericCentromeric banding,banding, CC--BBandinganding

DenaturedDenatured withwith saturatedsaturated Ba(OH)Ba(OH)22 thenthenDenaturedDenatured withwith saturatedsaturated Ba(OH)Ba(OH)22 thenthenstainedstained withwith GiemsaGiemsa..

SpeciallySpecially stainsstains centromerescentromeres andandt i tit i ticonstrictionsconstrictions..


Notice the large heterochromatic regions on chromosomes 1 and 9.

TelomereTelomere bandingbanding oror TerminalTerminal bandingbandingTelomereTelomere bandingbanding oror TerminalTerminal banding,banding,TT--BandingBanding

SpeciallySpecially stainsstains telomerestelomeres..

Study of telomeres can help us to obtain the structure and dynamics of chromosomes.


SilverSilver stainingstaining ofof NORNOR (( nucleolarnucleolar organizingorganizing regionsregions))SilverSilver stainingstaining ofof NORNOR (( nucleolarnucleolar organizingorganizing regionsregions)),,NN--BandingBanding

NORs appear darkly stained with AgNO3-As.

2012年4月6日星期五 Medical GeneticsStudy for distribution and activation of 18S+28SrDNA

SCESCE ( i t r( i t r hr tidhr tid x h )x h ) t i it i iSCESCE (sister(sister chromatidchromatid exchange)exchange) stainingstaining

ExchageExchage betweenbetween twotwo sistersister chromatidschromatidsExchageExchage betweenbetween twotwo sistersister chromatidschromatidsduringduring duplicationduplication ofof chromosomeschromosomes----SCESCE

BrdU 1 exchage

BrdUBrdU

2 exchages

1st cycle 2nd cycle

HighHigh--resolutionresolution bandingbanding techniquetechniqueAA newernewer techniquetechnique allowingallowing

examinationexamination ofof chromosomeschromosomes ininprometaphase,prometaphase, atat whichwhich timetime thethechromosomeschromosomes areare moremore extendedextended thanthaninin metaphasemetaphaseinin metaphasemetaphase..

MoreMore thanthan 850850 bandsbands perper haploidhaploid setsetcancan bebe distinguisheddistinguished byby thisthis techniquetechnique..


OtherOther stainingstaining andand bandingbanding techniquestechniquesOtherOther stainingstaining andand bandingbanding techniquestechniques

ShowShow ofof fragilefragile sitessites onon chromosomeschromosomesShowShow ofof fragilefragile sitessites onon chromosomeschromosomesFISHFISHSKYSKYSKYSKY

fragilefragile sitesite::f gf g

SitesSites whichwhich appearappear asas apparentapparent breaksbreaks ororii i li l ii (( b d)b d) ffgapsgaps inin specialspecial regionregion (or(or band)band) ofof

chromosomeschromosomes..FragileFragile XX isis associatedassociated toto mentalmentalFragileFragile XX isis associatedassociated toto mentalmental

retardationretardation (MR)(MR);;AutosomalAutosomal fragilefragile sitessites areare associatedassociated toto

cancercancer..

When peripheral blood lymphocyte cultures are deprived f f l t th idiof folate, or thymidine

metabolism is perturbed, these sites can be observed.


sites can be observed.

Chromosome PaintingChromosome PaintingSpectral Karyotyping, SKY Spectral Karyotyping, SKY

Chromosomal Polymorphism

Polymorphism:h it bl diff b ta heritable difference between

individuals in the same speciesp

PolymorphismPolymorphism

Presence in the general population of 2 or Presence in the general population of 2 or l i i h f fl i i h f fmore alternative variants; the frequency of more alternative variants; the frequency of

each of which cannot be maintained by each of which cannot be maintained by recurrent new mutations alone. recurrent new mutations alone. Polymorphism should follow the rulesPolymorphism should follow the rulesPolymorphism should follow the rules Polymorphism should follow the rules below:below:–– Having no remarkable heritable effect and Having no remarkable heritable effect and

cannot result in pathologic reactioncannot result in pathologic reaction–– According to Mendelian inheritanceAccording to Mendelian inheritance–– The rarest variant needs to have a frequency The rarest variant needs to have a frequency e a est va a t eeds to ave a eque cye a est va a t eeds to ave a eque cy≥≥1%1%

ForFor exampleexample::ABOABO bloodblood groupsgroupsABOABO bloodblood groupsgroupsImmunoglobulinsImmunoglobulinsMinorMinor variantsvariants inin chromosomechromosomestructurestructureDNADNA sequencesequence polymorphismpolymorphism

E tlE tl h ldh ld llll thth i ti tExactlyExactly wewe shouldshould callcall thesethese variantsvariantschromosomechromosome heteromorphismheteromorphism..pp

CommonCommon positionspositions andand formsforms ofofhh h t hih t hichromosomechromosome heteromorphismheteromorphism

SizeSize ofof YY longlong armarmSizeSize ofof YY longlong armarm

AboutAbout 1010%% ofof clinicallyclinically normalnormal malesmales havehave aayylongerlonger oror shortshort YY..

SizeSize ofof centromericcentromeric heterochromatinheterochromatinSizeSize ofof centromericcentromeric heterochromatinheterochromatin

relativelyrelatively frequentfrequent forfor chromosomechromosome 11,,99,,1616


SatelliteSatellite——presencepresence oror absence,absence, sizesize

mainlymainly onon 1313 1414 1515 2121 andand 2222mainlymainly onon 1313,,1414,,1515,,2121 andand 2222


ShortShort armsarms andand satellitessatellites ofof acrocentricacrocentricchromosomeschromosomes

LengthLength ofof shortshort armsarmsLengthLength ofof shortshort armsarmsSatelliteSatellite——presencepresence oror absence,absence, sizesizeFluorescenceFluorescence andand otherother stainingstaining variationvariationgg

SecondarySecondary constrictionconstriction ofof chromosomechromosome 11,, 99 andand 1616PresencePresence oror absence,absence, lengthlength

SomeSome autosomesautosomesSomeSome autosomesautosomesCentromereCentromere sizesize andand fluorescencefluorescence intensityintensity ofofchromosomechromosome 33,, 44

Chromosomal Chromosomal heteromorphismheteromorphismheteromorphismheteromorphism

A li tiA li tiApplication:Application:IndividualIndividual IdentificationIdentificationDetectingDetecting thethe originorigin ofof extraextra chromosomeschromosomesDetectingDetecting thethe originorigin ofof extraextra chromosomeschromosomesDetectingDetecting thethe originorigin ofof cellscellsGG iiGeneGene mappingmapping

GG ii D ffD ff bl dbl dGeneGene mappingmapping----DuffyDuffy bloodblood groupgroup

li k dli k d i h iti h itsexsex--linkedlinked inheritanceinheritancehemophiliahemophilia andand colorcolor--blindnessblindnesspp

InIn 19681968,, thethe firstfirst autosomalautosomal assignmentassignment ofofli kli k ddlinkagelinkage waswas mademade..Donahue,Donahue, anan associateassociate ofof McKusick,McKusick,observedobserved aa peculiarpeculiar microscopicallymicroscopically--visiblevisible stretchstretch ofof chromatinchromatin onon hishis ownownvisiblevisible stretchstretch ofof chromatinchromatin onon hishis ownownlargestlargest chromosomechromosome (Chromosome(Chromosome ##11))..

AA pairpair ofof numbernumber 11 chromosomeschromosomes fromfrom twotwo differentdifferentpaipai ofof numbenumbe ch omosomesch omosomes f omf om twotwo diffe entdiffe entindividualsindividuals

TheseThese studies,studies, donedone beforebefore thethe adventadvent ofofchromosomeschromosomes bandingbanding detectdetect aa remarkableremarkable increaseincreasechromosomeschromosomes banding,banding, detectdetect aa remarkableremarkable increaseincreaseinin lengthlength belowbelow thethe centromere,centromere, referredreferred toto asas aaheteromorphism,heteromorphism, inin oneone ofof thethe pairpair ofof chromosomeschromosomes

thth l ftl ftonon thethe leftleft..TheThe chromosomeschromosomes onon thethe rightright showshow thethe moremore usualusualmorphologymorphology..


LookingLooking atat aa numbernumber ofof bloodbloodLookingLooking atat aa numbernumber ofof bloodbloodfactorsfactors DonahueDonahue foundfound thatthat inin bothbothhimselfhimself andand hishis relati esrelati es anan alleleallele ofofhimselfhimself andand hishis relatives,relatives, anan alleleallele ofofthethe DuffyDuffy bloodblood factorfactor waswas linkedlinked totothisthis observableobservable physicalphysical changechange inintheirtheir chromosomechromosome ##11..

TheThe pedigreepedigree showsshows thethe patternpattern ofofinheritanceinheritance forfor thethe DuffyDuffy bloodblood groupgroupinheritanceinheritance forfor thethe DuffyDuffy bloodblood groupgroupandand thethe chromosomechromosome 11 heteromorphismheteromorphism

Ⅰ

ⅡⅡ

Ⅲ

ab bb bb ab ab ab bbbb ab

Ⅲ

Ⅳ

ab ab abaa aa bbbbbb

Ⅳ

aa ab abab

Presence of the a and bheteromorphism on

chromosome 1The Duffy blood group genotypes

ThTh "D ff ""D ff " i di d tt ChCh ##11ThusThus "Duffy""Duffy" waswas assignedassigned toto ChromosomeChromosome ##11..

2.2Mitosis and Meiosis

MITOSISMITOSISMITOSISMITOSIS

MitosisMitosisMitosisMitosis

The form of The form of cell divisioncell division by which a by which a eukaryotic eukaryotic somatic cellsomatic cell duplicates.duplicates.yy pp

MitosisMitosis is is asexual reproduction.asexual reproduction.MitosisMitosis is is asexual reproduction.asexual reproduction.

Cell divisionCell division is the continuation of life is the continuation of life based on the based on the reproduction of cellsreproduction of cells..pp

MitosisMitosisMitosisMitosis

Liver CellLiver Cell 2n=46

twoidentical

2n=46identicaldaughtercells

2n=46 Liver Cell

Cell CycleCell CycleCell CycleCell Cycle

The The dividingdividing and and nonnon--dividingdividing stages in the stages in the life of a celllife of a celllife of a cell.life of a cell.Phases:Phases:

11 I t hI t h th d DNA th d DNA 1.1. InterphaseInterphase: : growth and DNA growth and DNA replicationreplication

22 P hP h2.2. ProphaseProphase3.3. MetaphaseMetaphase4.4. AnaphaseAnaphase5.5. TelophaseTelophase

Mitotic divisionMitotic division

Cell CycleCell CycleCell CycleCell Cycle

Sphase

G1 G2interphase

Mitosis-prophase-metaphasemetaphase-anaphase-telophase

InterphaseInterphaseInterphaseInterphase

Comprises about Comprises about 90%90% of the of the cell cyclecell cycle..Cellular growthCellular growth::Cellular growthCellular growth::

a. a. protein synthesisprotein synthesisb b b li i i ib li i i ib. b. metabolic activitiesmetabolic activitiesc. c. DNADNA synthesissynthesis

Made up of Made up of three phasesthree phases::1. G1. G11 phasephase2. S phase2. S phase3. G3. G22 phasephase

Chromosome DNA

46 46

46 9246 92

46 46

Question:Question:Question:Question:

AA cellcell containingcontaining 2020 chromosomeschromosomes atatAA cellcell containingcontaining 2020 chromosomeschromosomes atatthethe beginningbeginning ofof mitosismitosis would,would, atat itsitscompletioncompletion prod ceprod ce cellscells containingcontainingcompletion,completion, produceproduce cellscells containingcontaininghowhow manymany chromosomeschromosomes eacheach??

AAAnswer:Answer:

2020 chromosomeschromosomes

MEIOSISMEIOSIS

MeiosisMeiosisMeiosisMeiosis

The form of The form of cell divisioncell division by which by which gametes, gametes, with with halfhalf the number of the number of chromosomes,chromosomes, are are produced.produced.

Di l id (2 ) Di l id (2 ) h l id ( )h l id ( )Diploid (2n) Diploid (2n) →→ haploid (n)haploid (n)

M i iM i i i i l d til d tiMeiosisMeiosis is is sexual reproduction.sexual reproduction.

T di i iT di i i (( i i I i i I d d i i IIi i II))Two divisionsTwo divisions ((meiosis I meiosis I and and meiosis IImeiosis II).).

Prophase IProphase IProphase IProphase I

Longest and most complex phase (90%)Longest and most complex phase (90%)Longest and most complex phase (90%).Longest and most complex phase (90%).

ChromosomesChromosomes condense.condense.

SynapsisSynapsis occurs: occurs: homologous chromosomes homologous chromosomes come come together to form a together to form a tetradtetrad..together to form a together to form a tetradtetrad..

TetradTetrad is two is two chromosomeschromosomes or four or four chromatidschromatids( i t d i t h tid )( i t d i t h tid )(sister and nonsister chromatids)(sister and nonsister chromatids)..

Prophase IProphase I SynapsisSynapsisProphase IProphase I -- SynapsisSynapsisH l hHomologous chromosomes

sister chromatids sister chromatidsTetrad

Crossing Over Crossing Over -- variationvariationCrossing Over Crossing Over -- variationvariationnonsister chromatids Tetrado s ste c o at ds

chiasmata: site of crossing over

variation

Sex ChromosomesSex ChromosomesSex ChromosomesSex Chromosomes

XX chromosome female XY chromosome maleXX chromosome - female XY chromosome - male

Prophase IProphase IProphase IProphase I

centriolesspindle fiber centrioles

asterasterfibers

M t h IM t h IMetaphase IMetaphase IShortest phaseShortest phaseShortest phaseShortest phase

TetradsTetrads align on the align on the metaphase platemetaphase plate..

INDEPENDENT ASSORTMENT OCCURS:INDEPENDENT ASSORTMENT OCCURS:1 Orientation of homologous pair to poles is 1 Orientation of homologous pair to poles is 1. Orientation of homologous pair to poles is 1. Orientation of homologous pair to poles is

random.random.2. Variation2. Variation2. Variation2. Variation3. 3. Formula: 2Formula: 2nn

Example:Example: 2n = 42n = 4Example:Example: 2n 42n 4then then n = 2n = 2

thusthus 222 2 = 4 = 4 thusthus 22 4 4 combinationscombinations

Metaphase IMetaphase IMetaphase IMetaphase I

OR

metaphase plate metaphase plate

Question:Question:Question:Question:

In terms of Independent Assortment In terms of Independent Assortment --how many different combinations of how many different combinations of how many different combinations of how many different combinations of sperm could a sperm could a humanhuman malemale produce? produce?

AnswerAnswerAnswerAnswer

F l 2F l 2Formula: 2Formula: 2nn

Human chromosomes:Human chromosomes: 2n = 462n = 46n = 23n = 23

222323 = ~8 million combinations= ~8 million combinations222323 = ~8 million combinations= ~8 million combinations

Anaphase IAnaphase IAnaphase IAnaphase I

H l hH l h d d Homologous chromosomesHomologous chromosomes separate and move separate and move towards the poles.towards the poles.

Sister chromatidsSister chromatids remain attached at their remain attached at their centromerescentromerescentromerescentromeres..

Telophase ITelophase Ipp

Prophase IIProphase IIProphase IIProphase II

same as same as prophaseprophase in in mitosismitosis

Anaphase IIAnaphase IIAnaphase IIAnaphase II

same as same as anaphaseanaphase in in mitosismitosisi h id i h id sister chromatids separatesister chromatids separate

Telophase IITelophase IITelophase IITelophase II

Same as Same as telophasetelophase in in mitosismitosis..

Nuclei form.Nuclei form.

CytokinesisCytokinesis occurs.occurs.

R bR b f h l id d ht ll f h l id d ht ll Remember:Remember: four haploid daughter cells four haploid daughter cells produced.produced.

gametes = sperm or egggametes = sperm or egg

Telophase IITelophase IITelophase IITelophase II

MeiosisMeiosisMeiosisMeiosisn=2

sex celln=2 n=2

sperm

2n=4

n=2

2n 4

n=2haploid (n)

diploid (2n) n=2

n=2

meiosis I meiosis II

VariationVariationVariationVariationImportant to population as the raw material for Important to population as the raw material for Important to population as the raw material for Important to population as the raw material for natural selectionnatural selection..Question:Question:Question:Question:

What are the three sexual sources ofWhat are the three sexual sources ofgenetic variation? genetic variation?

1. crossing over (prophase I)1. crossing over (prophase I)2. independent assortment (metaphase I)2. independent assortment (metaphase I)3 d f tili ti3 d f tili ti3. random fertilization3. random fertilization

Q tiQ tiQuestion:Question:AA cellcell containingcontaining 2020hh (di l id)(di l id) tt ththchromosomeschromosomes (diploid)(diploid) atat thethe

beginningbeginning ofof meiosismeiosis would,would, atat itsitscompletion,completion, produceproduce cellscellscontainingcontaining howhow manymanygg yychromosomeschromosomes??

AAAnswer:Answer:

1010 chromosomeschromosomes (haploid)(haploid)1010 chromosomeschromosomes (haploid)(haploid)

it i meiosismitosis meiosis

Somatic cellFuntion Producing germSomatic cell repreduction

Funtion Producing germ cells

No cells 1 2 1 4

No chr. 46 46 46 23

46XYSpermatogenesisSpermatogenesis

46XYMitotic period 46

XY

p gp g

46XY

46XY

46XY

46XY

46 Primary spermatocyteXY

23 23

Primary spermatocyte

Secondary23X

23YMeiotic period

Secondary spermatocytes

23X

23X

23Y

23Y spermatids

23X

23X

23Y

23Y

sperms

46XX Oogenesis Oogenesis OogenesisOogenesis XX

46XXMitotic period 46

XX

ggtakes place in takes place in ovaryovary

Ooge es sOoge es s

XX

46

Mitotic period XX

46 46 46XX

46

XX XX XX

Primary oocyte in 46XX

Primary oocyte in follicle Suspended in prophase

Ⅰuntil sexual maturity

23X

23XMeiotic period

Fertilization

Secondary oocyte

1st polar body

23X

23X

23X

23X

Fertilization

ovum 2nd polar bodies

FertilizationFertilizationFertilizationFertilizationThe fusion of a The fusion of a spermsperm and and eggegg to form a to form a zygotezygoteThe fusion of a The fusion of a spermsperm and and eggegg to form a to form a zygotezygote..

A t i f tili d A t i f tili d A zygote is a fertilized eggA zygote is a fertilized egg

n=23egg

spermn=23

egg

2 462n=46zygote

2.3 Sex Chromosomes2.3 Sex Chromosomes

T t i h ll tTortoiseshell cat

22 pairs of autoHuman males are the Human males are the heterogametic sex heterogametic sex

1 pair of sex chro.

heterogametic sex heterogametic sex with two different sex with two different sex chromosomes, (XY).chromosomes, (XY). 1 pair of sex chro.

XX or XYchromosomes, (XY).chromosomes, (XY).

H f l H f l Human females are Human females are the homogametic sex the homogametic sex (XX)(XX)(XX).(XX).

DNA content of human chromosomes

Chromosome Amount of DNA (Mb)

Chromosome Amount of DNA (Mb)

1 263 13 1141 263 13 1142 255 14 1093 214 15 1063 214 15 1064 203 16 985 194 17 925 194 17 926 183 18 857 171 19 677 171 19 678 155 20 729 145 21 509 145 21 50

10 144 22 5611 144 X 16412 143 Y 59

XX chromosomechromosome dosagedosage

Sex Chromosomes: females XX males XY

gg

Sex Chromosomes: females XX, males XYGenes on X: females 2 , males 1

HowHow toto createcreate equalequal amountamount ofof XX chromosomechromosome genegeneproductsproducts inin malesmales andand females?females?

Shouldn’tShouldn’t XXXX femalesfemales produceproduce twicetwice thethe amountamount ofofXX--linkedlinked genegene productsproducts (proteins)(proteins) asas XYXYgg pp pp

males?males?

Dosage CompensationDosage Compensation

decrease X gene products by half in femalesdecrease X gene products by half in females(e.g. humans called X-inactivation)

XX females “compensate” by inactivatingXX females “compensate” by inactivatingXX females compensate by inactivating XX females compensate by inactivating one of their X chromosomes to make a one of their X chromosomes to make a single “dosage” of Xsingle “dosage” of X--linked geneslinked genessingle dosage of Xsingle dosage of X linked genes.linked genes.

The Lyon Hypothesis of X InactivationThe Lyon Hypothesis of X Inactivation

In normal females, only one of the two In normal females, only one of the two X chromosomes is genetically activeX chromosomes is genetically activeX chromosomes is genetically activeX chromosomes is genetically activeX chromosome inactivation occurs X chromosome inactivation occurs

l i d l tl i d l t (l t bl t t (l t bl t t early in developmentearly in development (late blastocyst (late blastocyst stage of embryogenesis stage of embryogenesis -- 3232--64 cell 64 cell stage).stage).

Proposed by Mary Lyon and Liane Russell (1961)

X inactivation is random.X inactivation is random.The inactive X can either be maternal or paternal in The inactive X can either be maternal or paternal in origin and the choice is random in each cell and origin and the choice is random in each cell and ggindependent of the choice in other embryonic cells.independent of the choice in other embryonic cells.

X inactivation is irreversible in X inactivation is irreversible in somatic cellssomatic cells -- the inactive X in a the inactive X in a

l ll lll ll llparticular cell remains inactive in all particular cell remains inactive in all descendents of that cell.descendents of that cell.

Inactive X late replicatingInactive X late replicatingp gp g


XX--inactivationinactivation

Barr BodiesBarr BodiesBarr BodiesBarr Bodies

Murray Barr (1949)Murray Barr (1949)chromatin (inactive X) appears as a chromatin (inactive X) appears as a dense object in the nucleusdense object in the nucleusdense object in the nucleusdense object in the nucleus–– XX XX with one Barr bodywith one Barr body

XY XY –– XY XY chromatin negative (no Barr body)chromatin negative (no Barr body)

Inactive X chromosome is visible as Barr Inactive X chromosome is visible as Barr bodybody

Barr Bodies are Inactivated X Barr Bodies are Inactivated X Chromosomes in FemalesChromosomes in Females

Normal male,Turner female

Normal female,Klinefelter male

0 1Turner female Klinefelter male

2 3 # Barr bodies=N-1 rule

Inactive X chromosome is visible as Inactive X chromosome is visible as Barr bodyBarr bodyBarr bodyBarr body

XXX female XXXX female


XX--inactivation reveals alleles in cats inactivation reveals alleles in cats heterozygous for the fur color geneheterozygous for the fur color gene

ll bl kGenotype is Xyellow/Xblack

Yellow patches: black allele is inactive Xyellow/Xblack

X ll /Xbl kBl k t h ll ll l i i ti2012年4月6日星期五 Medical Genetics

Xyellow/Xblack Black patches: yellow allele is inactive

LyonLyon Hypothesis: XHypothesis: X inactivationinactivationA precursor cell to all coat color cells

LyonLyon--Hypothesis: XHypothesis: X--inactivationinactivationA precursor cell to all coat color cells

Random inactivation early in dev.

anhidrotic ectodermal dysplasia anhidrotic ectodermal dysplasia 无汗性外胚层发育不良无汗性外胚层发育不良

So if you get women with this "anhidrotic ectodermal So if you get women with this "anhidrotic ectodermal dysplasia" to exercise vigorously and generate a sweat, dysplasia" to exercise vigorously and generate a sweat, you can see the skin broken up into thousands ofyou can see the skin broken up into thousands ofyou can see the skin broken up into thousands of you can see the skin broken up into thousands of alternating little patches, each about one centimetre alternating little patches, each about one centimetre across.across.

One patch has skin with sweat, while the patch right One patch has skin with sweat, while the patch right O e pa c as s s ea , e e pa c gO e pa c as s s ea , e e pa c gnext to it has skin without sweat next to it has skin without sweat -- a mosaic pattern right a mosaic pattern right across the whole body. across the whole body.

This doesn't happen to men because they have only one This doesn't happen to men because they have only one X chromosome X chromosome

Inconsistencies between syndromes andyX inactivation

If normal XX female has one X inactivated, why i X T f l l?is a X Turner female not normal?

Similarly, if XXY male has one X inactivated, why does he have Klinefelter syndrome?why does he have Klinefelter syndrome?

i h h iExceptions to Lyon hypothesis

•Although X inactivation is usually random, aAlthough X inactivation is usually random, a structurally abnormal X, e.g., an X chromosome bearing a deletion is preferentially inactivated;bearing a deletion, is preferentially inactivated;

i i di id l i h l i i i•in individuals with X-autosome translocations, it is usually the normal X chromosome that is preferentially inactivated;

Although X inactivation is extensive, it is not complete, some genes are known to p gescape inactivation;

While x inactivation is permanent in most somatic cells, it must be reversible in the development of germ cells.in the development of germ cells.

S X t Some genes on X are not inactivated

G i d t l Genes in pseudo-autosomal regions PAR1 and PAR2.

XIST active only on the XIST, active only on the Inactive X.

Many Genes Escape X-InactivationMany Genes Escape X Inactivation

In a survey of 224 human X-linked

34 (15%)genes, 34 (15%)escaped X inactivationinactivation.

The genes escapingX-inactivationoccur primarily

Xon Xp.

From: Carrel et al Proc Natl Acad Sci 96(1999)14440 14444From: Carrel et al. Proc. Natl. Acad. Sci. 96(1999)14440-14444.

Nonrandom X Chromosome InactivationNonrandom X Chromosome Inactivation

The cells with deleted X: The cells with deleted X: Th ll XTh ll X t l t l tit l t l tiThe cells carry XThe cells carry X--autosomal translocationautosomal translocation

N d X Ch I i iNonrandom X Chromosome Inactivation

Normal X Deleted XNormal X

Nonrandom X Chromosome Inactivation

Normal X from X

from auto

Sex ChromatinSex Chromatin

X h ti Y h tiX-chromatin Y-chromatin

Origin Long arm of YInactivated XOrigin Long arm of YInactivated X

Feature Facultative ConstitutiveFeature Facultative Heterochromatin

ConstitutiveHeterochromatin

Number =N of Y=No X -1

Selection of one active X Selection of one active X chromosomechromosome

N l f l X d Xi N l f l X d Xi Normal females : one Xa and one Xi. Normal females : one Xa and one Xi.

H t di f i di id l ith H t di f i di id l ith t t However, studies of individuals with However, studies of individuals with extra extra copies of the X chromosomecopies of the X chromosome show that in cells show that in cells with more than two X chromosomes there is still with more than two X chromosomes there is still with more than two X chromosomes there is still with more than two X chromosomes there is still only one Xa, and all the remaining X only one Xa, and all the remaining X chromosomes are inactivated. chromosomes are inactivated.

Default state of the X chromosome in females is Default state of the X chromosome in females is i ti ti b t X h i l i ti ti b t X h i l inactivation, but one X chromosome is always inactivation, but one X chromosome is always selected to remain active.selected to remain active.

‘‘Blocking factorBlocking factor’’ modelmodelgg

It is hypothesized that there is an autosomallyIt is hypothesized that there is an autosomally--encoded 'blocking factor' which binds to the X encoded 'blocking factor' which binds to the X h dh dchromosome and prevents its inactivation. chromosome and prevents its inactivation.

The model postulates that there is a limiting The model postulates that there is a limiting blocking factor, so once the available blocking blocking factor, so once the available blocking g , gg , gfactor molecule binds to one X chromosome the factor molecule binds to one X chromosome the remaining X chromosome(s) are not protected remaining X chromosome(s) are not protected g ( ) pg ( ) pfrom inactivation. from inactivation.

This model is supported by :This model is supported by :

The existence of a single Xa in cells with The existence of a single Xa in cells with X h X h many X chromosomes many X chromosomes

The existence of two active X The existence of two active X h i ll li ith t i th h i ll li ith t i th chromosomes in cell lines with twice the chromosomes in cell lines with twice the

normal number of autosomes.normal number of autosomes.

Chromosomal componentChromosomal componentpp

The XThe X--inactivation center (XIC) on the X chromosome is inactivation center (XIC) on the X chromosome is necessary and sufficientnecessary and sufficient to cause Xto cause X--inactivation inactivation necessary and sufficientnecessary and sufficient to cause Xto cause X inactivation. inactivation. Chromosomal translocationsChromosomal translocations which place the XIC on an which place the XIC on an autosomeautosome lead to inactivation of the autosome, and X lead to inactivation of the autosome, and X h l ki th XIC t i ti t dh l ki th XIC t i ti t dchromosomes lacking the XIC are not inactivated.chromosomes lacking the XIC are not inactivated.

The XIC contains two nonThe XIC contains two non translatedtranslated RNARNA genes Xist genes Xist The XIC contains two nonThe XIC contains two non--translatedtranslated RNARNA genes, Xist genes, Xist and Tsix, which are involved in Xand Tsix, which are involved in X--inactivation. The XIC inactivation. The XIC also contains binding sites for both known and unknown also contains binding sites for both known and unknown ggregulatory proteinsregulatory proteins..

Xist and Tsix RNAsXist and Tsix RNAsThe XThe X--inactive specific transcript (inactive specific transcript (XistXist) gene is) gene isThe XThe X inactive specific transcript (inactive specific transcript (XistXist) gene is ) gene is located in the X inactivation center of Xq13 and is located in the X inactivation center of Xq13 and is transcribed only from transcribed only from the inactive the inactive X chromosome.X chromosome.yy

XIST mRNA transcripts are only detected in normalXIST mRNA transcripts are only detected in normalXIST mRNA transcripts are only detected in normal XIST mRNA transcripts are only detected in normal females, not normal males.females, not normal males.

It is a nonIt is a non--coding RNA, not translated into a protein.coding RNA, not translated into a protein.

The inactive X chromosome is coated by The inactive X chromosome is coated by XistXist RNA, RNA, whereas the whereas the XaXa is not. The is not. The XistXist gene is the only gene gene is the only gene which is which is expressedexpressed from the Xi but not from the from the Xi but not from the XaXa. . X chromosomes which lack the X chromosomes which lack the XistXist gene cannot be gene cannot be inactivated.inactivated.

Tsix geneTsix genegg

Lik Xi t th T i d l RNA Lik Xi t th T i d l RNA Like Xist, the Tsix gene encodes a large RNA Like Xist, the Tsix gene encodes a large RNA which is not believed to encode a protein. which is not believed to encode a protein. Th T i RNA i t ib d Th T i RNA i t ib d titi t Xi t t Xi t The Tsix RNA is transcribed The Tsix RNA is transcribed antisenseantisense to Xist, to Xist, meaning that the Tsix gene overlaps the Xist meaning that the Tsix gene overlaps the Xist gene and is gene and is transcribedtranscribed on the opposite strand of on the opposite strand of gene and is gene and is transcribedtranscribed on the opposite strand of on the opposite strand of DNADNA from the Xist gene. from the Xist gene. Tsix is a negative regulator of Xist; X Tsix is a negative regulator of Xist; X Tsix is a negative regulator of Xist; X Tsix is a negative regulator of Xist; X chromosomes lacking Tsix expression (and thus chromosomes lacking Tsix expression (and thus having high levels of Xist transcription) are having high levels of Xist transcription) are having high levels of Xist transcription) are having high levels of Xist transcription) are inactivated much more frequently than normal inactivated much more frequently than normal chromosomes.chromosomes.

Uses in experimental biology Uses in experimental biology p gyp gy

St l Mi h l G tlSt l Mi h l G tl d X h d X h Stanley Michael GartlerStanley Michael Gartler used X chromosome used X chromosome inactivation to demonstrate the clonal origin of inactivation to demonstrate the clonal origin of cancers. cancers. cancers. cancers. Examining normal tissues and tumors from Examining normal tissues and tumors from females heterozygous for isoenzymes of the sexfemales heterozygous for isoenzymes of the sex--yg yyg ylinked linked G6PDG6PD gene demonstrated that tumor cells gene demonstrated that tumor cells from such individuals express only one form of from such individuals express only one form of G6PD G6PD G6PD, G6PD, whereas normal tissues are composed of a whereas normal tissues are composed of a nearly equal mixture of cells expressing the two nearly equal mixture of cells expressing the two nearly equal mixture of cells expressing the two nearly equal mixture of cells expressing the two different phenotypes. This pattern suggests that different phenotypes. This pattern suggests that a single cell, and not a population, grows into a a single cell, and not a population, grows into a

cancer cancer

QuestionQuestionQQ

What are the possible mechanisms for a What are the possible mechanisms for a female being an XR affected?female being an XR affected?gg

Sex DeterminationS x

What determines gender?What determines gender?What determines gender?What determines gender?

XX (f l )Sex Chromosomes

XX (female)

XY ( l )XY (male)

Number of X chromosomes, or Number of X chromosomes, or Number of X chromosomes, or Number of X chromosomes, or presence of Y chromosome?presence of Y chromosome?

What determines gender?What determines gender?What determines gender?What determines gender?

Individuals with unusual chromosome combinations provide a clue:

X0 femaleXXY lXXY male

=> Y chromosome determines genderg

Y ChromosomeY Chromosome

mostly inert, very few genes, mostly repeat sequence DNA (high and middle).

PAR:PAR: pseudo-autosomal regions :: pseudo autoso a eg o s on tips of X and Y: homologous, contain some genes.

PAR1 has a required cross over for successful sperm development development.

Genes on the Y chromosomeGenes on the Y chromosomeThere are three classes of There are three classes of

genes on the Ygenes on the Ygenes on the Y.genes on the Y.

Genes shared with X Genes shared with X Ge es s a ed t Ge es s a ed t chromosome define the chromosome define the pseudoautosomal regions pseudoautosomal regions (PAR)(PAR)(PAR)(PAR)

Genes similar to X Genes similar to X Genes similar to X Genes similar to X chromosome genes are Xchromosome genes are X--Y Y homologshomologs

Genes unique to the Y Genes unique to the Y i l di SRY i l di SRY including SRY gene including SRY gene

Mapping the gene Mapping the gene responsible for maleness

Experimental paradigm: sexExperimental paradigm: sex--reversed reversed p p gp p gindividualsindividuals–– XY females (missing critical bit of Y)XY females (missing critical bit of Y)gg–– XX males (possessing critical bit of Y)XX males (possessing critical bit of Y)

Deletion mapping of Y coupled with Deletion mapping of Y coupled with pp g ppp g panalysis of sexanalysis of sex--reversed individuals and reversed individuals and ““chromosome walkingchromosome walking”” to get new to get new sequencessequences

Mapping the gene Mapping the gene responsible for maleness

In 1990, Sinclair and colleagues narrowed In 1990, Sinclair and colleagues narrowed the region to a 35,000 basethe region to a 35,000 base--pair domain of the pair domain of the small arm of the Y chromosome.small arm of the Y chromosome.

Sex-determining region Yg g


Sex determining region Yg g

Gene symbol : SRY Location : Yp11.3

SRY encodes a 223 amino acid zinc finger SRY encodes a 223 amino acid zinc finger SRY encodes a 223 amino acid zinc finger SRY encodes a 223 amino acid zinc finger transcription factor that is a member of the high transcription factor that is a member of the high mobility group (HMG)mobility group (HMG)--box family of DNA box family of DNA mobility group (HMG)mobility group (HMG) box family of DNA box family of DNA binding proteins. The protein is expressed binding proteins. The protein is expressed during testis development for only 2 days.during testis development for only 2 days.during testis development for only 2 days.during testis development for only 2 days.

Sequencing revealed a conserved Sequencing revealed a conserved if h ld h DNAif h ld h DNA bi di bi di motif that could have DNAmotif that could have DNA--binding binding

functionfunctionSRYSRY = = TDFTDF

XY sexXY sex reversed females have deletions or reversed females have deletions or –– XY sexXY sex--reversed females have deletions or reversed females have deletions or mutations of mutations of SRYSRY

i d l i d l XX XX SS l d l d –– transgenic mouse model transgenic mouse model -- XX + XX + SrySry leads leads to testis developmentto testis development

–– SRYSRY expressed in gonad, but only expressed in gonad, but only transiently, at the onset of differentiationtransiently, at the onset of differentiation

SRY gene on the Y chromosome was SRY gene on the Y chromosome was SRY gene on the Y chromosome was SRY gene on the Y chromosome was identified as the gene that codes for TDF:identified as the gene that codes for TDF:

–– SRY is translocated to X in rare XX malesSRY is translocated to X in rare XX males

–– SRY is absent from Y in rare XY femalesSRY is absent from Y in rare XY females

The “home run” experiment by Koopman et The “home run” experiment by Koopman et al. used transgenic mice.al. used transgenic mice.gg

Genotypically Female Mice TransgenicGenotypically Female Mice TransgenicGenotypically Female Mice Transgenic Genotypically Female Mice Transgenic for SRY are Phenotypically Malefor SRY are Phenotypically Maleyp yyp y

XY male XX male

Anne McLaren.

What makes a man a man?N t 1990 19 346(6281) 216 7Nature. 1990,19;346(6281):216-7.

Sexual developmentSexual developmentppAt the beginning of human development either male ordevelopment either male or female development is possiblepossible.

Unspecialized gonads and two sets of reproductive ducts exist until week 6.

An embryo develops as a An embryo develops as a male or female using male or female using male or female using male or female using information from the Y information from the Y chromosomechromosomechromosome.chromosome.

Effect of YEffect of Y

appearance of structuresthat will give rise to

external genitalia

appearance of “uncommitted” duct system

of embryo at 7 weeksEffect of YEffect of Y

ChromosomeChromosome

Y present

Y absent

7 weeks

Y present

Yabsentpresent absent present absent

10 weeks

testes ovaries

ovary


birth approaching testis

Downstream sexDownstream sex--determining determining Downstream sexDownstream sex determining determining genesgenes

Puzzling XY sexPuzzling XY sex--reversed females reversed females without detectable mutation in without detectable mutation in SRYSRYprovide evidence for additional genes, provide evidence for additional genes, p g ,p g ,including:including:DAX1DAX1 -- on X can suppress testison X can suppress testis--–– DAX1DAX1 -- on X, can suppress testison X, can suppress testis--formation in a dosageformation in a dosage--sensitive mannersensitive mannerSOX9SOX9 17 i d ith 17 i d ith SRYSRY f f –– SOX9SOX9 -- on 17q, required with on 17q, required with SRYSRY for for normal testis formationnormal testis formation

The EndThe End

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