biol 3301 - genetics ch10b - chromosome structure st

8/18/2019 BIOL 3301 - Genetics Ch10B - Chromosome Structure St

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The Importance of Watson-Crick

Proposal• Storage of genetic information

• Replication and inheritance

• Expression of the genetic message


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Chromosome Structure

• The genetic material in cells is organized

into structural and functional domains.

• To function, each chromosome need to hae

! "n origin of replication

! Centromere #for linear$

! Telomere #for linear$


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Prokar%otic Chromosomes

• " single nucleic acid molecule #&'" or R'"(linear or circular $

• )argel% deoid of associated proteins ! not true• Contain relatiel% little genetic information ! *ut

eer%thing the% need

• "*ilit% to package exceedingl% long &'"

molecules into a relatiel% small olume


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Chromosomes of +iruses

• " nucleic acid molecule #&'" or R'"$

• Either single- or dou*le-stranded

• )inear or circular ! ./ *acteriophage ! single-stranded circular &'"

! pol%oma irus ! dou*le-stranded circular &'"

! *acteriophage lam*da ! dou*le-stranded linear *ut iscircular in host( . µm long, packaged into 01 µm side

protein head


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Bacterial Chromosome

Structure• Prokar%otic cells #*acteria$ contain their

chromosome as circular &'"

• 2suall% the entire genome is a single circle,

*ut often there are extra circles called

plasmids

• The &'" is packaged *% &'"-*inding

proteins1


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3acterial Chromosomes

• &ou*le-stranded &'" al4a%s

• In a structure called nucleoid

• "ssociated 4ith &'"-*inding proteins

containing #5$ charged amino acids to *ind

#!$ charged phosphate groups

• Is not functionall% inert, can *e readil%

replicated and transcri*ed


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Bacterial Chromosome

Structure• The *acterial &'" is packaged in loops

• The *undled &'" is called the nucleoid1

• It concentrates the &'" in part of the cell, *ut it is not separated *% a nuclearmem*rane #as in eukar%otes$

• The &'" does form loops to a protein core,attached to the cell 4all1


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Supercoiled &'"

• 3oth iruses and *acteria

• 6igh speed centrifugation results in three

*ands of different densit% and compactness

• )east dense ! linear

• T4o more dense !

! Circular

! Circular supercoiled


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Eukar%otic Chromosomes

• &ou*le-stranded &'", linear

• 3inding 4ith proteins

• Seeral molecules of &'" ma% *e present

in cell forming chromosomes

• 'um*er of chromosomes characteristic for

each species


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Speciali7ed Eukar%otic Chromosomes

• )arge chromosomes, structure seen under

light microscope

! Pol%tene chromosomes

! )amp*rush chromosomes


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Pol%tene chromosomes

• 88, 3al*iani, 3al*iani rings

• Can *e seen in the nuclei of interphase cell

• 9an% rounds of replication 4ithout strand

separation or c%tokinesis

• The *anding pattern is distinctie to each

chromosome


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Pol%tene chromosomes

• 3ands and inter*ands

• Puffs ! regions of gene actiitt%

• " *and can contain up to 0. *ase pairs of

&'"


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)amp*rush Chromosomes

• 8:;, in ooc%tes of sharks ! the% are giant

chromosomes

• &irect the meta*olic actiities of the

deeloping egg

• Chromomeres, lateral loops

• "ctie in s%nthesis of R'"


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&ifferential Staining

• 9etaphase chromosomes differ from one another

in si7e and shape, and the a*solute length of an%

one chromosome aries depending on the stage ofmitosis in 4hich it 4as fixed1

• 6o4eer, the relatie position of the centromere is

constant, 4hich means that that the ratio of the

lengths of the t4o arms is constant for eachchromosome1


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&ifferential Staining

• Centromere position and arm ratios can assist inidentif%ing specific pairs of chromosomes, *utsome chromosomes still similar

• Certain d%es 4ould produce reproduci*le patternsof *ands 4hen used to stain chromosomes1

• Chromosome *anding has since *ecome astandard and indispensi*le tool for c%togenetic

anal%sis.


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Chromosome 3anding

• :.0s ! chromosome-*anding techni


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Chromosome 3anding

G banding> produced *% staining 4ith=iemsa after digesting the chromosomes

4ith tr%psin• C banding> chromosomes are treated 4ith

acid and *ase, then stained 4ith =iemsastain

• Q banding> chromosomes are stained 4itha fluorescent d%e such as


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Chromosome 3anding

• Each of these techni


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Chromatin

• In eukar%otic cells the &'" in the nucleus

is not naked *ut is associated 4ith protein

• This nucleoprotein complex of &'", R'",

and protein is referred to as chromatin1


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Chromatin

• Euchromatin and heterochromatin

• +isi*le chromosomes in mitosis !tightl%

coiled chromatin fi*ers

• Interphase ! chromatin is uncoiled

• " contraction in length of 0,000 times


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6eterochromatin and Euchromatin

• 6eterochromatin

! Tightl% packed, dark staining

! Transciptionall% inactie, late replication

! @acultatie or constitutie

• Euchromatin

! )ight staining, less tightl% packed

! Transciptionall% actie


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6eterochromatin

• Some parts of chromosome remain condensed

through cell c%cle, stain deepl% during interphase1

• 6eterochromatin ! geneticall% inactie, replicateslater during S phase

• "reas of centromeres, telomeres

•Whole chromosome inactiation ! 3arr *od%

• Position effect ! inactiit% in adAacent regions


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Chromatin

• &'" in E1coli ! ;00 µm long

• &'" in human chromosomes range from

:,000 to .B,000 µm

• "ll / chromosomes ! almost ; meters

• &iameter of a nucleus is D-0 µm


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How much chromatin or more precisely

DNA, does a cell hae!• &etermination of the amount of &'" per nucleus

can *e determined *% carr%ing out c%tochemical

tests, e1g1, "eulgen reaction, or Schiffs reagent1• @ollo4ing the specific staining of &'" in the

nucleus, the slide 4ith stained cells can *e ie4ed

4ith a special c%tophotometric microscope and the

amount of &'" per cell determined1


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C alue

• The amount of &'" per haploid genome is calledthe C alue1

• In eukar%otes the &'" content per haploidgenome is generall% expressed in picograms #pg$1

pg F 0: *p of &'"

pg of &'" F a*out B cm of &'"

1


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&'" Packaging

• 6umans hae B1D pgGhaploid genome

• Thus, the human diploid nucleus has

greater than ####################### of&'"1

• Het the nucleus has an aerage diameter of

onl% m1• 6o4 does all this &'" get packaged into

such a tin% spaceJ


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Beads on a String

• "n aerage si7ed human chromosome possesses &'" thatis a*out D cm in length 4hen stretched out1 6o4eer, atmetaphase of mitosis, it is D um, a 0,000-fold reduction1

• @olding of the &'" is made possi*le *ecause the &'" is KKKKKKKKKKKKKKKKKKK -- to form chromatin1

• Experimental methods hae *een deeloped to spreadchromatin on an E9 grid and isuali7e its structure1

• If nuclei are l%sed in dilute salt and the chromatin spread,one sees 4hat is referred to as the ?*eads-on-a-string?structure, 4ith *eads eenl% spaced out1


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'ucleosome

• 6o4 much &'" is associated 4ith one repeating unit or *eadJ

• 'ucleases cleae exposed &'" *et4een *eads1 Isolate

*eads and electrophorese &'" on a gel1 @ragments area*out /0*p

• That much &'" is 4rapped around the *ead1

• The linker &'" aries from species to species #for sea

urchins, it is a*out 00 *p( in humans, it is a*out 0 *p$1• The protein *ead is the *asic packing unit of chromatin

and is called a nucleosome


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Chromatin proteins

• Proteins associated 4ith &'" are histones

and nonhistones

• Positiel% charged histones *ondelectrostaticall% to the negatiel% charged

phosphates( fie t%pes ! 6, 6;", 6;3,

6B, 6/• )ess positiel% charged - nonhistones


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'ucleosome

• The *ead proteins are histone proteins1

• The histones are a*out 00 - D0 amino acids in length andare all highl% *asic1 This gies them a net #5$ charge,allo4ing them to *ind to &'"1

• The *ead histones> ! 6;"

! 6;3

! 6B

! 6/• These range in 9W from 000 ! /000 &a

• 6, the fifth histone, is larger #a*out ;,000 &a 9W$ andis not part of the *ead1


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6istones

• @or 6;", 6;3, 6B, and 6/, one half of the amino

acids making the protein is highl% *asic

#5charged$ 4hile the other half is h%dropho*ic• The *asic end is for interacting 4ith &'", 4hile

the h%dropho*ic end is more important for

protein-protein interactions1

• 6 has *asic residues at the t4o ends and is more

h%dropho*ic and acidic in the middle1


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'ucleosome Core

• The nucleosome core, the ?*ead? structure consists

of t4o each of 6;", 6;3, 6B, and 6/, to form an

octamer #8$ protein core1• Wrapped around each octamer is a*out /0*p

&'"1

• 6 is associated 4ith the &'" linker and seres

to link adAacent nucleosomes1 Thus, there is onl%

one 6 histone per nucleosome1

• When treating chromatin 4ith diltule salt1 6 is

remoed, so that explains 4h% the L*eads on a


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$% nm chromatin &iber -- the natie

chromatin structure• In the a*sence of salt and 4ith careful l%sis on an

E9 grid, most of the chromatin is seen as a fi*er4ith a diameter of *et4een ;0-B0 nm, commonl%kno4n as the $%nm chromatin &iber

• The nucleosomes in the B0 nm chromatin fi*er are packed in a spiral manner, 4ith -. cucleosomes per turn1

• 6 allo4s for the interaction *et4een nucleosomesand is responsi*le for the formation of the helicalshape


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'ooped domains • Packing of the &'" in B0 nm chromatin fi*ers onl%

reduces the D cm of &'" do4n to Aust oer KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK1

• The pro*a*le next leel of folding 4as originall%

suggested *% lamp*rush chromosomes and pol%tenechromosomes, 4hich appear to *e organi7ed

KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK

• " looped domain is simpl% a B0 nm chromatin fi*er that is *ent to KKKKKKKKKKKKKKKKKKKKKKKK1

• " single looped domain ma% hae *et4een ;0,000 -00,000 *p of &'"1

• " chromosome 4ould *e organi7ed in a series of loopeddomains1


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)ooped &omains

• This formation of looped domains reduces

our ?aerage? chromosome to a*out

KKKKKKKKKKKKKKKKKKK #a KKKKKKKKKKKKKfurther reduction than the B0 nm fi*er$1

• This looped domain structure for

chromosomes is pro*a*l% ho4 a t%picalinterphase chromosome is organi7ed1


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'ucleosome

• Chromatin consists of repeating units #M ;00*p$ KKKKKKKKKKKKKKKKKKKKKKKKKKKK

• Chromatin fi*ers are composed of linear arra%s of

spherical particles ! KKKKKKKKKKKKKKKKKKKKKKK • Each nucleosome consists of #6;"$;•#6;3$; and

#6B$;•#6/$; tetramers in association 4ith a*out ;00 *p of&'"

• 'ucleosome core particle consists of /. *p, the rest is

linker &'" associated KKKKKKKKKKKKKKKKKKKKKK • http>GG4441Aohnk%rk1comGchromosomestructure1html - animation

• http>GG4441*iostudio1comGdemoKfreemanKdnaKcoiling1htm


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&'" packaging

• ; nm &'" molecule is coiled into a nm in diameter

nucleosome ! first leel, GB reduction

• @urther packaged into a B0 nm structure ! solenoid !

second leel, spiral 4ith -. nucleosomes per turn( 6

clamp holds the helical shape ! D0-fold reduction

• @orms a series of loops and further condense into B00 nm

in diameter chromatin fi*er ! 0-fold from preious leel

• @orm chromosome arms that constitute a chromatid .00

nm in diameter ! 6 phosphor%lation


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Scaffold

• &igestion of &'" a4a% from metaphase

chromosomes reeals a protein net4ork, or

scaffold, shaped like the chromosome, that ma% *eresponsi*le for maintaining

KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK1

• &'" folding is precise and al4a%s occurs the

same 4a% for a chromosome, as can *edemonstrated *% KKKKKKKKKKKKKKKKKKKKKKKKKK


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'uclear Scaffold

• 'uclear scaffold s%stem, termed the nuclear matrix,

determines the shape of the nucleus

• Proides a B-dimensional lattice for organi7ing the &'"

into specific functional units1

• The nuclear matrix is a ri*oprotein structure that organi7es

D0,000 loop domains of &'" of approximatel% 0

kilo*ase lengths each

• &uring replication the loops are reeled through fixed

replicating sites attached to the nuclear matrix


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Histone (odi&ications

• Phosphor%lation of histones ma% *e important in KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK1

• "cet%lation of histones ma% KKKKKKKKKKKKKKKKKhistones affinit% for &'" #l%sine most commonl%is acet%lated, resulting in decreased charge$1

• "cet%lation of histones ma% *e important in

KKKKKKKKKKKKKKKKKKKKKKKKKKK #allo4inggreater accessi*ilit% of &'" *% R'"

pol%merase$1


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Chromatin Structure o& Actie

Genes

• Interphase chromatin can *e diided into

t4o classes> ! ###################### - packaged

similarl% as mitotic chromatin #tightl%$, er%little transcription of &'"

! ###################### - packaged aslooped domains, this is ?actie chromatin?,containing genes that are transcri*ed1


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6o4 do genes that are in the process

of transcription lookJ• "ctie genes are still associated 4ith

KKKKKKKKKKKKKKKKKKKKKKKKKKKKKK

• "s a rule, actie genes are h%pometh%lated atC%tosine residues1

• "ctie genes, as a rule, hae more KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK

• The promoter region of actie genes is

h%persensitie to &'"se I #suggesting fe4erhistones here to allo4 for R'" pol%merase toinitiate transcription more easil%$1


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'uclear "rchitecture

• In the eukaryotic nucleus, chromosomes occupyindividual, nonoverlapping territories and reactions ofDNA and RNA metabolism are confined to discretestructures in the nuclear interior

• The highest order packing may result from attachment ofchromosomes to a nuclear matrix or nuclear scaffold

• The loop organization of DNA in interphase seems to bemaintained by anchoring of specific DNA sequences(MAR/SAR) to a protein network of the nucleoskeleton.

• MAR – matrix attachment region

• S"R ! scaffold attachment region


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Conclusions• 'ucleus has a er% high leel of structural and functional

organi7ation• In the interphase, &'" is

KKKKKKKKKKKKKKKKKKKKKKKKKKK then during celldiision *ut each chromosome has a specific location

4ithin nucleus and is anchored *% specific se

biol 3301 - genetics ch10b - chromosome structure st

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