Lewin Benjamin (ed.) Genes IX

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Band

p22.3

p22.2

p22.1

p21

p11.4

p11

p11.2

cenlromere

q12

q13

q21

q22

q23

q24

q25

q26

q27

q28

6"k

4o/o

2Y"

30 40

60%

G-C

content

f iiri.ifii:

ili:l.t

The

human X

chromosome

can

be divided

into distinct regions

by its

banding

pattern.

The

short

arm

and the long arm

q,'

each

arm is

djvjded into

larger

regions

that

are

further

subdivided. This

map

shows

a [ow resotution

structure;

at higher

resolution,

some

bands

are

further

subdivided

into

smatter bands

and inter-

bands,

e.9.,

p21

divided into

p21.1.,

p27.2,

and

p21,.3.

Iower

G-C content

than the interbands.

This is

peculiar

result.

If there

are

-10

bands on

large

chromosome

with

a total

content of

-100

Mb,

this means

that the

chromosome is

divided

into regions

-5

Mb in length

that

alternate

between

low

G-C

(band)

and high

G-C

(interband)

content.

There is

a tendency

for

genes (as

identified

by hybridization

with

mRNAs)

to be located

in the

interband regions.

All of this

argues for

some long-range

sequence-

dependent organization.

The human

genome

sequence

confirms

the

basic observation.

:'i*r-i"!ti;

;*.::.i

shows

that there

are distinct fluctuations

G-C

content when

the

genome

is divided

into

small tranches

(DNA

segments

or lengths).

The ave rage

of 4lo/o

G-C

common to mammalian genomes.

There

are

regions

as low as

30o/o or as

high as 650/o.When

longer

tranches

are examined,

there is less

vari-

ation. The

average length

of regions

with>4joh

G-C is 200 to 250 kb.

This makes

it clear

that tne

band/interband

structure

does not

represent

homogeneous

segments

that

alternate in

G-C

content, although

the bands

do contain a higher

content of low

G-C segments.

Genes

are con-

centrated in regions

of higher

G-C content.

have

yet

to understand

how the

G-C content

affects

chromosome

structure.

ai{;iJfii:

llir.ili

There

are large

ftuctuations

G-C con-

tent over

short djstances.

Each bar shows the

percent

20 kb fragments with the

given

G-C content.

Lampbrush Chromosomes

Are Extended

Sites of

gene

expression

on lampbrush

chromosomes shc,w loops that are extended

from

the chromosomaI axis.

would be extremely useful to visualize

gene

expression

in its natural state

in order to see

what

structural chranges

are associated with tran-

scription. The couLpression of

DNAin chromatin.

coupled with the difficulty of

identifying

partic-

ular

genes

within

it, makes it impossible to visu-

alize the transcription of individual active

genes.

Gene expres:;ion can

visualized

directly

certain unusual situations

which

the chro-

mosomes

are found

highly extended form

that allows indivjdual loci

(or groups

of loci) to

be distinguished. Lateral differentiation of struc-

ture is evident h many chromosomes

when

they first

appear

for meiosis.

this stage,

the

chromosomes resemble a series of

beads on a

string. The

beads

are densely staining

granules,

properly

known;rs chromomeres.

general,

though,

there

is little

gene

expression

at meio-

sis, and it is not

practical

use this material to

identify

the

activities of

individual

genes.

exceptional situation that allows

the material

to be examined is

presented

by lampbrush

chromosomes, which

have been best

charac-

terized in certain amphibians.

Lampbrush

chromosomes

are

formed dur-

ing an unusually extended

meiosis, which

can

last up to several months!

During this

period,

the chromosomc's are

held in a stretched-out

form in which thr:y can be

visualized

in the light

microscope. At

later

point

during

meiosis, the

chromosomes re\.ert to their

usual compact size.

28.9 Lampbrush Chromosomes

Are Extended 741

Lampbrush

chromosome is a

meiotic

bivaLent in which the two

Dairs

of sister chromatids are

heLd together at chiasmata

(indicated

by arrows).

Photo

courtesy

of Joseph

G. Gatt, Carnegie Institution.

[ampbrush chromosome

loop is sur-

rounded

by a matrix of

ribonucteoprotein.

Reproduced

from

GatL, J. G., et al.

MoL. BioL.

Cell.

December, 1999. 10:

4385-4402. Photo

courtesy of Joseph G. GaLt, Carnegie

Institution.

Thus

the extended state essentially

proffers

unfolded

version of the normal condition of the

chromosome.

The lampbrush

chromosomes are meiotic

bivalents, each consisting

two

pairs

sister

chromatids. shows an example in

which the

sister chromatid

pairs

have mostly

separated

that they

are

held

together only

by chiasmata. Each sister

chromatid

pair

forms

a series of ellipsoidal chromomeres,

-l

diameter, which are connected by a very fine

thread. This

thread contains the two sister

duplexes

DNA and runs

continuously along

the chromosome, through the

chromomeres.

The iengths

the individual lampbrush

chromosomes

in the newt Notophthalmus viri-

descens range from 400

to 800

pm,

compared

with the range

to 20

seen later in meio-

sis. Thus the lampbrush

chromosomes are

-30

times

less tightly

packed.

The total length

of the

entire lampbrush

chromosome set is 5 to 6 mm

and is

organized into

-5000

chromomeres.

CHAPTER 28

Chromosomes

The

lampbrush chromosomes take their

name from the

lateral loops that extrude from

the chromomeres

at certain

positions. (These

resemble a lampbrush,

which is an extinct

object.)

The loops extend

pairs,

one

from

each

sister chromatid.

The loops are continuous with

the axial thread,

which suggests that they rep-

resent chromosomal

material extruded from its

compact

organization

the chromomere.

The loops are surrounded

by a matrix of

ribonucleoproteins

that contain nascent RNA

chains. Often, a

transcription unit can be defined

by the increase

the

length of the RNP mov-

ing

around

the loop.

example

shown

j,l,ri,ijl

r]:i

Lr.

Thus the loop is an extruded segment of

DNA that is being actively

transcribed. In some

cases, loops corresponding

particular genes

have been identified.

For these

cases, the struc-

ture of

the transcribed

gene-and

the nature

of the

product-can

be scrutinized in situ.

Polytene Chromosomes

Form Bands

Polytene chromosomes of dipterans

have

a series

of bands that can be used as a cytotogicaI

map.

The interphase nuclei of some tissues of the lar-

vae oI dipteran

flies contain chromosomes that

are

greatly

enlarged

relative

to their usual con-

dition.

They

possess

both increased diameter

and

greaterlength.

;i,r.ri-ri;:

r.; :r

shows an exam-

ple

of a chromosome set

from

the salivary

gland

of.

D. melanogaster. The members of this set are

called

polytene

chromosomes.

Each member of the

polytene

set consists

of a

visible

series of bands

(more

properly,

but

rarely, described as chromomeres). The bands

range in size from the largest, with a breadth of

-0.5

pm,

to the smallest, at

-0.05

pm.

(The

small-

est can be distinguished only under an electron

microscope.) The bands contain most of the mass

DNA and

stain

intensely with

appropriate

reagents. The regions between them

stain more

lightly and are called interbands. There

are

-5000

bands

in the D. melanlgaster

set.

The centromeres of all four chromosomes

of. D melanogaster aggregate to form a chromo-

center that consists largely of heterochromatin.

(In

the male it includes the entire Y

chromo-

some.)

Allowing for

this,

-75o/o

of the haploid

DNA set is organized into alternating

bands

and interbands. The length of the

chromosome

742

iri{il-tfti:

lii.lri: The

polytene

chromosomes

of D. melono-

gasterform

an atternating

series

ofbands and interbands.

Photo

courtesy ofJos6 Bonner,

Indiana

University.

set is

-2000

pm.

The DNA

extended form

would

stretch for

-40,000

pm,

so the

packing

ratio is

-20.

This

demonstrates

vividly

the

extension of the

genetic

material

relative to

the usual

states

interphase

chromatin

mitotic

chromosomes.

What is

the structure

of these giant

chro-

mosomes? Each

produced

by the

successive

replications

of a

synapsed

diploid

pair.

The

repli-

cas do not

separate, but instead

remain

attached

each other in

their extended

state. At the

start of

the

process,

each synapsed

pair

has

DNA

content

(where

C represents

the

DNA

content

of the individual

chromosome).

This

amount

then doubles

up to

nine times, at

its maximum giving

a content

of 1024C.

The

number

of doublings is

different

in the various

tissues of the D.

melanogasterlarva.

Each

chromosome

can be visualized

as a

large number

parallel

fibers running

longitu-

dinally

that are tightly

condensed

in the

bands

and less so in

the interbands.

It is likely

that each

fiber

represents

a single

(C)

haploid

chromo-

some. This

gives

rise

to the name

polytene:

The

degree of

polyteny

the number

of haploid

chro-

mosomes contained

in the

giant

chromosome.

The

banding

pattern

characteristic for

each strain

Drosophila.

The

constant number

and linear arrangement

of the bands was

first

noted in

the

I930s,

when

it was realized

that

they form aqttologicalmap

olthe chromosomes.

Rearrangements-such

deletions, inversions,

or duplications-result

alterations

of the order

of bands.

The

linear array

of bands

can be equated

with the linear

array

genes.

Thus

genetic

rearrangements,

seen in a linkage

map, can

be correlated with

structural rearrangements

of the cytological map.

Ultimately,

particular

mutation can

located

particular

band.

The

total number

genes

in D.

melanogaster

Freez:e in dry ice

Wasl- with ethanol

Dip ir

agar

solution

Denature DNA

Add radioactive

probe

Wasl-

ofi

unreacted

probe

Autoradiography

iarget

cells

squashed

on slide

Black areas a1e silver

grains

that identify sites

where

probe

hyhridized

ll:ii:

i.J

fi f ,,il:i,,:

Indi viduaI ba nds contai

g particutar

genes

can be identified by in situ hybridization.

exceeds the numller of bands, so there are

prob-

ably multiple

gerles

in most or all bands.

The

positions

particular genes

the

cytological map

(

an be determined directly by

the technique

situ

hybridization. The

protocol

is summarized

4iiii!.i*{

i:*:.51i}. A

radioactive

probe

representing a

gene (most

often a labeled cDNA clone derived

from the

mRNA) is hybridized

with

the denatured

DNA

of the

polytene

(hromosomes

in situ.

Autora-

diography identilies the

position

positions

the correspondirg

genes

the

superimposi-

tion

grains

at a

particular

band or bands.

example is

shown

iit,l,f 'rtii ;:*",iJi:l. More recently,

fluorescent

probes

have replaced

radioactive

probes.

With this type of

technique at hand,

possible

determine directly the band

within

which

particule

sequence

lies.

Polytene Chromosomes

Expand at Sites of

Gene

Expres;sion

Bands

that are sil.es

gene

expression on

polytene

chromosomes expand

give

"puffs."

One of the intriguing

features of the

polytene

chromosomes

is t hat

active

sites can be visual-

ized.

Some of the rands

pass

transiently

through

28.11 Potvtene Chromosomes

Expand

Sites of Gene

Expression 743

;'.:

::i.:i .::..:..

A magnified

view

of bands 87A

and 87C

shows their hybridization

sifu

with Labeted RNA extracted

irom heat-shocked

ce[ts.

Photo

courtesv ofJos6

Bonner,

Indiana

Universitv.

an expanded state

in which they appear like a

puff

on the chromosome, when chromosomal

material is

extruded

from the axis. Examples of

some very large

puffs

(called

Balbiani rings) are

shown in

:::i:r;iii

'li::

j j

What is

the

nature

the

puff?

It consists

of a region in which the chromosome

fibers

unwind from their usual state of

packing

in the

band. The fibers remain continuous with those

in the chromosome axis. Puffs usually emanate

from single bands, although when they are

very

.arge, as typified by the Balbiani rings, the

swelling

may

be so extensive as to obscure

the

underlying array of bands.

The

pattern

puffs

is related to

gene

expres-

sion. During larval development,

puffs

appear

and regress in a definite, tissue-specific

pattern.

characteristic

pattern

puffs

is found in

each

tissue at any

given

time. Puffs are induced by

the hormone

ecdysone that controls Drosophila

development. Some

puffs

are induced directly

by the hormone; others are induced indirectly

by the

products

of earlier

puffs.

The

puffs

are sites where RNA is

being synthe-

sized

The

accepted view of

puffing

has been that

expansion

of the band

a consequence of the

need to relax its

structure

order to synthesize

RNA. Puffing has therefore been viewed as a

con-

sequence of transcription. A

puff

can be

generated

a single active

gene.

The sites of

puffing

differ

CHAPTER 28 Chromosomes

iilii

Hl- ;i *. ll I Chromosome

of the

nsect C. tentans

has

three

Batbjani rings'in the salivary

gtand.

Reproduced

from

Cell,

vo[. 4, Danehott, 8., et at., Transciption in

poLy-

tene

chromosomes

pp.

1-9.

with

permission

from Etsevier.

Photo

courtesy of

BertiI Dane-

hoLt, MedicaI

NobeI Institute.

from

ordinary bands

in accumulating additional

proteins,

which

include RNA

polymerase

and

other

proteins

associated with transcription.

The

features

displayed

by lampbrush and

polytene

chromosomes suggest a

general

con-

clusion.

order to be

transcribed,

the

genetic

material is dispersed from

its

usual, more tightly

packed

state.

The

question

keep in

mind is

whether this dispersion at the

gross

level

of the

chromosome

mimics the events that occur at

the molecular level within the mass of ordinary

interphase euchromatin.

Do the bands of a

polytene

chromosome

have a functional significance, that is, does each

band correspond

to some type of

genetic

unit?

You might thinkthat the answerwouldbe imme-

diately evident

from

the sequence of the fly

genome,

because by mapping interbands

to the

sequence it should be

possible

to determine

whether a band

has

any

fixed

type of identity.

Thus far, though, no

pattern

has been found that

identifies a functional significance for

the bands.

The Eukaryotic

Chromosome

Segregation

Device

eukaryotic chromosome

hetd on the mitotic

spindte by the attachment of mjcrotubules to

the

kinetochore

that forms

in its

centromeric region.

Centromeres

often

have heterochromatin

that is

rich in

sateltite DNA seouences.

744

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TCACATGATGATATTTGATTTTATTATATTTTTAMMMGTAAAAAATAAAAAGTAGTTTATTTTTAAMMTAMAT]-TAAMTATTTCACMMTGATTTCCGAA

AGTGTACTACTATAAACTMAATAATATAAMATTTTTTTCATTTTTTATTTTTCATCAAATAAAMTTTTTTATTTTMATTTTATAAAGTGTTTTACTAAAGGCTT

CDE-ll

8O-9O

bo.

>90%

A + T

l:Gil5iltS.il

ThreeconseryedregionscanbeidentifiedbythesequencehomotogiesbetweenyeastCE/Vetements.

Centromeres

Have

Short

DNA

Sequences

cerevisiae

CEN etements

are identified

5. cerevisiae

bu the

abitity

to al.l.ow

ptasmid

to segregate

accuritety

at mitosis.

CEN etements

consist

of the short

conserved

sequences

CDE-I and

CDE-III that ftank

the A-T-

rich region

CDE-II.

If a centromeric

sequence

of DNA

is responsi-

ble for segregation,

any molecule

of DNA

pos-

sessing

this sequence

should

move

properly

cell division,

whereas

any

DNA lacking

should

fail to

segregate.

This

prediction

has

been used

isolate

centromeric

DNA in

the

yeast,

S. cerevisiae. Yeast

chromosomes

do not

display visible

kinetochores

comparable

those

of higher

eukaryotes,

but

otherwise

divide

at mitosis

and segregate

at meiosis

the

same mechanisms.

Genetic

engineering

has

produced

plasmids

yeast

that are

replicated

chromosomal

sequences

(see

Section 15.8,

Replication

Ori-

gins

Can Be Isolated

in Yeast).

They

are unsta-

ble at mitosis

and meiosis,

though,

and

disappear

from

a majority

of the cells

because

they segre-

gate

erratically.

Fragments

chromosomal

DNA

containing

centromeres

have

been isolated

their ability

to confer

mitotic

stability on

these

plasmids.

centromeric DNA

regions (CEN)

frag-

ment

is identified

as the minimal

sequence that

can confer

stability

upon

such a

plasmid.

Another

way to

characterize

the function

such sequences

is to modify

tlnelriL

in vitro and

then reintroduce

them

into the

yeast

cell,

where

they replace

the

corresponding

centromere

the chromosome.

This

allows the

sequences

required for

CENfunction

to be defined

directly

the context of the

chromosome.

CENfragmt:nt derived from one chromo-

some

can replact: the centromere of another

chromosome

with no apparent consequence.

This

result suggests that centromeres are inter-

changeable

. Thel are used simply to attach the

chromosome

to the spindle, and

play

no role in dis-

tinguishing

one

chromosome

from

another.

The

sequences

required for centromeric

function

fall within a stretch of

-I20

bp. The

centromeric region is

packaged

into a nuclease-

resistant

structure and binds

single

micro-

tubule.

We may ttrerefore

look

to the S. cerevisiae

centromeric regio.c to identify

proteins

that bind

centromeric DNA

and

proteins

that connect the

chromosome to the spindle.

summarizr:d

in i'3{*:i"ifif,:,

'J*.JS,

three types

of sequence elenrent can be distinguished

the CEN region:

cell cycle-dependent element

(CDE)-I

is a

sequence

of 9 bp that

conserved

with minc,r variations

at the left bound-

ary of all centromeres.

CDE-II is

,t >90o/o

A-T-rich sequence of

80 to 90

found in all centromeres;

its

function

could depend

on its Iength rather

than exact sequence.

Its

constitution

reminiscent of some

short tandemly

repeated

(satellite)

DNAs

(see

Section

6.I2,

Arthropod

Satellites

Have Very Short Iden-

tical Repe;rts).

Its

base

composition may

Cduse SorrLe characteristic

distortions of

the DNA oouble

helical structure.

CDE-III isan I I bp sequence

highly

con-

served at

the right boundary of all cen-

tromeres. Sequences

on either side of

the element are

less well conserved, and

may also be needed

for centromeric

function.

:CDE-III

could be

longer than

I I bp if il turns out

that the flanking

sequences are essential.)

Mutations in CDE-I or CDE-II

reduce, but

do not inactivatt:,

centromere

function, but

point

mutations

the

central CCG of CDE-III

completely inactivate the centromere.

28.14 Centromeres

Have Short

DNA

Sequences

in S. cerevisioe 747

Protein Complex

The Centromere

Binds

A specialized

protein

comptex that

atternative to the

usuaI chromatin structure

formed at

CDE-IL

The

CBF3

protein

comptex

that binds to CDE-III

essentiaI

for

centromeric

function.

The

proteins

that connect

these two comptexes

may

provide

the connection to

microtubules.

Can

identify

proteins

that are

necessary for

the

function

of CEN sequences?

There are

sev-

eral

genes

in which

mutations affect chromo-

some segregation,

and whose

proteins

are

localized at centromeres.

The contributions of

these

proteins

to the centromeric

structure are

summarized in

FISUftE I8.tS.

specialized

chromatin structure

is built

by binding the CDE-II

region to a

protein

called

Cse4,

which resembles one of the histone

pro-

teins that comprise the basic subunits

of chro-

matin

(see

Section 3I.3, Heterochromatin

Depends

Interactions with Histones).

pro-

tein called Mif2

may

also be

part

this com-

plex,

or at least connected to

it.

Cse4 and

Mif2

have

counterparts

that are localized at higher

eukaryotic centromeres, called CENP-A

and

CENP-C, which suggests that this

interaction

may be a universal aspect of centromere

con-

struction. The

basic

interaction consists of bend-

ing

the DNA of the CDE-II

region arounci a

protein

aggregate; the reaction is

probably

assisted

by the occurrence

of intrinsic bending

the CDE-II sequence.

CDE-I is bound by the

homodimer

CBFI;

this interaction

not essential

for

centromere

function, but in its absence the fidelity of chro-

mosome

segregation

is reduced

-10x.

A 240 kD

complex of four

proteins,

called CBF3, binds to

CDE-I CBFl

Microtubules

cDE-rl Cse4

ctf19

Okpl

cDE-il1

f3t:iifi[

tii, t$ The DNA at CDE-II

'is

wound around a

pro-

tein aggregate including

Cse4p, CDE-III

bound to CBF3,

and

CDE-I

bound to CBF1.

These

oroteins are connected

by the

group

of Ctf19, Mcm21,

and Okp1.

CHAPTER 28

Chromosomes

CDE-III.

This interaction

is essential for cen-

tromeric

function.

The

proteins

bound

at CDE-I and CDE-III

are connected

to each other

and also to the

pro-

tein structure bound

at CDE-II by another

group

proteins

(Ctf

19, Mcm2l, Okpl).

The

con-

nection

to the microtubule

mav be made bv this

complex.

The overall

model suggests that the complex

is localized

at the centromere

by a

protein

struc-

ture

that resembles

the normal building block

of chromatin

(the

nucleosome).

The bending

DNA at this structure

allows

proteins

bound

to the

flanking elements

become

part

of a

single complex.

Some components of the com-

plex (possibly

not those

that bind directly to

DNA) link the centromere

to the microtubule.

The construction

of kinetochores

probably

fol-

lows a similar

pattern,

and uses related compo-

nents,

in a wide variety of organisms.

Telomeres

Have Simple

Repeating

Sequences

The telomere is

required for the stability of the

chromosome

end.

A tetomere consists

of a simple repeat where a

C+A-rich strand

has the sequence C1(A/I)L4.

Another essential feature

in all

chromosomes

the telomere,

which

"seals"

the end. We

know

that the telomere

must

a special structure,

because

chromosome ends

generated

by break-

age are

"sticky"

and tend to react with other

chromosomes, whereas

natural ends are stable.

We can apply

two criteria in identifying a

telomeric sequence:

It must lie at the end of

chromosome

(or,

at least at the end of an authentic lin-

ear DNA molecule).

It must confer stability on a linear

molecule.

The

problem

of finding a system that offers

an assay for function again

has

been brought

to the

molecular level by using

yeast.

All the

plasmids

that

survive

yeast

(by

virtue

pos-

sessing

ARS and CEN elements) are

circular

DNA

molecules. Linear

plasmids

are unstable

(because

they are degraded). Could an authen-

tic telomeric DNA sequence confer stability on

a Iinear

plasmid?

Fragments from

yeast

DNA

that

prove

to be located at chromosome ends

can be identified by such an assay, and a region

from

the end of

a known natural linear DNA

748

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FIGURE 28.29

A loop forms

at the end of chromosomal

DNA. Photo

courtesy ofJack Griffith,

University of

North

Carolina

at Chaoel Hilt.

What feature of the telomere is responsi-

ble

for

the stability of

the

chromosome end?

FIGURE 28.29

shows that a

loop

of DNA forms at

the telomere. The absence of any free end may

be the crucial feature that stabilizes

the end of

the chromosome. The average length of the

loop in animal cells is 5 to l0 kb.

FIGURE

28,30

shows that the loop is

formed

when the 3' single-stranded end of the telo-

mere

(TTAGGG)"

displaces the same

sequence

an upstream region of the telomere. This con-

verts the

duplex

region into

a structure like a

D-loop, where a series of TTAGGG repeats

are

displaced to form a single-stranded region,

and

the tail of the telomere

naired with the homol-

ogous strand.

The reaction is catalyzed

by the telomere-

binding

protein

TRF2, which together with

other

proteins

forms a complex that

stabilizes the

chromosome

ends.

Its importance

protect-

ing

the ends is indicated by the fact

the

deletion of TRF2 causes chromosome

rearrange-

ments

to occur.

TRF2

FIGURF

?8.30 The

3'single-stranded

end ofthe tetomere

(TTAGGG)"

disptaces

the homologous repeats from

duplex

DNA

to form

a t-[oop. The reaction is

catatyzed by TRF2.

formed from

the second

guanine

each

repeat-

ing unit.

A series of

quartets

could

be stacked

Iike

this in a helical

manner. Although

the for-

mation

of this structure

attests to

the unusual

properties

of the

G-rich sequence

invitro, it does

not of course

demonstrate whether

the

ouartet

forms

in vivo.

Chromosomes

Te[omeres

Are

Synthesized by a

Ribonucleoprotein

Enzyme

Telomerase

uses the 3'-0H ofthe

G+T telomeric

strand to

prime

synthesis of tandem TTGGGG

reDeats.

The RNA

component of tetomerase has

a sequence

that

pairs

with the C+A-rich repeats.

One of the

protein

subunits is

reverse

transcriptase

that uses the RNA as temptate

to synthesis

the G+T-rich sequence.

The telomere has

two

functions:

One is to

protect

the chromosome

end.

Any other DNA

end-for example,

the

end

generated

by a

double-strand

break-becomes

a target for repair

sys-

tems. The cell has

to be able to

distin-

guish

the telomere.

The

second is to allow

the telomere

be extended.

If it is not

extended, it

becomes

shorter with

each replication

cycle

(because

replication

cannot start

at the very end).

Proteins

that bind to

the telomere

provide

the solution for

both

problems.

yeast,

differ-

ent sets of

proteins

solve each

problem,

but both

750

CHAPTER 28