Lewin Benjamin (ed.) Genes IX

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from

the removal

of exeLctly

3.7 kb

of DNA, the

precise

distance between

the

cx,l and u,2

genes.

It appears to have

been

generated

by unequal

crossing-over between

the

o,1 and

genes

themselves. This

precisely

the situation

depicted in Figure

6.13,,

Depending

on the

diploid

combination

thalassemic chromosomes,

an affected indi-

vidual may have

any number

of o, chains

from

zero to

three.

There

are few

differences

from

the wild type

(four

genes)

in individuals

with three

or two o

genes.

If an individual

has

only one o,

gene,

though,

the excess

chains

form

the unusual tetramer

F+,

which causes

hemoglobin H

(HbH)

disease.

The complete

absence

of cr

genes

results

in hydrops

fetalis,

which

fatal at or

before birth.

The same

unequal crossing-over

that

gen-

erated the thalassemic

chromosome

should also

have

generated

a chromosome

with three

genes.

Individuals with

such chromosomes

have

been identified

in several

populations.

some

populations,

the frequency

of the

triple o, Iocus

is about

the same as that

of the single

locus;

in others, the triple

genes

are

much /ess com-

mon

than single cr,

genes.

This suggests

that

(unknown)

selective factors

operate in

differ-

ent

populations

to adjust the

gene

levels.

Variations in

the number

of u

genes

are

found relatively frequently,

which

argues that

unequal crossing-over in

the cluster must

fairly common. It

occurs more

often in the a

cluster than in

the

cluster.

possibly

because

the

introns in

genes

are

much shorter

and

therefore

present

Iess irnpediment

mispair-

ing

between

nonhomologous genes.

The deletions

that cause

B-thalassemias

are

summarized

:;.!.iiiJ*,i

-i:.tF.

some

(rare)

cases,

only the

gene

is affected.

These have

a dele-

tion

600

bp, extending from

the second intron

through the l'flanking regions.

the other

cases,

than one

gene

of the cluster is

affected. Many of the

deletions are

very

long.

extending from the 5'end indicated

the map

for

>50

kb toward

the

right.

The Hb Lepore

type

provided

the classic

evidence that deletion c'an result

from unequal

crossing-over between linked

genes.

The

and

genes

differ only

-7"1>

sequence. Unequal

recombination

deletes the material

between the

genes.

thus

fusing

them together

(see

Fig-

ure 6.13). The fused

gene

produces

a single

p-like

chain that consists of the

N-terminal sequence

of 6

joined

to the C-terminal

sequence of

Several types

Hb Lepore

now are known,

the difference between

them lying in the

point

f :.irii=:1,:

ii,.i

tha[assemias

resutt from

various detetions

in the a-gl.obin

gene

c[uster.

tJ{r;4"iil{i

ii":.:, Deletions

in the

B-gtobin

gene

cluster cause

severaI types of tha[assemia.

of transition

from 6 to

sequences.

Thus when

the 6 and

genes pair for unequal

crossing-

over, the exact

point

recombination

deter-

mines the

position

at which

the switch

from

sequence

occurs

in the amino

acid

chain.

The reciprocal of

this event

has

been found

in the form of

Hb anti-Lepore,

which

pro-

duced by a

gene

that

has the N-terminal

part

and the C-terminal

part

6. The

fusion

gene

lies between

normal

5 and

genes.

Evidence that

unequal

crossing-over

can

occur

between

more distantly

genes

provided

by the identification

Hb Kenya,

another fused hemoglobin.

This

contains the

6.7

UnequaI

Crossing-over

Rearranges

Gene Clusters

t7l

N-terminal

sequence of the

\gene

and the C-

terminal sequence of

the

gene.

The fusion

must have resulted from

unequal crossing-over

between

and

which differ

-20%

sequence.

From

the differences between

the

globin

gene

clusters

various mammals,

we see that

duplication

followed

(sometimes)

variation

has

been an important feature

in the evolution

of each

cluster. The human thalassemic

dele-

tions

demonstrate that

unequal crossing-over

continues

to occur in

both

globin gene

clusters.

Each

such

event

generates

a duplication as well

as the

deletion, and we must

account

for

the

fate

of both recombinant

loci in the

population.

Deletions

can also occur

(in

principle)

by recom-

bination

between homologous

sequences

lying

the same chromosome. This

does not

gener-

ate a corresponding

duplication.

It is

difficult to estimate

the natural fre-

quency

of these

events, because

selective

forces

rapidly

adjust the levels

of the variant clusters

in the

population.

Generally a contraction in

gene

number is likely

to be deleterious and

selected

against. However, in

some

populations,

there

may be a

balancing advantage that main-

tains the

deleted form at a low frequency.

The

structures of the

present

human clus-

ters show

several duplications

that attest to the

importance

of such mechanisms.Th'e

functional

sequences

include

two o,

genes

coding the same

protein,

fairly

well related

and 6

genes,

and

two

almost identical

genes.

These

compara-

tively recent independent

duplications have

survived in

the

population,

not

to mention the

more distant

duplications

that originally

gen-

erated

the various

types of

globin

genes.

Other

duplications

may have

given

rise to

pseudo-

genes

or have been lost.

We expect

continual

duplication

and deletion

to be a feature

of all

gene

clusters.

Genes for rRNA

Form

Tandem

Repeats

Ribosomal

RNA is coded

by a large number

identicaL

genes

that are tandemty

repeated to form

one or more

clusters.

Each rDNA

ctuster is

organized so

that

transcription

units

giving

joint

precursor

to the

major rRNAs

alternate

with nontranscribed

spacers.

In the

cases

have

discussed

thus far, there

are

differences

between

the individual

mem-

CHAPTER

6 Ctusters

and Repeats

bers of a

gene

cluster that allow selective

pres-

sure to

act independently upon each

gene.

contrast

provided

two

cases of

large

gene

clusters that contain

many identical

copies of

the same

gene

genes.

Most

organisms con-

tain multiple copies of the

genes

for the histone

proteins

that are a

major

component of the chro-

mosomes, and there are almost always multi-

ple

copies of the

genes

that code for the

ribosomal RNAs. These situations

pose

some

interesting

evolutionary

questions.

Ribosomal

RNA is

the

predominant prod-

uct of transcription, constituting

some

80%-90% of the total

mass

of cellular RNA in

both eukaryotes and

prokaryotes.

The number

of major rRNA

genes

varies

from seven in

E. coli, 100 to 200

in lower

eukaryotes, to sev-

eral hundred in higher eukaryotes. The

genes

for

the

large and small rRNA

(found

the large

and small subunits of the ribosome, respec-

tively) usually

form a

tandem

pair. (The

sole

exception is the

yeast

mitochondrion.)

The lack of any detectable

variation

the

sequences of the

rRNA molecules

implies that

all the copies of each

gene

must

identical,

least must have

differences below the level

of detection in rRNA

(-I%\.A

point

of major

interest is what mechanism(s)

are used to

pre-

vent variations from accruins in

the individual

sequences.

In bacteria, the multiple rRNA

gene pairs

are

dispersed.

In most

eukaryotic nuclei, the rRNA

genes

are contained in a tandem

cluster or clus-

ters.

Sometimes

these regions

are called rDNA.

(In

some cases, the

proportion

of IDNA in

the

total DNA, together with its atypical

base com-

position,

great

enough to allow its isolation

as a separate fraction directly from

sheared

genomic

DNA.) An important

diagnostic fea-

ture of a tandem cluster is that it

generates

circular restriction map, as shown in

FiSlJftf, S.3S.

Suppose

that each

repeat

unit has

three

restriction

sites. When we map

these fragments

by conventional means,

find

that A is next

to B, which is next to C,

which is next to A,

generating

the circular map.

If the

cluster is

Iarge, the internal fragments

(A,

B, and

C) will

present

in much

greater quantities

than the

terminal fragments

and Y),

which connect

the

cluster to adjacent DNA.

In a

cluster of 100

repeats, X

and Y would be

present

at I

the

Ievel

B. and C. This

can make it difficult

to obtain

the ends of a

gene

cluster for map-

pmg purposes.

The region

of the

nucleus

where rRNA

syn-

thesis

occurs has a characteristic

appearance,

tt2

l:.l.rii::

t-:.:.r'

tandem

gene

ctuster has

an alternation of transcription

unit and nontranscribed

spacer and

generates

a cjrcular restriction

map.

with a core

fibrillar

nature

surrounded

by a

granular

cortex. The fibrillar

core is

where the

rRNA is

transcribed from

the

DNA template,

and the

granular

cortex is formed

the ribonu-

cleoprotein

particles

irLto

which

the rRNA is

assembled. The

whole

area is called

the nucle-

olus. Its characteristic

.morphology

evident

lfl

!ir:i::r1: r:

The

particular

chromosomal

regions

asso-

ciated with a nucleolus

are

called nucleolar

organizers. Each

nucleolar

organizer

corre-

sponds

to a cluster

of tandemly repeated

rRNA

genes

on one chromosome.

The

concentration

of the tandemly repeated

rRNA

genes,

together

with their very intensive

transcription, is respon-

sible for creating the

characteristic

morphology

the nucleoli.

The

pair

of major rRNAs

is transcribed

as a

single

precursor

in both

bacteria

and eukary-

otic

nuclei.

Following

transcription,

the

precur-

sor is cleaved to release

the individual

rRNA

molecules. The

transcription

unit is

shortest in

bacteria and is longest in

mammals

(where

it is

known as 45S RNA,

accrrrding

to its rate of

sed-

imentation). An rDNA

cluster

contains many

transcription

units, each

separated from

the

by a nontranscribed

spacer. The

alter-

nation of transcription

unit

and nontranscribed

spacer can be

seen directly in

electron micro-

graphs.

The

example shown in

i-ii,i:itt

ii,

iiii

taken from

the newt Not|opthalmus

viridescens,in

which

each transcription

unit is intensively

i'ii

i.t;r;1

,:1,

The nucleo[ar core

identifies

rDNA

under

transcription, and the surrounding

granutar

codex consists

of assembling ribosomaI subunits.

This thin sectjon shows

the nucteotus ofthe

newt NotopthoLmus viidescens.

Photo

courtesy of 0scar

Mitter.

expressed,

that many RNA

polymerases

are

simultaneously engaged

in transcdption on

one

repeating

unit.

The

polymerases

are

so closely

packed

that the

RNA transcripts

form a char-

acteristic matrix displaying

increasing

length

along the transcription

unit.

6.8 Genes

for

rRNA Form Tandem Repeats

773

:j:..i=:*i:

r;.

i:-i

Transcription of rDNA ctusters

generates

series of

matrices, each corresponding to one transcrip-

tion unit and separated

from

the

the nontran-

scribed spacer.

Photo

courtesy of 0scar

Mi[ter.

:i....,--ij::=.i:j

ThenontranscribedspacerofX. laevrsrDNAhasaninternatly

repetitious

structure that is responsibte

for its variation

in [ength. The Bam

is[ands

are short constant sequences that separate the repetitious

regions.

The

Repeated Genes

for rRNA Maintain

Constant Sequence

The

genes

rDNA

cluster a[[

have

identical

sequence.

The nontranscribed

spacers consist of shorter

repeating

units whose number varies

that the

lengths of individuaI spacers are different.

The nontranscribed

spacer

varies

widely

Iength

between and

(sometimes)

within

species. In

yeast

there is a short nontranscribed

spacer that is relatively

constant in length. In

D. melanogaster

Ihere is almost a twofold vari-

;:,Ti:ii:iT:*:j;[::?ilTl:;:Hil'ffi:

A similar

situation

seen in X. laevis.In each

of these

cases, all of the repeating units are

present

as a single tandem cluster on one

particular

chromosome.

(In

the example of

CHAPTER 6 Ctusters

and Repeats

D. melanogaster,Ihis

happens to

the

sex

chro-

mosome. The cluster on the X chromosome

Iarger than

the

one

on the Y chromosome, so

female flies

have more copies of the rRNA

genes

than male flies.)

In mammals the repeating unit is very much

larger, comprising the transcription unit of

-13

kb and a nontranscribed spacer of

-30

kb.

Usually. the

genes

lie in several dispersed

clusters-in the

case of man and mouse the

clusters reside on

five

and six chromosomes,

respectively. One interesting

(but

unanswered)

question

is how the corrective

mechanisms

that

presumably

function within a single cluster to

ensure constancy of

rRNA

sequence are able to

work when there are several clusters.

The

variation

in length of the nontran-

scribed spacer in a single

gene

cluster contrasts

with

the conservation of sequence of the tran-

scription unit.

spite

of this variation,

the

sequences of longer

nontranscribed

spacers

remain homologous with those of the shorter

nontranscribed spacers. This implies that each

nontranscribed spacer

internally repetitious, so

that the variation

in length results from

changes

in the number of repeats of some subunit.

The

general

nature of the nontranscribed

spacer

is illustrated

the example

laevis.

ft+UFt[

1':*

illustrates the situation. Regions that

are fixed in length alternate with regions that

vary.

Each

of the three

repetitious regions

com-

prises

a variable number of repeats of a rather

short sequence. One type of repetitious region

has repeats of. a 97 bp sequence; the other, which

occurs

two

locations, has

repeating

unit

found in two forms, 60 bp and 8l bp long. The

variation in the number of repeating

units

the repetitious regions accounts for the overall

variation in spacer length. The repetitious

regions are separated by shorter constant

sequences

called Bam islands.

(This

descrip-

tion takes its name

from

their isolation

via the

use of

the BamHI restriction

enzyme.) From

this type of organization, we see that the clus-

ter has evolved by duplications involving

the

promoter

region.

We need to explain the lack

variation

the expressed copies of the

repeated

genes.

One

model would suppose that there is

quantita-

tive demand

for

a certain

number

of.

good"

sequences. This would, however,

enable

mutated

sequences to accumulate up

to a

point

at which their

proportion

of the cluster is

great

enough

for

selective

pressure

to be exerted.

can

exclude such models because of the lack

such variation in

the cluster.

Repetitious

Bepetitious

Repetitious

region 1

8"n.,

,"gion,

B",

region

island island

ffiffiffi

-500

-300

97 bp

repeats

60/81

bp repeats

60/81 bp repeats

Variable length

774

The lack

of variation

implies

the

existence

of selective

pressure

some

form

that is

sensi-

tive

individual

variations.

One model

would

suppose

that the entire

cluster is

regenerated

periodically

from

one or from

a very

few

mem-

bers. As

practical

matter,

any mechanism

would

need

to involve

regeneration

every

generation.

We can

exclude

such models

because

a regen-

erated cluster would

not

show variation

the

nontranscribed

regions

of the individual

repeats.

are

left

with

a dilemma.

Variation

the

nontranscribed

regions

suggests

that there

frequent

unequal

crossing-over.

This

will change

the size

of the cluster,

but

will not

otherwise

change the

properties

of the individual

repeats.

So how

are mutations

prevented

from

accumu-

lating?

We'll see in

the next

section

that

con-

tinuous

contraction

and

expansion

of a

cluster

may

provide

a mechanism

for

homogenizing

its copies.

functions

between

embryonic and adult

globin

genes.

Yet there

are instances

which duplicated

genes

retain

the same function, coding for the

identical

or nearly identical

proteins.

Identical

proteins

are

coded by the

two human

cr-globin

genes,

and

there is only a single amino acid dif-

ference

between the two

y-globinproteins.

How

selective

pressure

exerted to

maintain

their

sequence

identity?

The most

obvious

possibility

is that

the two

genes

not

actually

have identical functions,

but instead

differ in some

(undetected) property,

such as time

place

of expression.

Another

possibility

is that the need for two copies is

quan-

titative, because neither by itself

produces

a suf-

ficient

amount

protem.

In more

extreme cases of

repetition, how-

ever, it is

impossible to avoid the conclusion

that no single copy

the

gene

is essential. When

there

are many copies of a

gene,

the

immedi-

ate

effects of mutation in any one copy must be

very slight. The

consequences

of an individual

mutation

are diluted by the

large number

copies of the

gene

that retain the wild-type

sequence.

Many mutant copies could

accumu-

late

before a lethal effect is

generated.

Lethality becomes

quantitative,

a conclu-

sion reinforced

the observation that half of

the units

of the rDNA cluster of.

X. laevis or

melanogaster canbe deleted without

ill

effect.

So how

are these units

prevented

from

gradu-

ally accumulating

deleterious

mutations?

What

chance is

there

for

the

rare favorable mutation

display its advantages

the cluster?

The basic

principle

of models to explain the

maintenance

identity among repeated copies

to suppose that nonallelic

genes

are

not inde-

pendently

inherited, but rather must be con-

tinually regenerated from one of the copies of a

preceding generation.

In the simplest

case of

two identical

genes,

when a mutation occurs

one copy, either

it is

chance eliminated

(because

the

sequence

of the other copy

takes

over), or it is spread to both

duplicates

(because

the mutant

copy becomes

the dominant ver-

sion). Spreading exposes a

mutation to selection.

The result is that the two

genes

evolve

together

as though

only a single

locus existed.

This is

called coincidental evolution

or concerted

evolution

(occasionally

coevolution).

It can

be applied

to a

pair

identical

genes

(with

further

assumptions) to a cluster

containing

many

genes.

One mechanism supposes

that the se-

quences

of the

nonallelic

genes

are directly

Crossover

Fixation

Could

Maintain

ldenticaL

Repeats

UnequaI crossing-over

changes

the size

of a ctuster

of tandem repeats.

Individual

repeating

units

can be eliminated

can spread through

the cluster.

The

same

problem

encountered

whenever a

gene

has been

duplicated.

How

can selection

be imposed

prevent

the accumulation

of dele-

terious mutations?

The duplication

of a

gene

is likely

ro result

in an immediate

relaxation

of the evolutionary

pressure

on its

sequence.

Now that

there are

two identical

copies, a

change in

the sequence

of either one will not

deprive

the

organism of

functional protein,

because

the

original amino

acid

sequence continues

be coded

by the

other copy. Then

the

selective

pressure

the

two

genes

is diffused,

until

one of

them mutates

sufficiently

away from

its

original function

refocus

all the

selective

pressure

the other.

Immediately

following

gene

duplication,

changes might accumulate

more rapidly in

one

of the copies, leading

eventually

a new func-

tion

(or

to its

disuse in

the form

of a

pseudo-

gene).

If a new function

develops,

the

gene

then

evolves

at the same,

slower rate

characteristic

of the original function.

Probably

this is

the sorr

mechanism

responsible

for

the separation

6.10

Crossover

Fixation Coutd Maintain

ldentical Repeats 115

compared with one

another and

homogenized

by enzymes that

recognize any differences.

This

can be done by exchanging

single strands

between them

to form

genes,

one

of whose

strands derives

from

one

copy, and one

from

the other copy. Any differences

are revealed as

improperly

paired

bases, which attract

atten-

tion from enzymes able to

excise and replace

base,

so that only A-T and G-C

pairs

survive.

Ihis type of event

is called

gene

conversion

i^}.t,il#,ffi :"5i.Tj'1.?".i"',l..;:::S?:i:f;;

events by comparing the sequences

of dupli-

cate

genes.

they

are subject to concerted

evo-

lution. we should

not

see

the accumulation of

silent site substitutions

between them

(because

the homogenization

process

applies

to these as

well

to the replacement sites). We

know that

the extent of the

maintenance mechanism

need

::,'.','Ti'.i;',?#JT"Tff

'ffi?,:",i"Ti,T;

sequences are entirely

different. Indeed, we

may see abrupt boundaries that

mark the ends

of the

sequences

that were homogenized.

We must remember that the existence

such mechanisms can

invalidate

the

determina-

tion of the history of such

genes

via their diver-

?ffii;lf iLTi,|,i;!:,:::,,:;,i;;!;:::;1,:l;:'::,

the original dup lication.

The

crossover

fixation model supposes

that an entire cluster

subject

to continual

rearrangement by the

mechanism

of unequal

crossing-over. Such

events can explain the con-

certed evolution of

multiple

genes

if unequal

crossing-over causes all the copies to be

regen-

erated

physically

from one copy.

Following the sort of event depicted

Figure 6.13, for example, the chromosome car-

rying a triple locus could suffer deletion of one

of the

genes.

Of the two remaining

genes,

represent

the sequence of

one

the original

copies;

only k of

the

sequence of

the other orig-

inal

copy

has

survived.

Any mutation in the

first region now exists in both

genes

and

sub-

ject

to selective

pressure.

Thndem

clustering

provides

frequent

oppor-

tunities for

"mispairing"

genes

whose

sequences

are the same. but that

lie in

different

positions

their clusters. By continually

expanding and contracting the number of units

via

unequal crossing-over, it is

possible

for all

the units in one cluster to be derived from rather

a small

proportion

those in

an ancestral

clus-

ter. The variable lengths of the spacers are con-

sistent with the idea that unequal crossing-over

CHAPTER

6 Ctusters and Repeats

events take

place

in spacers

that are internally

mispaired.

This can explain the

homogeneity

of the

genes

compared

with the variability

the spacers.

The

genes

are

exposed to selection

when

individual

repeating units are amplified

within the

cluster;

however, the spacers are

irrelevant and can

accumulate changes.

In a region of

nonrepetitive DNA, recom-

bination occurs

between

precisely

matching

points

on the two

homologous chromosomes,

thus

generating

reciprocal

recombinants. The

basis for this

precision

is the ability of two

duplex

DNA sequences

to align exactly. We

know that

unequal

recombination

can occur when there

are

multiple copies of

genes

whose exons are

related, even though

their flanking and inter-

vening sequences

may differ. This

happens

because

of the mispairing

between correspon-

ding exons

in nonallelic

genes.

Imagine

how much more frequently

mis-

alignment must occur

in a tandem cluster of

identical or

nearly identical

repeats. Except at

the

very ends of the

cluster, the close relation-

ship between

successive

repeats makes it impos-

sible even

to define the exactly corresponding

repeats! This has two

consequences: there is

continual

adjustment of the size of the cluster;

and there is homogenization

of the repeating

unit.

Consider

a sequence consisting of a

repeat-

ing unit

"ab"

with

ends

"x"

and

"y."

If we rep-

resent one chromosome

in black and the other

color,

the exact alignment between

"allelic"

sequences would be:

xababababababababababababababababy

xabababa

ba ba baba babababa babababy

is likely, however, IhaI any sequence ab

in one chromosome could

pair

with any

sequence ab

intL'e

other

chromosome. In a mis-

alignment

such as:

xababababababababababababababababy

xababababa ba ba ba ba ba ba ba ba ba ba ba by

the region of

pairing

is no less

stable than in the

perfectly

aligned

pair,

although it is

shorter. We

do not know very much about how

pairing

initiated

prior

recombination,

but very likely

it starts between short corresponding regions

and then spreads. If it starts within satellite DNA,

it is more

likely

than

not to involve repeating

units

that do not have exactly

corresponding

Iocations in their clusters.

Now

suppose that a

recombination

event

occurs within the unevenly

paired

region. The

recombinants

will

have different numbers

776

repeating

units.

In one

case,

the

cluster has

become longer;

the

other, it

has

become

shorter,

ffi&#&&HbdhH$nbghabababa

bababa

baby

Xr,

ba ba ba

baba ba

b'c&iiFghti'&d

dsa,bb,bfr:

xabababababalaUan*a

la na ba

ba ba ba

bababa bv

xababababababababababababababy

where

"x"

indicates

the

site

of the

crossover.

If this type

of event

common,

clusters

tandem repeats

will

undergo

continual

expan-

sion

and contraction.

This

can

cause a

particu-

lar

repeating

unit to

spread

through

the cluster,

as illustrated

iri::ljfitr

r.r.;ii.

Suppose

that the

cluster

consists initially

of a sequence

abcde,

i.i{:iiiir r:.li:

Unequal recombination

altows

one

partic-

utar

repeating

unit

to occupy the

entire ctuster. The num-

bers

indicate

the Length

of the repeating

unit

at each

stage.

where each letter represents

a repeating unit.

The

different repeating units are closely enough

one another to

mispair for recombi-

nation.

Then by a series of unequal recombi-

nation

events,

the size of the

repetitive region

increases

or decreases, and one unit spreads to

replace

all

the others.

The

crossover fixation model

predicts

that

any

sequence of DNA that is not under selective

pres-

sure

will

be taken

over by

a series of identical tan-

dem

repeats

generated

in this way. T):re critical

assumption is

that the

process

of crossover fix-

ation is

fairly rapid relative to mutation, so that

new

mutations either are eliminated

(their

repeats

are lost)

or come

to take over the entire

cluster. In

the case of the

rDNA

cluster, of course,

further

factor is imposed by selection for an

effective

transcribed seouence.

.ifficdcde

affide

abbbbbcdcde-

dmmluitude

ffiSlffftdcde

bbbbbr:&de

Satellite

DNAs

0ften

Lie

Heterochromatin

HightV

repetitive DNA has a very short

repeating

sequence

and no coding

function.

It occurs in large blocks that can

have

distinct

physicaI properties.

is often the major constituent of centromeric

heteroch romati n.

Repetitive

DNA is defined

by its

(relatively)

rapid

rate

of renaturation. The component

that rena-

tures most rapidly in a eukaryotic

genome

called highly repetitive DNA and consists of very

short sequences repeated many times

tan-

dem in large

clusters.

As a result of

its

short

repeating

unit, it is sometimes

described as sim-

ple

sequence DNA. This type of component

present

in almost all

higher eukaryotic

genomes,

but its overall amount

is extremely

variable.

In mammalian

genomes

it is typically

<l0o/o,

but in

(for

example)

Drosophila virilis, it

amounts to

-5O"/o.In

addition

to the large clus-

ters in which this type of sequence was

origi-

nally

discovered, there

are smaller clusters

interspersed

with nonrepetitive

DNA. It typi-

cally consists of short sequences

that are repeated

in identical

related copies

the

genome.

The

tandem

repetition of a short sequence

often creates a fraction with

distinctive

physical

properties

that can

be used to

isolate it. In some

cases, the repetitive sequence

has a base com-

position

distinct from the

genome

average, which

allows it to form a separate

fraction by

virtue

its distinct buoyant density.

A fraction of this sort

6.11

Sate[tite

DNAs

0ften

Lie in Heterochromatin

777

Mouse

DNA

seoarated

into a main band

and a sateltite by centrifugation

through a density

gra-

dient of CsCL.

called satellite

DNA. The term satellite DNA

essentially synonymous with simple

sequence

DNA. Consistent with its simple sequence,

this

DNA

not transcribed or translated.

Thndemly repeated sequences are especially

liable

to undergo

misalignments

during

chromo-

some

pairing,

and thus the sizes of tandem

clus-

i;T:

11"'*:

*T#i : :ililffi il

"-,:

:,'f

smaller clusters of such sequences

can be used

to characterize individual

genomes

in the tech-

rique of

"DNA

fingerprinting"

(see

Section 6.14,

Minisatellites Are Useful for Genetic Mapping).

The

buoyant

density

a duplex DNA

depends on

its

G-C content according

to the

emoirical formula

1.660

0.00098

(%c-cr

g-cm-r

Buoyant density usually is determined by

centrifuging DNA through a density

gradient

of CsCl. The DNA forms a band at the

position

corresponding to

its

own density. Fractions of

:,il13'i'"",:ii,:i :" #:::ri',f,i Jran

u s u

When eukaryotic DNA is

centrifuged

on a

density

gradient,

two types of material may be

distinsuished:

Nlurt

of the

genome

forms a continuum

fragments

that appear as a

rather

broad

peak

centered on the buoyant

density corresponding to the average G-

C content of the

genome.

This is called

the main

band

Sometimes an additional, smaller

peak

(or peaks)

seen at a different value.

This material is

the satellite

DNA.

CHAPTER

6 Ctusters

and Reoeats

Satellites

are

present

in many eukaryotic

genomes.

They

may be either heavier or

lighter

than the

main band, but

it is uncommon

for

them to represent

>5%

of the total

DNA. A clear

example

provided

by mouse

DNA.

shown

i ii,riirr:: r:;,.i ,.

The

graph

is a

quantitative

scan of

the bands

formed when mouse

DNA is

cen-

trifuged

through a

CsCl density

gradient.

The

main band contains

92'k of the

genome

and is

centered on

a buoyant density

of 1.70I

g-cm-;

(corresponding

to its average G-C of

42%,

typ-

ical

for

mammal).

The

smaller

peak

represents

8'/. of.

the

genome

and

has a distinct buoyant

density of

1.690

g-cm-;. It

contains

the mouse

satellite

DNA,

whose G-C content

(30%)

much lower than any

other

part

of the

genome.

The behavior of satellite

DNA on density

gradients is

often

anomalous. When the

actual

base

composition of a satellite

is determined, it

is different

from the

prediction

based on

its

buoyant density.

The reason is that

is a

func-

tion not

just

of base composition,

but of the

constitution

in terms of nearest

neighbor

pairs.

For simple sequences,

these are likely to devi-

ate from the

random

pairwise

relationships

needed to obey the equation

for buoyant den-

sity.

In addition, satellite

DNA may

meth-

ylated,

which

changes its density.

Often,

most

the highly repetitive DNA

genome

can be

isolated in the form

of satel-

lites.

When a highly repetitive DNA component

does not separate

as a satellite, on isolation its

properties

often

prove

to be similar to those of

satellite

DNA. That

to say,

highly repetitive

DNA consists

of multiple tandem repeats with

anomalous centrifugation.

Material isolated in

this manner is sometimes

referred to as a

cryp-

tic satellite. Together

the cryptic and appar-

ent satellites usually account

for

all the

large

tandemly repeated blocks of

highly repetitive

DNA.

When

genome

has more than one type

of highly repetitive

DNA,

each exists

in its

own

satellite block

(although

sometimes different

blocks are adjacent).

Where

the

genome

are the

blocks of

highly

repetitive DNA located? An

extension

of nucleic acid

hybridization techniques

allows

the

location

satellite sequences to

be deter-

mined directly in the chromosome complement.

In the technique of

situ

hybridization,

the

chromosomal DNA

denatured by treating cells

that have been squashed on

cover slip. Next,

a solution containing

a radioactively

labeled

DNA

RNA

probe

is added. The

probe

hybridizes

with

its complements in the

dena-

tured

genome.

The location of the

sites of

118

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