Lewin Benjamin (ed.) Genes IX

f}*tJ*il

3.$ Comparison of the restriction maps of cDNA and

genomic DNA for

mouse

B

globin

shows that the

gene

has two introns that are

not

present

in

the cDNA. The

exons can be aliqned exactlv between cDNA and

qene.

.Fg*LiRg

3"$

An intron

is a sequence

present

in the

gene

but absent

from the

mRNA

(here

shown

in

terms ofthe cDNA sequence). The reading frame is

indicated by the

atternating

open and shaded blocks; note that altthree

possible

reading frames

are btocked

by termi-

nation

codons in the intron.

Ultimately, a comparison

of the

nucleotide

sequences of the

genomic

and mRNA sequences

precisely

defines the introns. As indicated in

ft$#ftE

s"$,

an intron usually has no

open

read-

ing frame. An intact reading frame

is created

in

the mRNA sequence by the removal of the

introns.

The structures of eukaryotic

genes

show

extensive variation. Some

genes

are uninter-

rupted, so that the

genomic

sequence is colin-

ear with

that of the mRNA. Most higher

eukaryotic

genes

are interrupted,

but the

introns

vary enormously

in

both number and size.

All classes of

genes

may

be interrupted:

nuclear

genes

coding for

proteins,

nucleolar

genes

coding for rRNA, and

genes

coding Ior

IRNA.

Interruptions also are found in mito-

chondrial

genes

in

lower

eukaryotes and in

chloroplast

genes.

Interrupted

genes

do

not

appear

to be excluded from any class of eukary-

otes and

have

been

found in

bacteria

and

bac-

tedophages.

They are. however,

extremely

rare

in

prokaryotic genomes.

Some

interrupted

genes

possess

only one

or a few introns.

The

globin

genes

provide

an

extensively

studied

example

(see

Section 3.10,

The Members of a Gene

Family Have

a Common

Organization).

The

two

general

types

of

globin

gene,

o and

B,

share

a

common

type of

struc-

ture.

The consistency

of

the organization

of

mammalian

globin

genes is evident

from the

structure of

the

"generic" globin gene

summa-

rized in

l;i;i.iltF, :. i.

Interruptions

occur

at

homologous

posi-

tions

(relative

to

the coding

sequence)

in all

known

active

globin

genes,

including

those

of

mammals,

birds, and

frogs.

The

first intron

is

always fairly short,

and

the

second

usually

is

longer, but the

actual

lengths

can vary.

Most

of

the variation

in overall

lengths

between

differ-

ent

globin genes results

from the

variation in

the second

intron.

In

the

mouse, the

second

intron in the

cr-globin

gene is only

150 bp

long,

so the overall

length of

the

gene

is 850

bp, com-

pared

with the

major

p-globin

gene

for which

the intron

length of

585

bp

gives

the

gene

a

Exon

Blocked Blocked

Blocked

Gene sequence

mRNA

seouence

Ser Leu Leu Ser Arg Asn Ser Trp Cys

Ph€

3.4

0rganization

of

Interrupted

Genes

May

Be Conserved

4l

Intron length

116-130

.+

Exon length 142-145

I

Y

Contains 5'UTR

+

coding 1-30

222

V

Amino

acids

31

-1

04

216-255

I

v

Coding

105-end

+

3' UTR

iI{iJR[

3.?

At[ functionaL

gtobin genes

have an interrupted structure

with three exons. The

lengths

indicated

in the figure

appty to the mammalian

p-gtobin

genes.

iiGL;FA

-:.S

Mammatian

genes

for

DHFR have

the same

retative

organization

of rather

short exons

and

very

[ong

jntrons,

but vary

extensively in

the lengths

of

introns.

total length

of

1382

bp. The

vadarion in length

of

the

genes

is

much

greater

than the range

of

lengths

of the

mRNAs

(u-globin

nRNA

=

585

bases;

B-globin

nRNA

=

620 bases).

The

example

of DHFR,

a somewhat larger

gene,

is

shown in

FIilURf

l.*. The mammalian

DHFR

(dihydrofolate

reductase) gene

is

orga-

nized into

six

exons that correspond

to

the

2000-

base mRNA.

They

extend over

a much

greater

Iength

of DNA

because the introns

are

very

long.

In

three mammals

the

exons remain

essentially

the

same, and

the relative

positions

of the

introns

are

unaltered. The

lengths

of

individ-

ual introns

vary

extensively,

though, resulting

in a variation

in

the length

of the

gene

from25

ro 3l

kb.

The

globin

and

DHFR

genes present

exam-

ples

of a

general

phenomenon:

Genes that

are

have related

organizations

with conservation

of the

positions

0f

(at

least

slme)

of the introns.

Variations

in the lengths

of the

genes

are

primarily

determined

by the

lengths of the

introns.

12

3 4

5

6Exons

051015202530

KO

Exon

Sequences

Are

Conserved

but Introns

Vary

o

Comparisons of retated

genes

in

different

species

show that the sequences

of the corresponding

exons are usua[[y conserved

but the sequences

of

the introns

are

much

less we[[ related.

r

Introns

evotve much more rapidly

than

exons

because of the lack of selective

Dressure to

produce

a

protein

with

a useful sequence.

Is a

structural

gene

unique in its

genome?

The

answer can be ambiguous. The

entire length

of

the

gene

is

unique as such, but its

exons

often

are related to

those of other

genes.

As

a

gen-

eral rule,

when two

genes

are related.

the rela-

tionship between their

exons is closer

than the

relationship

between

their introns.

In

an

extreme case, the

exons of two

genes

may

code

for the

same

protein

sequence,

whereas

the

introns

may be different.

This implies

that

the

two

genes

originated

by a duplication

of

some

common ancestral

gene.

Then

differences

accu-

mulated between

the copies,

but they

were

restricted

in the

exons by the need

to code

for

protein

functions.

As

we will see later

when we

consider

the

evolution

of the

gene,

exons

can be considered

basic building

blocks that

are assembled

in var-

ious combinations.

A

gene

may have

some

exons that

are related to exons

of another gene,

but

the other exons may

be unrelated.

Usually

the introns

are not related

at all in

such cases.

Such

genes

may

arise by duplication

and

translo-

cation of individual

exons.

The relationship

between

two

genes

can

be

plotted

in

the form of the

dot matrix

compari-

son

of rifiLJft.fl

"]"s.

A

dot

is

placed

to indicate

each

position

at which the

same sequence

is found

42

CHAPTER

3

The

Interrupted

Gene

In corresponding

introns,

the

pattern

of

divergence involves

both changes

in size

(due

to deletions

and insertions)

and base

substitu-

tions. Introns evolve

much

more rapidly

than

exons.

When

a

gene

is compared

in different

species, there are

times

when its exons

are

homologous but

its introns

have diverged

so

much

that

corresponding

sequences

cannot be

recognized.

Mutations occur

at the same

rate

in both

exons and

introns, but

are removed

more effec-

tively from the exons

by adverse

selection.

How-

ever, in the absence

of the constraints

imposed

by a coding

function,

an intron

is able

quite

freely to accumulate

point

substitutions

and

other changes.

These changes

imply that the

intron does

not have a sequence-specific

func-

tion. Whether

its

presence is at all

necessary

for

sene

function is

not clear.

Genes

Show

a Wide

Distribution

of

Sizes

o

Most

genes

are

uninterrupted

in

yeasts,

but are

interrupted in higher eukaryotes.

.

Exons are usua[[y

short,

typicaLty

coding

for

<100

amino acids.

r

Introns are short

in lower

eukaryotes,

but

range

up to several

10s of kb

in Length

in higher

eukaryotes.

o

The overa[[ length

of a

gene

is determined [argety

by

its introns.

I'i{;r-ifiil

;1

,1i:

shows

the overall

organization

of

genes

in

yeasts, insects, and

mammals.

In

Saccharomyces

cerevisiae,

the

great

majority

of

genes

(>96%)

are not

interrupted,

and

those

that have exons usually

remain

reasonably

com-

pact.

There are virtually

no S. cerevisiae

genes

with

more than

four exons.

In insects

and

mammals

the situation

is

reversed. Only

a

few

genes have uninterrupted

coding sequences

(6%

in mammals).

Insect

genes

tend

to have

a

fairly small

number

of

exons-typically

fewer than

10.

Mammalian

genes

are split

into

more

pieces,

and

some

have

several

l0s

of

exons.

Approximately

50%

of

mammalian

genes

have

>10 introns.

Examining

the consequences

of

this type

of. organization

for the

overall

size of

the

gene,

we see

in iir"-lJFli.

.l.': i that

there

is a striking

dif-

ference between

yeast

and the

higher

eukary-

otes.

The average

yeast

gene

is

1.4 kb

long, and

ni.{'{.Jftil

},.;;

The sequences

of the

mouse

omar- and am,n-gtobin

genes

are ctosely related in

coding

regions

but differ in the

fLanking regions and long

jntron.

Data

provided

by Phitip Leder,

Harvard MedicaI Schoo[.

in

each

gene.

The

dots form a line at an angle

of,45" if two sequences are identical. The line

is broken by regions that lack

similarity and is

displaced

laterally

or vertically by deletions or

insertions in one sequence relative

to the other.

When the two

p-globin

genes

of the mouse

are compared, such a line extends

through the

three

exons

and through

the small intron. The

line

peters

out

in

the flanking regions and in

the large intron. This is a typical

pattern,

in

which coding sequences are well related and

the relationship can extend

beyond the bound-

aries of the exons.

The

pattern

is lost, though,

in

longer introns

and the regions on either side

of the

gene.

The

overall

degree

of divergence between

two exons is related to the differences between

the

proteins.

It is caused mostly by base substi-

tutions. In the translated regions,

the

exons are

under

the constraint

of

needing

to code

for

amino

acid sequences, so they

are

limited in

their

potential

to change sequence.

Many

of

the changes do not affect codon meanings,

because they change one codon into another

that represents the same amino acid. Changes

occur

more freely in nontranslated regions

(cor-

responding to the 5'leader and 3'trailer of the

mRNA).

3.6

Genes

Show

a Wide

Distribution

of Sizes

BO

60

40

20

OJU

fro

10

15

10

5

12345

678910

12 14

20

<40 >60

r

'':'

Most

genes

are uninterrupted

in

yeast,

but most

genes

are interrupted

in flies

and mammals. (Uninterrupted

genes

have

only one exon

and are totaled in the

leftmost cotumn.)

Yeast

genes

are

short, but

genes

in flies

and

mammals

have a

dispersed distributjon

extending

to very

[ong sizes.

very

few are longer than

5

kb.

The

predomi-

nance

of

interrupted

genes

in

high eukaryotes,

however, means

that the

gene

can

be

much

larger

than the unit that codes for

protein.

Rel-

atively

few

genes

in flies or mammals

are shorter

than 2 kb, and many have lengths

between

5 kb and 100 kb. The average

human

gene

is

27 kb long

(see

Figure

5.11).

The

switch from largely uninterrupted

to

largely interrupted

genes

occurs in the lower

eukaryotes. In fungi

(except

the

yeasts),

the

majority of

genes

are interrupted,

but they have

a relatively

small

number

of exons

(<6)

and are

fairly

short

(<5

kb). The switch

to

long

genes

occurs within the higher

eukaryotes, and

genes

become significantly larger

in the insects.

With

this increase in

the

length

of the

gene,

the rela-

tionship

between

genome

size

and organism

complexity is lost

(see

Figure 4.5).

As

genome

size increases,

the tendency is

for introns

to become rather large,

whereas

exons remain

quite

small.

i

l.ij

-iiii

'"

i

,r shows that the

exons coding for

stretches of

protein

tend

to be fairly small.

In

higher

eukaryotes, the average

exon

codes

for

-50

amino acids,

and the

general

distribution

fits well

with the idea that

genes

have evolved

<0.5

<1 <2

<5 <10<25<50<100>100

Size of

gene

(kb)

CHAPTER

3

The

Interrupted

Gene

Exons coding for

proteins

usuatly are

short.

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q$ue1-ye6

Exon 1

lntron Exon 2 lntron

Exon 3

I

RNA

synthesis

v

Only

introns

spliced out

5',

J

Long

protein

has all 3 exons

lntrons

+

exon

2

soliced out

s',

3',

5',

3,mRNA

c

Short

protein

has only exon

1+

exon

3

fC&**f

3

"

t ? Alternative

spticing uses the

same

pre-m

RNA to

generate

m RNAs

that have different

combinations

of exons.

Sometimes

two

pathways

operate simulta-

neously, with

a certain

proportion

of the RNA

being spliced

in

each way. Sometimes the

path-

ways are

alternatives that are expressed under

different

conditions-one

in

one cell type and

one

in

another

cell type.

So alternative

(or

differential) splicing

can

generate

proteins

with overlapping sequences

from a single

stretch of DNA. It is curious that

the higher eukaryotic

genome

is

extremely

spa-

cious and

has large

genes

that are often

quite

dispersed, but at

the

same

time it may make

multiple

products

from

an

individual Iocus.

Alternative splicing expands the

number of

pro-

teins relative

to the number of

genes

by

-I5%

in flies and worms, but

has much

bigger

effects

in man,

for

which

-60'h

of

genes

may have

altemative

modes of expression

(see

Section 5.5,

The Human Genome

Has Fewer Genes Than

Expected). About 80% of the alternative

splic-

ing

events

result in a change

in

the

protein

seouence.

How

Did Interrupted

Genes

Evolve?

o

The major evotutionary

question

is whether

genes

originated as

sequences

interrupted

by introns

or

whether they

were

originatty

uninterrupted.

r

Most

protein-coding

genes

probably

originated

in

an interrupted

form.

but

interrupted

genes

that

code

for

RNA

may

have originatly

been

uninterruoted.

.

A special

class of

introns

is

mobile and

can insert

itsetf into

genes.

The

highly interrupted

structure

of

eukaryotic

genes

suggests

a

picture

of

the eukaryotic

genome

as a sea

of

introns

(mostly but not

exclu-

sively unique

in sequence),

in which

islands

of

exons

(sometimes very

short)

are

strung

out in

individual

archipelagoes

that

represent

genes.

What

was the

original

form of

genes

that

todav

are interrupted?

3.8

How

Did Interrupted

Genes

Evo[ve?

47

.

The

"introns

early" model

proposes

that

introns

have always

been an integral

part

of the

gene.

Genes originated

as

interrupted

structures, and

those with-

out introns

have lost

them in the

course

of evolution.

.

The

"introns

late" model

proposes

that

the

ancestral

protein-coding

units

con-

sisted

of uninterrupted

sequences

of

DNA.

Introns

were

subsequently

inserted

into them.

A test

of the models

is to ask

whether the

difference

between

eukaryotic and

prokaryotic

genes

can be accounted

for by

the acquisition

of introns

in

the eukaryotes

or by the loss

of

introns

from

the

prokaryotes.

The

"introns

early" model

suggests

that the

mosaic

structure

of

genes

is

a

remnant

of an

ancient

approach

to the reconstruction

of

genes

to make

novel

proteins.

Suppose

that an

early

cell had

a number

of separate

protein-coding

sequences:

One aspect

of its evolution

is likely

to have

been

the

reorganization

and

juxtapo-

sition of

different

polypeptide

units

to build

up

new

protelns.

If

the

protein-coding

unit

must be

a con-

tinuous

series

o{ codons,

every such reconstruc-

tion

would

require a

precise

recombination

of

DNA

to

place

the two

protein-coding

units in

register,

end to end in

the same reading

frame.

Furthermore,

if

this combination is

not success-

ful, the cell has

been damaged

because it has

lost

the original

protein-coding

units.

If an

approximate recombination

of DNA

could

place

the two

protein-coding

units within

the same transcription

unit,

splicing

patterns

could be tried

out at the level

of RNA

to com-

bine the

two

proteins

into

a single

polypeptide

chain. If these

combinations are

not successful,

the

original

protein-coding

units remain

avail-

able for further trials.

Such an approach

essen-

tially allows the

cell to try

out controlled

deletions in RNA

without suffering

the

damag-

ing instability

that could

occur from

applying

this

procedure

to DNA. This argument

is

sup-

ported

by the fact that we

can find related

exons

in different

genes,

as though

the

gene

had

been

assembled

by mixing and

matching

exons

(see

Section

3.9, Some Exons

Can Be Equated

with

Protein

Functions).

fl5$iJF*

-:.iS illustrates

the outcome

when

a

random

sequence

that includes

an

exon is

translocated

to a new

position

in the

genome.

Exons

are

very

small relative

to introns,

so it

is

Iikely

that the exon

will find itself

within

an

intron.

Only

the sequences

at the exon-intron

junctions

are required for

splicing,

and as

a

result

the exon is likely

to be flanked

by functional

3'

Introns

are much longer

than

exons

5' splice

junction

+

rntron

i:I*{J#il

":t..1*

An exon

surrounded

by ftanking

sequences

that is translocated

into

an

intron

may

be

sp[iced into

the RNA

product.

CHAPTER

3 The

Interrupted

Gene

and 5'

splice

junctions,

respectively.

Splicing

junctions

are

recognized

in

pairs;

thus the

5'

splicing

junction

of the original

intron is likely

to interact with the 3' splicing

junction

intro-

duced by

the new exon, instead

of

with

its orig-

inal

partner.

Similarly, the 5'

splicing

junction

of the new exon will interact with the 3'splic-

ing

junction

of the original intron. The result is

to insert the

new

exon into the RNA

product

between the original two exons. As long as the

new

exon

is in the same

coding

frame

as the

original exons, a new

protein

sequence will be

produced.

This

type of event could have been

responsible

for

generating

new combinations

of exons during evolution.

Note

that the

prin-

ciple of this

type

of event

is

mimicked by the

technique of exon trapping that is used to screen

for functional exons

(see

Figure 4.1 I

).

Alternative forms

of

genes

for rRNA

and

IRNA are sometimes found, both with and with-

out introns. In the case of the tRNAs, for which

all the molecules conform to the

same

general

structure,

it

seems unlikely that evolution

brought

together the two regions

of the

gene.

After all, the different

regions

are involved

in

the base

pairing

that

gives

significance to the

structure.

So here it must

be that the

introns

were inserted

into

continuous

genes.

Organelle

genomes

provide

some striking

connections

between the

prokaryotic

and

eukaryotic worlds.

There

are

many

general

sim-

ilarities between mitochondria or chloroplasts

and bacteria, and because of this it seems likely

that the

organelles originated

by an

endosym-

biosis in

which

an early bacterial

prototype

was

inserted

into

eukaryotic cytoplasm.

Yet in

con-

trast

with the resemblances with bacteria-for

example,

as seen in

protein

or RNA synthesis-

some organelle

genes

possess

introns and there-

fore resemble eukaryotic

nuclear

genes.

Introns are

found in

several chloroplast

genes,

including some that have homologies

with

genes

of

E. coli. This

suggests

that the

endosymbiotic event occurred before

introns

were

lost from the

prokaryotic

line. If a suitable

gene

can be

found, it may be

possible

to trace

gene

lineage back to the

period

when endosym-

biosis

occurred.

The mitochondrial

genome presents

a

par-

ticularly striking

case. The

genes

of

yeast

and

mammalian

mitochondria

code

for virtually

identical

mitochondrial

proteins

in spite of

a

considerable

difference in

gene

organization.

Vertebrate

mitochondrial

genomes

are very

small, with an extremely

compact organization

of continuous

genes,

whereas

yeast

mitochon-

drial

genomes

are

larger and

have some

com-

plex

interrupted

genes.

Which

is the ancestral

form? The

yeast

mitochondrial

introns

(and

cer-

tain other

introns) can

have

the

property

of

mobility-they are

self-contained

sequences

that can splice out

of the

RNA

and insert

DNA

copies elsewhere-which

suggests

that they

may have arisen

by

insertions

into the

genome

(see

Section

27 .5, Some

Group

I Introns

Code

for Endonucleases

That Sponsor

Mobility

and

Section 27.6, Group

II Introns

May

Code for

Multifunction

Proteins).

Some

Exons

Can

Be

Equated

with

Protein

Functions

r

Facts suggesting

that exons

were the buitding

btocks of evotution

and

the

first

genes

were

interrupted are:

r

Gene structure

is conserved

between

genes

in

very distant

species.

.

Many exons

can be

equated

with

coding

for

protein

sequences

that

have

particutar

functions.

.

Retated exons

are found

in different

genes.

If current

proteins

evolved

by

combining

ances-

tral

proteins

that

were

originally

separate,

the

accretion

of units

is

likely to

have

occurred

sequentially

over some

period

of

time, with

one

exon

added at

a time.

Can

the different

func-

tions from which

these

genes were

pieced

together

be seen

in their

present

structures?

In

other words,

can

we equate

particular functions

of current

proteins with

individual

exons?

In some cases,

there

is a clear

relationship

between

the structures

of the

gene

and

the

pro-

tein.

The example

par

excellence

is

provided

by

the immunoglobulin

proteins, which

are coded

by

genes

in which

every

exon

corresponds

exactly

with a

known

functional

domain

of the

protein. it*LlRf 3.?*

compares

the

structure

of

an immunoglobulin

with

its

gene.

An immunoglobulin

is a

tetramer

of two

light chains and

two

heavy

chains,

which

aggre-

gate

to

generate

a

protein with

several

distinct

domains.

Light

chains

and

heavy

chains

differ

in structure,

and there

are

several

qpes

of

heavy

chain.

Each

type of

chain

is

expressed

from a

gene

that

has a series

of

exons

corresponding

with

the structural

domains

of

the

protein.

3.9 Some

Exons

Can

Be Equated

with

Protein

Functions

49

L

exon V-J

exon

C exon

Light chain

Leader Variable

Constant Hinge

Constant 2

Constant 3 Protein

domains

Heavy

chain

L exon

V-D-J exon

C1 exon Hinge exon

C2 exon

C3 exon

ffGiJ&t

3.19 Immunoglobulin

light chains and heavy

chains are coded by

genes

whose structures

(in

their expressed forms)

correspond

with the

distinct domains in the

protein.

Each

protein

domain

corresponds

to

an exon; introns

are

numbered

1

to 5.

In many

instances,

some

of the exons

of a

gene

can be identified

with

particular

functions.

In

secretory

proteins,

the first

exon, coding for

the N-terminal

region

of the

polypeptide,

often

specifies

the signal

sequence

involved

in mem-

brane

secretion.

An

example is insulin.

The view

that

exons are

the functional

building

blocks

of

genes

is

supported

by cases

in which

two

genes

may have

some

exons that

are related

to one another,

whereas other

exons

are found

only in

one

of the

genes.

ftSL!ft1

3.tr*

summarizes

the relationship

between

the receptor

for human

LDL

(plasma

low

density

lipoprotein)

and other

proteins.

In

the center

of

the LDL receptor gene

is a

series

of exons

exons

of the

gene

for

the

precursor

for

EGF

(epidermal

growth

fac-

tor). In

the

N-terminal

part

of the

protein,

a

series

of

exons codes

for

a sequence

blood

protein

complement

factor

C9.

So the

LDL receptor gene

was created

by

assembling

modules

for

its various

functions.

These

modules

also are

used in

different

com-

binations

in

other

proteins.

Exons

tend

to be fairly

smali

(see

Fig-

ure 4.ll),

around

the size

of the

smallest

polypeptide

that

can assume

a

stable folded

structure (-20

to 40 residues).

Perhaps proteins

were

originally

assembled

from

rather

small

modules.

Each

module

need

not necessarily

correspond

to

a current

function;

several

mod-

ules could

have

combined

to

qenerate

a func-

CHAPTER

3 The

Interrupted

Gene

LDL receptor

Cg complement

EGF

precursor

homology

EGF

precursor

FIGUft{

}.*S

The

LDL receptor

gene

consjsts

of 18 exons.

some

of which

are

retated

to EGF

precursor

exons and

some

of which are related

to the

C9 btood comDtemenr

gene.

Triangles

mark the

positions

of introns.

tion. The number

of

exons in a

gene

tends

to

increase

with the length

of its

protein,

which is

consistent

with the view

that

proteins

acquire

multiple functions

by successively

adding

appro-

priate

modules.

This

idea might

explain

another

feature

of

protein

structure.

It appears

that

the sites

rep-

resented

at exon-intron

boundaries

often are

located

at

the surface

of a

protein.

As

modules

are added

to a

protein,

the

connections-at

least

of the

most recently

added

modules-could

tend

to lie

at the surface.

Lewin Benjamin (ed.) Genes IX

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