Lewin Benjamin (ed.) Genes IX

because only the second

codon has been

changed.

But a mutation that inserts

or deletes a single

base will change the triplet

sets

for

the entire

subse-

quent

sequence.

A

change of this

sort

is

called a

frameshift. An insertion

might take the form:

UUU AAX AGG

GCC C

aal aa5 aa6 aa7

Because the new

sequence of triplets is com-

pletely

different from

the old one, the entire

amino acid sequence

of the

protein

is altered

beyond the site of

mutation.

So the function

of

the

protein

is likely to be lost

completely.

Frameshift mutations

are induced by the

acridines, compounds that

bind to DNA and

distort the structure of the double helix,

caus-

ing additional bases to be incorporated

or omit-

ted during replication. Each

mutagenic event

sponsored by an acridine results

in the addition

or

removal

of a single base

pair.

If an acridine mutant

is

produced

by, say,

addition of a nucleotide, it

should

revert

to wild

type by deletion of the nucleotide.

However,

reversion also

can be caused by deletion of a

different base at a site close to the first.

Combi-

nations of such mutations

provided

revealing

evidence about the nature of the

genetic

code.

i:iilii!-li:

iri.::

illustrates

the

properties

of

frameshift mutations.

An insertion or deletion

::;iiii:ir .:.:1, Frameshift mutations show that the

genetic

code

is read in triptets from a fixed starting

point.

changes

the

entire

protein

sequence

following

the site of mutation.

However,

the combina-

tion of an insertion

and a deletion

causes

the

code to be

read incorrectly

only between

the

two sites of mutation;

correct

reading

resumes

after the second

site.

In 1961,

genetic analysis of

acridine

muta-

tions

in

the

rII region of

the

phage

T6 showed

that all the mutations

could

be classified

into

one of two sets,

described

as

(+)

and

(-).

Either

type of mutation by

itself causes

a

frameshift:

the

(+)

type by virtue

of a base

addition,

and

the

(-)

type by

virtue of

a base deletion.

Dou-

ble mutant combinations

of the types

(+ +)

and

(-

-)

continue to show

mutant

behavior.

How-

ever, combinations

of the

types

(+

-)

or

(-

+)

suppress one

another,

giving

rise to a descrip-

tion in which one

mutation

is described

as

a

frameshift supressor

of the

other.

(In

the con-

text of this work,

"suppressor"

is

used

in an

unusual sense

because

the second

mutation

is

in the same

gene

as

the

first.)

These

results show

that the

genetic

code

must be read as a

sequence

that

is fixed by the

starting

point.

Thus

additions

or deletions

com-

pensate

for each other,

whereas

double

addi-

tions or double

deletions

remain mutant.

However, this does

not

reveal

how many

nucleotides

make up each

codon.

When

triple

mutants are

constructed,

only

(+

+ +

)

and

(-

- -)

combinations

show

the wild

phenotlpe,

whereas

other

combinations

remain

mutant. If we take

three additions

or

three dele-

tions to correspond

respectively

to the addition

or omission

overall

of a single

amino

acid, this

implies that the

code

is read

in triplets.

An incor-

rect amino acid

sequence

is

found between

the

two outside sites

of mutation

and

the sequence

on either side

remains

wild

type, as

indicated

in

Fig:ure 2.9.

Every Sequence

Has

Three

Possible

Reading

Frames

.

Usualty onty one

reading

frame'is

translated

and

the other two

are blocked

by

frequent

termjnation

signa[s.

If

the

genetic

code

is

read in

nonoverlapping

triplets, there

are three

possible

ways

of trans-

lating any

nucleotide

sequence

into

protein,

depending

on the starting

point. These are

called

reading

frames.

For the

sequence

ACGACGACGACGACGACG

Wild type

Ala

Ala,

'Ala

,Ala

,

,Ala

Ala ,i Ala.","',,

'_._]

GCU

AGC

UGC UGC UGC UGC UGCIU-

\-

,,{16;,:r:"1413:,,

Ser Cys Cys

Cys Cys Cys

Deletion

G

-.GCU

GCU GCU GCU GCU CUG CUG CUG

Ala

Ala Ala Ala Ala

Leu Le u Leu

-

GCU GCU

AGC UGC

UGCUCU GCU GCUFr

Ala

A,la

Ser Cys Cys Ser

Ala

AIa Ata

Cys Cys [4et His Ala Ala

Wild-type

sequence

Mutant

sequence

2.9

Every Sequence

Has

Three Possibte

Reading

Frames

31

the three

possible

reading frames

are

ACG

ACG ACG ACG

CGA CGA CGA

CGA

GAC GAC

GAC GAC GAC GAC

GAC

A reading

frame that

consists exclusively

of triplets representing

amino

acids

is

called

an open reading

frame

or ORF. A sequence

that is

translated into

protein

has a reading

frame

that starts with

a special initiation

codon

(AUG)

and then extends

through a

series

of triplets representing

amino acids

until

it

ends at

one of three types

of termination

codon

(see

Chapter 7, Messenger

RNA).

A

reading frame

that

cannot be read

into

protein

because termination

codons occur

frequently

is said to be

blocked.If a sequence

is

blocked in

all three reading

frames, it

cannot have

the function

of coding for

protein.

When the

sequence of a DNA region

of

unknown function

is obtained,

each

possible

reading

frame

is analyzed

to determine

whether

it is

open or blocked.

Usually no more

than

one of the three

possible

frames

of

read-

ing is

open in any

single stretch

of DNA.

ri$-

URf t.t*

shows

an example

of a sequence

that

can be read

in only one reading

frame

because

the

alternative reading

frames

are blocked

by

frequent

termination

codons. A long

open

reading frame

is unlikely

to exist

by chance;

if it

were not translated

into

protein,

there

would have

been no

selective

pressure

to

pre-

vent

the accumulation

of termination

codons.

So the identification

of a

lengthy

open read-

ing frame

is taken

Io be

prima

facie

evidence

that

the sequence

is translated

into

protein

in

that frame.

An

ORF

for

which no

protein

prod-

uct has

been identified

is sometimes

called an

unidentified

reading

frame

(URF).

Only one open reading

frame

Second reading

frame

is blocked

Third reading

frame is

blocked

Termination

FiGgJftil

2.1*

An

open

reading

frame

starts

with AUG

and contin-

ues in

triptets

to a termination

codon.

Blocked reading

frames may

be interrupted

frequently

by termination

codons.

Prokaryotic

Genes

Are

Co[inear with

Their Proteins

r

A

prokaryotic gene

consists of a continuous

[ength

of 3fi nucteotides that codes for fi

amino acids.

.

The

gene,

mRNA,

and

protein

are a[[ cotinear.

By comparing the nucleotide

sequence

of a

gene

with the amino acid sequence

of a

protein,

we

can determine

directly whether the

gene

and the

protein

are colinear; that is,

whether

the

sequence of nucleotides in

the

gene

corresponds

exactly

with the sequence of amino

acids in the

protein.

In bacteria and their

viruses, there is

an

exact

equivalence. Each

gene

contains a contin-

uous stretch of DNA whose

length is

directly

related to the number

of amino acids in

the

pro-

tein that it represents.

A

gene

of 3N bp is

required

to code for a

protein

of N amino acids,

according to the

genetic

code.

The

equivalence of the

bacterial

gene

and

its

product

means

that a

physical

map of DNA

will

exactly match an amino

acid map

of the

protein.

How well

do these maps fit

with the

recombination map?

The

colinearity

of

gene

and

protein

was

originally investigated

in the tryptophan

syn-

thetase

gene

of E. coli.

Genetic distance

was

measured

by the

percent

recombination

between mutations;

protein

distance

was mea-

sured by the number

of amino acids

separating

sites

of

replacement. FI€UftH

?.3.1

compares

the

two maps. The

order of seven sites

of mutation

is

the same as the

order of the

corresponding

sites

of

amino

acid replacement,

and the recom-

bination

distances are relatively

similar

to the

actual distances in

the

protein.

The

recombina-

tion map

expands the distances

between

some

mutations,

but otherwise there

is little

distor-

tion

of the recombination

map relative

to the

physical

map.

The

recombination

map makes

two further

general points

about

the organization

of the

gene.

Different

mutations

may cause

a wild-

type amino

acid to be replaced with

different

substituents.

If two

such mutations

cannot

recombine,

they must

involve

different

point

mutations

at the

same

position

in

DNA. If

the

mutations

can

be separated

on the

genetic

map,

but affect

the same amino

acid

on the upper

map

(the

connecting lines

converge

in the fig-

ure), they must

involve

point

mutations

at dif-

32

CHAPTER 2

Genes

Code for Proteins

DNA consists of

two base-paired

strands

top strand

5'

ATGCCGTTAGACCGTTAGCGGACCTGAC

3' TACGGCAATCTGGCAATCGCCTGGACTG

r

bottom strand

I

RNA

I

*

sYnthesis

5' AUGCCGUUAGACCGUUAGCGGACCUGAC

3

RNA has same

sequence

as DNA

top strand;

is

complementary

to Dl{A bottom

strafld

SeveraI

Processes

Are

Required to Express the

Protein Product

of a Gene

.

A

prokaryotic gene

is expressed by transcription

into mRNA

and then by transtation of the mRNA

into

orotein.

.

In eukaryotes, a

gene

may contain internal

regions that are not represented in

protein.

.

InternaI regions are removed from

the

RNA

transcript by RNA spticing

to

give

an

mRNA

that

is colinear with the

protein product.

e

Each mRNA

consists of a

nontranstated

5' leader, a

coding

region,

and a nontranstated 3'traiter.

In comparing

gene

and

protein,

we are restricted

to dealing with the sequence

of

DNA

stretching

between

the

points

corresponding to the ends

of the

protein.

However, a

gene

is not directly

translated

into

protein,

but is expressed via the

production

of a rnessenger RNA

(abbreviated

to mRNA),

a nucleic acid intermediate

actu-

F;t-{;l,iii:i;;:,'i; RNA

is

synthesized

by using one strand

of

DNA

as a temptate

for complementary

base

pairing.

ally used to synthesize

a

protein

(as

we see

in

detail in Chapter

7, Messenger

RNA).

Messenger

RNA is synthesized

by

the same

process

of complementary

base

pairing

used

to

replicate DNA.

with the

important

difference

that

it

corresponds

to

only one

strand of

the

DNA double

helix.

flt{"illfii:

l:":.;i

shows

that the

sequence of

mRNA

is complementary

with the

sequence of one

strand

of DNA

and is

identical

(apart

from the

replacement

of T with

U) with

the other strand

of

DNA. The

convention

for

writing DNA sequences

is that the

top strand

runs 5'-+3', with the

sequence

that

is the same

as RNA.

The

process

by which

a

gene

gives

rise to a

protein

is called

gene

expression.

In bacteria,

it

consists of two

stages.

The

first stage

is tran-

scription, when an

nRNA

copy

of one strand

of the DNA

is

produced. The second

stage

is

translation of the

nRNA

into

protein. This is

the

process

by

which

the sequence

of

an mRNA

is read in triplets

to

give

the series

of

amino

acids that

make the

corresponding

protein.

A mRNA

includes

a sequence

of

nucleotides

that corresponds

with the sequence

of

amino

acids in the

protein.

This

part

of

the nucleic

acid

is

called the

coding

region.

However,

the

mRNA includes

additional

sequences

on either

end;

these sequences

do

not directly

represent

protein.

The 5' nontranslated

region

is called

the leader, and

the 3'

nontranslated

region

is

called the trailer.

The

gene

includes

the

entire sequence

rep-

resented in

messenger

RNA.

Sometimes

muta-

tions impeding

gene

function

are found

in the

additional,

noncoding

regions,

confirming

the

view that

these comprise

a legitimate

part

of

the

genetic

unit.

Different

mutations

at same

position

i6nt

"r

I

Mutations

can be

separated but

aflect

same

position

i:ii:;:,iF!-: ;:.i 1 The

recombination map

of the tryptophan

synthetase

gene

corresponds

with the amino acid sequence

of the

protein.

ferent

positions

that

affect the same amino acid.

This happens because the unit

of

genetic

recom-

bination

(actually

I

bp)

is

smaller than the unit

coding for the amino

acid

(actually

I bp).

2.11

SeveraI

Processes

Are Required

to

Express the

Protein

Product

of a Gene

33

Leader I

rraiter

RNA

Length

of RNA defines region

of

gene

I,'

rufficprotein

Protein

defines coding region

FIGURf

?.13 The

gene

may be longerthan

thesequence

coding for

protein.

Transcription

Translation

FIGURf

i.14

Transcription

and transtation take

ptace

in the same

comoartment

in

bacteria.

F1GURE

2.13

illustrates

this

situation, in which

the

gene

is

considered

to comprise

a continu-

ous stretch

of DNA, needed

to

produce

a

par-

ticular

protein.

It includes

the sequence

coding

for

that

protein,

but

also includes

sequences

on

either

side of the

coding region.

A

bacterium

consists

of only a

single com-

partment,

so

transcription

and translation

occur

in the

same

place,

as illustrated

in

FIfiURI

f .14.

In eukaryotes

transcription

occurs in

the

nucleus,

but

the RNA

product

must

be trans-

portedto

the cytoplasm

in order

to be translated.

For

the

simplest

eukaryotic

genes (just

like in

bacteria)

the transcript

RNA is in

fact the nRNA.

However,

for

more

complex

genes,

the imme-

diate transcript

of the

gene

is

a

pre-rnRNA

that

requires

processing

to

generate

the mature

mRNA.

The

basic stages

of

gene

expression in

a eukaryote

are

outlined in frl#{Jf{f,

*.iS.

This

results

in a

spatial separation

between

transcrip-

Genes

Code for

Proteins

tion

(in

the nucleus)

and translation

(in

the

cytoplasm).

The most

important

stage in

processing

is

RNA

splicing. Many

genes

in

eukaryotes

(and

a majority in higher

eukaryotes)

contain inter-

nal regions that

do not code for

protein.

The

process

of splicing removes

these regions

from

the

pre-mRNA

to

generate

an RNA

that has

a

continuous

open reading frame

(see

Figure

3. I

).

Other

processing

events that

occur at this

stage

involve

the modification

of the

5'and 3'ends

of the

pre-mRNA (see

Figure 7.16).

Translation

is accomplished

by a

complex

apparatus

that includes

both

protein

and RNA

components.

The actual

"machine"

that under-

takes the

process

islhe

ribosome,

alarge complex

that includes

some

large

RNAs

(ribosomal

RNAs,

abbreviated to rRN/s)

and many

small

proteins.

The

process

of recognizing

which

amino acid

corresponds

to a

particular

nucleotide

triplet

requires

an intermediate

transfer RNA

(abbrevi-

ated

to IRNA); there is

at least one

IRNA species

for every

amino acid. Many

ancillary

proteins

are involved.

We

describe translation

in

Chap-

TRNAY

5',

CYTOPLASM

FIGUftE 3.15

Gene expression is

a multistage

process.

34

CHAPTER 2

ter 7, Messenger RNA,

but note for now

that

the

ribosomes

are the large structures

in Figure

2.I4that move along

the mRNA.

The important

point

to

note

at this stage is

that the

process

of

gene

expression involves

RNA not

only

as the

essential substrate, but also

in

providing

components of the apparatus. The

rRNA and IRNA components

are coded by

genes

and

are

generated

by the

process

of transcrip-

tion

(just

like mRNA, except

that there

is

no

subsequent stage of translation).

Protei ns Are Tra

n

s-acti

ng,

but Sites on

DNA

Are

Crs-acting

.

At[

gene products

(RNA

or

proteins)

are trans-

acting.

They

can act on any copy of a

gene

in the

ceIt.

r

crs-acting mutations identify sequences

of

DNA

that are targets for recognition

by trons-acting

products.

They

are not expressed as RNA

or

protein

and affect only the contiguous stretch of DNA.

A

crucial step

in

the definition of the

gene

was

the realization that all its

parts

must

be

present

on one contiguous stretch of DNA. In

genetic

terminology, sites that

are

located

on the same

DNA are said to be in c/s. Sites that are located

on two different molecules

of

DNA

are described

as

being

in trans.

So

two mutations

may be

in

crs

(on

the same DNA) or in trans

(on

different

DNAs). The complementation

test uses this con-

cept to determine whether two mutations are

in the same

gene (see

Figure 2.3 in Section 2.1,

Mutations in the Same Gene Cannot Comple-

ment).

We

may now

extend the concept of the

difference between cis and trans eff-ects from

defining the coding

region

of a

gene

to describ-

ing the interaction between regulatory elements

and a

gene.

Suppose that the ability of a

gene

to be

expressed

is controlled by a

protein

that binds

to the

DNA

close

to the

coding

region. In

the

example depicted in

Fi*LtF.{

t.tS, nRNA can be

synthesized

only when the

protein

is bound to

the DNA. Now suppose

that

a

mutation

occurs

in the DNA sequence to which this

protein

binds, so that the

protein

can no longer recog-

nize

the

DNA. As a result, the DNA

can

no longer

be expressed.

So a

gene

can be inactivated either

by

a mutation

in a control site or by

a

mutation in

a

coding

region.

The

mutations cannot be

distinguished

genet-

ically, because both

have

the

property

of

act-

RNA

Two types of

DNA

ir{*j-i,-iI

i]".i *; Control sites

in DNA

provide

binding

sites

for

proteins;

coding

regions are

expressed

via the synthe-

sis of RNA.

l'i{it.iRir

li

I

i A crs-acting

site controts

the

adjacent

DNA,

but does

not influence the

other a[te[e.

ing only on the

DNA

sequence

of

the single

allele

in which they

occur.

They

have

identical

properties

in the complementation

test,

and a

mutation in a control

region

is therefore

defined

as comprising

part

of

the

gene

in the

same

way

as a

mutation in

the coding

region.

f li:;tittf, ii.: r shows

that

a deficiency

in the

control site

affects

only

the coding

region

to which

it is connected;

it does

not affect

the

ability of

the other

allele to be expressed.

A mutation

that acts

solely

by affecting

the

properties of

the contiguous

sequence of

DNA

is called

cis-acting.

Both alleles

synthesize

RNA

in wild type

,,

,:'''";,

,,,'i

'..,,_.,'it-'

;.,

,,it

"r.

RNA synthesis

continues

from allele

2

,

r.1..,t'

,.,

'

,,1

o,

i!

NO RNA SYNTHESIS

FROM

ALLELE 1

2.12

Proteins

Are

lrans-acting,

but Sites

on

DNA

Are

Cis-acting

The

active

protein

acls on both

alleles

tl

Mutanl

protein

cannot

bind

to either allele

S--Mutant

protein

NO RNA

SYNTHESIZED FROM

ALLELE 1

NO BNA

SYNTHESIZED

FROM ALLELE 2

i-iLi;-:$:i.

.t.:i:

A trons-acting

mutation in

a

protein

affects

both

atteles of a

gene

that it controts.

We

may

contrast the

behavior of the czs-

acting

mutation

shown in Figure

2.17

with the

result

of

a

mutation

in

the

gene

coding for the

regulator

protein.

lii:i"iFI

i.5*

shows that the

absence

of regulator

protein

would

prevenl

both

alleles from

being

expressed.

A mutation

of this

sorl is

said

tobe trans-acting.

Reversing

the

argument, if

a mutation is

trans-acting,

we know

that its

effects must

be

exerted through

some

diffusible

product (typ-

ically

a

protein)

that

acts on multiple

targets

within

a

cell. However,

if a mutation

is cli-act-

ing,

it must

function

via affecting

directly

the

propefties

of the contiguous

DNA,

which means

that it is

not expressed

in the

form

of RNA

or

protein.

Su m mary

A

chromosome

consists

of an

uninterrupted

length

of duplex

DNA

that contains

many

genes.

Each

gene

(or

cistron)

is transcribed

into

an RNA

product,

which in

turn is

trans-

lated

into

a

polypeptide

sequence

if the

gene

codes

for

protein.

An RNA

or

protein

product

of a

gene

is said Io

be

trans-acting.

A

gene

is

defined

as a unit of a single stretch

of DNA by

the complementation test. A

site on DNA

that

regulates the activity

of an adjacent

gene

is

said

to be czs-acting.

When a

gene

codes

for

protein.

the reia-

tionship

between the sequence

of DNA and

sequence

of the

protein

is

given

by the

genetic

code.

Only one of the two strands

of DNA

codes

for

protein.

A codon consists

of three nucleotides

that represent

a single amino

acid. A

coding

sequence of DNA

consists of a series

of codons,

read from

a fixed starting

point.

Usually

one of

the three

possible

reading

frames

can be trans-

Iated into

protein.

A

gene

may have multiple

alleles. Reces-

sive alleles are

caused by loss-of-function

muta-

tions

that

interfere with

the function

of the

protein.

A null allele has

total loss-of-function.

Dominant alleles

are caused

by

gain-of-func-

tion mutations

that create a new

property

in

the

protein.

References

The

Genetic

Code

Is Triolet

Review

Roth, J. R.

(

1974).

Frameshift mutations.

Annu.

Rev.

GeneL 8, Jl9-346.

Resea rc h

Benzer,

S. and Champe,

S.P.

(1961).

Ambivalent

rII

mutants of

phage

T4.

Proc

Natl. Acad

Sci

usA 47, 40)-416.

Crick, F. H.

C., Barnett, L., Brenner,

S., and Watts-

Tobin, R.

J.

(

I

96 I

).

General nature

of

the

genetic

code for

proteins.

Nature 192,

t227-1,2)2.

Prokaryotic

Genes Are Cotinear

with Their

Proteins

Yanofsky,

C.,

Drapeau,

G.R.,

Guest, J. R.,

and

Carlton, B.C.

,1967).

The

complete

amino

acid sequence

of the tryptophan

synthetase

A

protein (p

subunit) and its

colinear relation-

ship

with the

genetic

map

of

the A

gene.

Proc.

Natl.

Acad.

Sci.

USA 57, 2966-2968.

Yanofsky,

C. etal.

(19641.

On the colineariry

of

gene

structure

and

protein

structure.

Proc

Natl.

Acad

Sci USA, 51.266-272.

Researc

h

36

CHAPTER

2 Genes

Code for

Proteins

Interrupted

Gene

CHAPTER

OUTLINE

Introduction

An Interrupted

Gene Consists

of

Exons

and

Introns

o

Introns are removed

by the

process

of RNA spLicing, which

occurs only in

crs on an

jndividual

RNA motecu[e.

.

Onty

mutations

in exons

can affect

protein

sequence;

how-

ever. mutations in introns

can affect

processing

of the RNA

and therefore

prevent production

of

protein.

Restriction Endonucleases Are

a

Key Too[ in Mapping

DNA

.

Restriction endonucleases

can be

used

to cteave

DNA into

defined fragments.

.

A map can

be

generated

by using the overlaps between the

fragments

generated

by different

restriction

enzymes.

0rganization of Interrupted

Genes

May Be

Conserved

r

Introns can

be detected by the

presence

of additional

regions when

genes

are compared with their RNA

products

by

restriction

mapping or electron microscopy. The uttimate

definition, though, is based on comparison

of

sequences.

.

The

positions

of introns

are usually conserved

when homol-

ogous

genes

are

compared between different organisms.

The lengths of the corresponding introns may vary

greatty.

o

Introns usualty

do

not

code

for

proteins.

Exon

Sequences

Are

Conserved but

Introns

Vary

r

Comparisons of retated

genes

in different species show that

the sequences of the corresponding exons are usualty con-

served but the seouences of the introns are much less we[[

retated.

o

Introns evolve much more rapidly

than exons

because of

the lack of selective

pressure

to

produce

a

protein

with a

usefuI sequence.

Genes Show a Wide Distribution of Sizes

r

Most

genes

are uninterrupted

in

yeasts,

but are

interrupted

in higher eukaryotes.

r

Exons are

usuatty short, typicatty coding

for

<L00

amino

ac'ids.

r

Introns are short in lower eukaryotes, but range up to sev-

era[

10s

of

kb

in [ength in higher eukaryotes.

r

The overatl Length

of a

gene

is

determined largeLy

by its

introns.

Some DNA Sequences

Code

for More Than One Protein

.

The

use of atternative initiation or termination codons

atlows two

proteins

to be

generated

where one

is

equivatent

to a fragment of the

other.

o

Nonhomologous

protein

sequences

can be

produced

from

the

same sequence

of DNA when

itis read

in

different

read-

ing frames by two

(overtapping)

genes.

.

Homotogous

proteins

that differ by

the

presence

or

absence

of certain

regions can

be

generated

by differential

(atterna-

tive) spticing

when certain

exons are

included or excluded.

This may take

the form of

including

or excluding

individuaL

exons or of

choosing between

atternative

exons.

How

Did Interrupted

Genes

Evolve?

.

The major evolutionary

question

is whether

genes

origi-

nated as sequences

interrupted by

introns or

whether they

were originatly

uninterrupted.

o

Most

protein-coding

genes

probab[y

originated

in an inter-

rupted form, but

interrupted

genes

that

code for RNA

may

have origina[y

been uninterrupted.

.

A speciaI class

of introns

is mobile and

can

insert itself into

genes.

Some

Exons Can

Be

Equated with

Protein

Functions

.

Facts suggesting

that

exons were

the buitding

blocks of

evotution

and the

first

genes

were interrupted

are:

o

Gene

structure

is conserved

between

genes

in very

distant species.

.

Many exons

can be equated

with coding

for

protein

sequences

that

have

particutar

functions.

r

Retated

exons are

found

in different

genes.

The

Members of a

Gene

Famitv Have a

Common

0rganization

o

A common

feature in a set

of

genes

is assumed to

identify a

property

that

preceded

their separation

jn

evolution.

.

Att

gtobin genes

have a common

form of organization

with

three exons

and two

introns,

suggesting

that they

are

descended

from a singte

ancestral

gene.

Is A[[ Genetic

Information

Contained

in DNA?

o

The definition

of the

gene

has

reversed

from

"one

gene

:

one

protein"

to

"one protein

: one

gene."

r

PositionaI

information

is

also

important

in

development.

Summary

37

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Genomic DNA

Exon 1 Intron 1 Exon 2

AB

lntron 2

Exon 3

A-B B-C

mRNA

Exonl Exon2 Exon3

|

L----r---------r+

A-B B-C

rtri.i;liitir.:i"ii

ExonsremaininthesameorderinmRNAasinDNA,butdistances

along the

gene

do not conespond to distances along the

mRNA or

protein

prod-

ucts. The distance from A-B in the

gene

is smatter than the

distance

from

B-C;

but the

distance

from A-B in

the

mRNA

(and

protein)

is

greater

than

the djs-

tance

from B-C.

between

the corresponding

points

in the

pro-

tein. The length of the

gene

is defined by the

length

of

the initial

(precursor)

RNA instead of

by the

length of the messenger RNA(nRNA).

All of the exons are

represented

on the same

molecule of

RNA,

and their splicing together

occurs only

as anintramolecular reaction. There

is usually no

joining

of exons carried by dffir-

er7lRNA

molecules,

so

the mechanism excludes

any splicing together of sequences

represent-

ing

different

alleles. Mutations located in dif-

ferent exons of

a

gene

cannot complement

one

another; thus they continue to be defined as

members of the same complementation

group.

Mutations that directly affect the sequence

of a

protein

must lie in exons. What are the

effects of

mutations in

the

introns? The introns

are not

part

of the

messenger RNA, thus muta-

tions

in them cannot directly affect

protein

struc-

ture. However, they can

prevent

the

production

of the

messenger RNA-for example, by inhibit-

ing the splicing together of exons.

A mutation

of this sort

acts only on the allele that carries

it.

As a result,

it fails to complement any other

mutation in that allele and constitutes

part

of

the same complementation

group

as the exons.

Mutations that affect splicing are

usually

deleterious.

The majority

are single-base

sub-

stitutions

at the

junctions

between

introns and

exons.

They may cause an exon to be

left

out

of the

product,

cause an

intron to be included,

or

make splicing occur at an aberrant site.

The

most common

result is to introduce a termina-

tion codon

that results in truncation of

the

pro-

tein sequence.

About l5'h of the

point

mutations that

cause human diseases are caused

by disruption

of splicing.

Eukaryotic

genes

are not necessarily

inter-

rupted. Some correspond

directly with the

pro-

tein

product

in the same

manner

as

prokary-

otic

genes.

In

yeast, most

genes

are

uninter-

rupted.

In higher eukaryotes

most

genes

are

interrupted,

and the

introns

are

usually

much

longer than exons.

This creates

genes

that

are

noticeably larger

than

their coding

regions.

Restriction

Endonucleases

Are a

Key

Toot

in Mapping

DNA

r

Restriction endonucleases

can be

used to

cleave

DNA into defined

fragments.

o

A map can be

generated by using

the overlaps

between

the

fragments

generated

by different

restriction enzymes.

The characterization

of

eukaryotic

genes

was

made

possible

by

the development

of techniques

for

physically mapping

DNA.

The

techniques

can be extended

to

(single-stranded)

RNA

by

making a

(double-stranded)

DNA

copy

of the

RNA.

A

physical map of

any

DNA

molecule

can

be obtained

by

breaking

it at defined

points

whose distance

apart

can be

accurately

deter-

mined. Specific

breaks

are

made

possible by the

ability of

restriction

endonucleases

to

rec-

ognize

rather short

sequences

of double-

stranded

DNA

as targets

for cleavage.

Each restriction

enzyme

has

a

particular

target in duplex

DNA,

usually

a

specific

sequence

of

four to six base

pairs. The enzyme

cuts

the

DNA at every

point

at

which

its target

sequence

occurs.

Different

restriction

enzymes

have dif-

ferent target

sequences,

and

a large

range

of

these activities

(obtained from

a wide

variety

of bacteria)

now

is available.

3.3

Restriction

Endonucteases

Are a

Key Too[

in Mapping

DNA 39

A lri ,1.

ll A

1000 200

1900

600

800 500

f,:*i:S:L

-!.* A restriction map

is a [inear

sequence

of sites

separated by defined

distances on DNA. The

map identi-

fies the

sites cteaved by enzymes

A and B,

as defined

by

the individuaI

fragments

produced

by

the single and

dou-

b[e digests.

molecule in the form

shown in

{l$ijftU

J.*.

The

map

shows the

positions

at

which particular

restriction

enzymes

cut DNA;

the distances

between

the sites of cutting

are measured

in

base

pairs.

So the DNA

is divided

into a

series of

regions

of defined lengths

that lie

between

sites rec-

ognized by the

restriction enzymes.

An important

feature

is that a restriction

map can

be

obtained for any

sequence

of DNA,

irrespec-

tive of whether

mutations

have been

identified

in

i/,

or, indeed,

whether we

have any

knowl-

edge of its function.

;i

ji:

Jii:

.-1..1 Fragments

generated

a restriction

endonuctease

can be

to their

sizes.

by cleaving DNA

with

separated according

A restriction

map

represents

a

linear

sequence

of the

sites at

which

particular

restric-

tion

enzymes

find

their targets.

For

short dis-

tances,

the distance

along

such

maps is

measured

directly

in

base

pairs

(bp).

Longer

distances

are

given

in

kilobases

(kb),

which

correspond

to kilobase

(

103)

pairs

in DNA

or to

kilobases

in RNA.

At the level

of

the chromo-

some,

a map is

described

in megabase pairs

(l

Mb

=

106 bp).

When

a DNA

molecule

is cut

with a

suit-

able restriction

enzyme,

it is

cleaved into

dis-

tinct fragments.

These

fragments

can

be

separated

on the basis

of their

size

by

gel

elec-

trophoresis,

as

shown in ti**=iEt

3."1.

The cleaved

DNA

is

placed

on top

of a

gel

made

of agarose

or

polyacrylamide.

When

an electric

current is

passed

through

the

gel,

each fragment

moves

down

at a rate

that is inversely

of its

molecular

weight. This

movement pro-

duces

a

series

of bands. Each

band corresponds

to a fragment

of

particular

size,

decreasing

down

the

gel.

By

analyzing

the restriction

fragments

of

DNA, we

can

generate

a map

of the

original

CHAPTER

3 The

Interrupted

Gene

0rganization

of

Interrupted

Genes

May

Be

Conserved

r

Introns

can be detected

by the

presence

of

additionaI regions

when

genes

are

compared with

their

RNA

products

by

restrict'ion

mapping

or

electron microscopy.

The

uttimate

definition,

though. is

based on

comparison

of sequences.

.

The

posjtions

of

introns

are

usuatty

conserved

when

homotogous

genes

are

compared

between

different

organisms. The

lengths

of the

corresponding

introns

may vary

greatty.

o

Introns

usuatly

do not

code

for

proteins.

When

a

gene

is

uninterrupted,

the

restriction

map

of

its

DNA

corresponds

exactly

with

the

map

of its mRNA.

When

a

gene possesses

an intron,

the

map

at each

end of the

gene

corresponds

with

the

map

at each

end of the

message

sequence.

Within the

gene,

though,

the

maps

diverge

because

additional

regions

that are

found in

the

gene

are not represented

in the

message.

Each

such region

corresponds

to

an intron.

The

exam-

ple

of

F3*{}F*

3,} compares

the restriction

maps

of a

B-globin

gene

and nRNA.

There

are

two

introns,

each of

which contains

a

series of restric-

tion

sites

that are absent

from

the

cDNA.

The

pattern

of restriction

sites in

the exons

is

tne

same in

both the

cDNA and

the

gene.

40

Lewin Benjamin (ed.) Genes IX

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