Lewin Benjamin (ed.) Genes IX

RNA

polymerase

cannot initiate

transcription

without assistance

from

an ancillary

protein.

Such

proteins

are

positive

regulators,

because

their

presence

is

necessary to switch

on

the tran-

scription unit.

T\.pically,

the activator

overcomes

a deficiency in the

promoter,

for example, a

poor

consensus sequence

at

-35

or

-I0.

One of the

most

widely acting activators is

a

protein

called

CRP activator that controls

the activity of a

large

set of operonsinE. coli.The

protein

is a

positive

control factor

whose

pres-

ence

is necessary

to

initiate

transcription at

dependent

promoters.

CRP is

active only in

the

presence

of

qclic

AMP,

which behaves as a classic

small-molecule

inducer for

positive

control

(see

i::i-,:+,

r:'..:

;

Uppef

fight).

Cyclic AMP is synthesized

by the enzyme

adenylate cyclase.

The

reaction uses ATP as

substrate

and introduces a 3'-5'link

via

phos-

phodiester

bonds,

which

generates

the struc-

ture drawn

in

i:irl..i

ji:i:

t,i ilri.

Mutations in the

gene

coding for adenylate cyclase

(cya-)

do

not

respond to changes

in

glucose

levels.

The level of cyclic AMP is inversely related

to the level of

glucose.

The

basis for this effect

lies with the same component of the Pts system

that

is responsible for

controlling lactose uptake.

The

phosphorylated

form of

protein

IIAGI'stim-

ulates adenylate

cyclase.

When

glucose

is

imported, the dephosphorylation of IIAGI' leads

to a

fall in

adenylate cyclase activity.

ir,',::;:i

.

..

shows that reducing the Ievel

of cyclic

AMP renders the

(wild-type)

protein

unable to bind

to

the control

region, which in

turn

prevents

RNA

polymerase

from initiating

transcription.

Thus

the effect of

glucose

in reduc-

ing

cyclic

AMP levels is to deprive the relevant

operons of a control

factor necessary for

their

expression.

TNDUCED

I

nepnesseo

INDUCED

REPRESSED

INDUCED

I

REPRESSEIJ

INDUL;EU

.

----l

--

Corepressor

INDUCED

REPRESSED

CRP

Functions in

Different

Ways

in

Different Target

0perons

.

CRP introduces a 90o bend

into

DNA at

its

bindinq

site.

o

CRP-binding

sites lie at highty variable [ocations

retative to the

promoter.

o

CRP

interacts with RNA

potymerase,

but the detaits

of the

interaction depend on the relative locations

of the CRP-bjnding

site and the

promoter.

The

CRP

factor binds to DNA, and complexes of

cyclic

AMP.CRP DNA can be isolated at each

iriljitirtl

j,.

r..r Controtcircuitsareversatiteandcanbedesignedtoatlowpositiveorneg-

ative control

of

induction or

repression.

I

ii,i,iiii

,r r;'r CycLic

AMP has a

singte

phosphate

group

connected to both

the 3'

and 5'

positjons

of the

sugar

n ng.

promoter

at

which

it functions.

The

factor is

a

dimer of two

identical

subunits

of.

22.5 kD,

which

can be activated

by

a single

molecule

of

cyclic

AMP. A CRP

monomer

contains

a DNA-bind-

ing region and

a transcription-activating

region.

12.2! CRP

Functions

in Different

Ways

in

Different

Target 0perons

Adenine

\l

ll

/

_!

6'

327

t

cl

I

Glucose

I

V

Reduced

cAMP

I

V

Inactive

CRP

Nrsrsffiffi

No transcription

ts

Active

CRP

I

*t,

irIGUfiS

1?.?$

By reducing

the

[eve[ of cyclic AMP,

gtu-

cose inhibits

the

transcription of

operons that require

CRP

activitv.

Transcription

+

Highly

conserved

Less conserved

pentamer

fi6iJftt

1*.3*

The consensus

sequence for

CRP contains

the wet[

conserved

pentamer

TGTGA and

(sometimes)

an

inversion

of this sequence

(TCANA).

Center ot

dyad

symmetry

f,gl'#l?il

:t.]i

CRP bends DNA >90o

around the

centerof

symmetry.

A

CRP dimer binds to a

site of

-22bp

at a

responsive

promoter.

The

binding

sites include

variations

of the consensus

sequence

given

in

FIfiLiRf;

t*.3il.

Mutations

preventing

CRP

action

usually are located

within the well-conserved

TGTGA

pentamer

;;;;i,

which appears

to

be the

essential element

in recognition.

CRP binds most

strongly to sites that contain

two

(inverted)

ver-

sions of the

pentamer,

because this

enables both

subunits of the dimer to bind

to the DNA.

Many

binding sites lack

the second

pentamer,

how-

ever, and in

these the second

subunit must

bind

a different sequence

(if

it binds

to DNA). The

hierarchy

of binding affinities for

CRP helps

to

explain why different

genes

are activated

by

different Ievels

of cyclic AMP in vivo.

CRP introduces

a large

bend when it

binds

DNA.

In Ihe lac

promoter,

this

point

lies at the

center

of dyad symmetry. The

bend

is

quite

severe,

>90o,

as illustrated

in

the model

of

FIfiUft[

1e.53.

There is, therefore,

a

dramatic

change in the organization

of the DNA

double

helix

when CRP

protein

binds. The

mechanism

of bending

is to introduce

a sharp kink

within

the TGTGA consensus

sequence.

When

there

are inverted repeats

of the

consensus,

the two

kinks in

each copy

present

in

a

palindrome

cause

the overall90"

bend. It is

possible

that the

bend

has some direct

effect upon transcription,

but it

could

be the case that it is needed

simply to allow

CRP to contact RNA

polymerase

at the

promoter.

The

action of CRP has

the curious

feature

that its

binding sites lie

at different

locarions

relative

to the startpoint

in the various

operons

that it regulates.

The TGTGA

pentamer

may lie

in

either orientation.

The three

examples

sum-

marized in FIG{Jftil 1f,3f

encompass

the range

of locations:

.

The

CRP-binding

site is adjacent

to

the

promoter,

as in

the lacoperon,

in which

the region

of DNA

protected

by

CRP is

centered on

-61.

It is

possible

that

two

dimers

of CRP are

bound. The

binding

pattern

is consistent

with

the

presence

of CRP largely

on one face

of DNA,

which is the

same face

that is

bound by

RNA

polymerase.

This

location

would

place

the two

proteins

just

about

in

reach

of each other.

o

Sometimes

the

CRP-binding

site lies

within

the

promoter,

as in

tine

gallocus,

where

the CRP-binding

site is

centered

on

-41

. It is likely

rhat

only a single

CRp

dimer is

bound,

probably

in

quite

inti-

mate

contact with

RNA

polymerase,

322

CHAPTER

12 The

Operon

because the CRP-binding

site extends

well

into

the region

generally protected

by

the RNA

polymerase.

.

In other operons, the CRP-binding site

lies

well upstream of the

promoter.

In

tll'e ara region, the binding

site

for a sin-

gle

CRP is the farthest from

the start-

point,

centeredat-92.

Dependence on CRP is related

to the

intrin-

sic efficiency of the

promoter.

No

CRP-depend-

ent

promoter

has a

good

-3

5

sequence and some

also lack

good

-10

sequences.

In fact,

we

might

argue

that effective control by

CRP

would be

difficult if the

promoter

had effective

-35

and

-10

regions that interacted independently with

RNA

polymerase.

There are in

principle

two ways in

which

CRP might activate transcription: it could

inter-

act directly with RNA

polymerase,

or it could act

upon

DNA to change its

structure

in

some

way

that assists RNA

polymerase

to bind. In fact,

CRP

has effects upon both RNA

polymerase

and

DNA.

Binding sites

for

CRP at most

promoters

resemble either lac

(centered

at

-6I

)

or

gal

(cen-

tered at

-

I bp). The basic difference between

them

is that in the first type

(called

class I) the

CRP-binding

site is entirely upstream of the

pro-

moter, whereas

in

the second type

(called

class

II) the CRP-binding site overlaps the binding

site

for RNA

polymerase. (The

interactions at

the ara

promoter

may

be

different.)

In both types of

promoter,

the CRP bind-

ing site

is

centered

an integral number of turns

of

the double helix

from

the startpoint.

This

suggests

that CRP is bound to the same face of

DNA as

RNA

polymerase.

The nature of the

interaction between CRP and RNA

polymerase

is, however, different

at the two types of

promoter.

When the cr

subunit

of

RNA

polymerase

has a deletion

in

the C-terminal end, transcrip-

tion appears

normal except for the loss of abil-

ity to be activated by CRP. CRP

has

an

"activating

region" that

is required for activating both types

of its

promoters.

This activating region, which

consists

of an exposed

loop

of

-

I 0 amino

acids,

is

a small

patch

that interacts directly with

the

cr subunit

of RNA

polymerase

to stimulate the

enzyme.

At

class

I

promoters,

this interaction is

sufficient.

At class II

promoters,

a second

inter-

action is required,

which involves another

region of CRP

and the

N-terminal

region of the

RNA

polymerase

cr subunit.

Experiments

using CRP dimers in which

only one

of the subunits

has

a

functional tran-

Startpoint

gal

Iac

ara

-Promoter+

€

CRP-binding

locations+

.F'Ifi{-!RH 1 f .3 E

The

CRP

protein

can bind

at different

sites

relative to RNA

potymerase.

scription-activating

region

shows

that, when

CRP is bound attine

lacpromoter,

only

the acti-

vating

region of

the subunit

nearer the

start-

point

is

required,

presumably because

it touches

RNA

polymerase.

This offers

an

explanation

for

the

lack of dependence

on the

orientation

of

the binding

site: the

dimeric

structure

of

CRP

ensures that one

of the

subunits

is available

to

contact

RNA

polymerase, no matter

which

sub-

unit binds to

DNA and

in which

orientation.

The effect

upon

RNA

polymerase

binding

depends on the

relative

locations

of the

two

proteins.

At class

I

promoters, where

CRP binds

adjacent to

the

promoter, it increases

the rate

of

initial binding

to

form a closed

complex.

At

class

II

promoters, where

CRP

binds

within the

promoter,

it

increases

the

rate of transition

from

the closed to open

complex.

Translation

Can

Be Regulated

o

A repressor

protein

can

regulate translation

by

preventing

a ribosome

from binding

to an

initiation codon.

o

Accessibility of

initiation

codons

in

a

polycistronic

mRNA can be

controlled

by changes

in the

structure of the

mRNA

that

occur as

the

result

of transtation.

Ttanslational control

is a notable

feature of

oper-

ons coding

for components

of

the

protein

syn-

thetic

apparatus.

The operon

provides

an

arrangementf.or

coordinale

regulation

of

a

group

of structural

genes. Further

controls

superim-

posed

on the

operon,

though,

such

as those

at

the

level of translation,

may

create

differences

tn

1.2.22

TransLation

Can

Be Regulated

323

the

extent to

which individual genes

are

expressed.

A

similar type

of

mechanism

is

used to

achieve

translational

control in

several systems.

Repressor

function

is

provided

by a

protein

that

binds

to a target

region on wRNA

to

prevent

ribosomes

from

recognizing

the initiation

region. Formally

this is

equivalent

to a

repressor protein

binding

to

DNA

to

prevent

RNA

polymerase

from

utilizing

a

promoter.

aiSilRil

::.3:

illustrates

the most

common

form

of this interaction,

in

which the

regulator

protein

binds directly

to a sequence

that includes

the AUG initiation

codon, thereby

preventing

the ribosome

from

binding.

Some

examples

of translational

repressors

and

their targets

are summarized

in ijl**ft{ ti..:i4.

A

classic

example is the

coat

protein

of the RNA

phage

Rl7; it

binds to a hairpin

that

encom-

passes

the ribosome-binding

site in

the

phage

mRNA.

Similarly, the T4

RegA

protein

binds

to

a consensus

sequence

that includes

the AUG

initiation

codon in several

T4 early

mRNAs, and

T4

DNA

polymerase

binds to

a sequence in

its

own

mRNA

that includes

the Shine-Dalgarno

element

needed for ribosome

bindins.

€

Regulato,r

binding site+

55{;tji?l

.:il"i}

A regutator

protein

may

btock transtation

by binding

to a

site on mRNA

that overtaps

the ribosome-

binding

site at the initiation

codon.

Another form

of translational

control occurs

when

translation of one

cistron requires

changes

in

secondary structure

that depend

on transla-

tion of a

preceding

cistron. This

happens

dur-

ing translation

of the RNA

phages,

whose

cistrons always

are expressed in

a set

order.

F{*#R[

if-3*

shows that the

phage

RNA

takes

up a secondary

structure in

which only

one ini-

tiation

sequence is accessible;

the

second can-

not be recognized

by ribosomes

because

it is

i::fiilltf tI=3S

Secondary structure

can

control initia-

tion.

0nty one initiation

sjte

js

avaitable

in

the RNA

phage.

but translation

ofthe first

cistron

chanqes the

conforma-

tion of the

RNA so that

other initjatio-n

site(s)

become

avai [abte.

il3{"i#Flil

1t";1.{

Proteins

that

bind to sequences

within

the initiation

regions

of

mRNAs

may

function

as translationaI

reoressors.

Only one initiation

site

is

available

initially

Second initiation

site is blocked

First initiation

site

is accessible

Repressor

R17

coat

protein

T4

RegA

T4 DNA

polymerase

T4

p32

Target

Gene

R17 replicase

early T4 mRNAs

T4 DNA

polymerase

gene

32

Site of Action

hairpin

that includes ribosome

binding site

various

sequences

including initiation

codon

Shine-Dalgarno

sequence

single-stranded

5' leader

324

CHAPTER

12

The

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VNUTU

I

OG

Binding to:

ssDNA

P32

mRNA

10

8

10

7

10

6

10

5

1O-4

1O-3

Protein concentration

(Mola0

+

A feature of autogenous

control

is that each

regulatory interaction

is unique:

a

protein

acts

only on the

nRNA

responsible

for

its own syn-

thesis.

Phage

T4

provides an example

of

a more

general

translational

regulator,

coded

by the

gele

regA, which

represses

the

expression

of

several

genes

that

are transcribed

during

early

infection.

RegA

protein

prevents the transla-

tion

of mRNAs

for these

genes

by competing

with 30S subunits

for the

initiation

sites on

the

mRNA. Its action

is a direct

counterpart

to the

function of

a repressor

protein

that

binds

mul-

tiple operators.

i;:.li:liili:

,i.i.:i:,:

6.n.32 orotein binds to various sub-

strates

wjth different affinities, in the order singte-stranded

DNA, its own mRNA, and other mRNAs. Binding to

its

own

mRNA

prevents

the [eve[ of

p32

from rising

>10-6 M.

lation of its own

nRNA. The

effect

is mediated

directly

by

p32

binding to mRNA to

prevent

ini-

tiation of translation.

In

all

likelihood

this occurs

at an

A-T-rich region that surrounds the ribo-

some

binding site.

TWo features of the binding of.

p32

to the

site on

nRNA are required to make the control

loop work effectively:

.

The affinity of

p32

for the site on

gene

32

mRNA

must

be significantly

lower than

its affinity for single-stranded DNA.

The

equilibrium constant

for

binding

RNA is

in fact almost two orders of

magnitude

below that

for

single-stranded

DNA.

.

The affinity ofp32 for the mRNA,

how-

ever,

must

be significantly

greater

than

the affinity

for

other

RNA

sequences.

It

is influenced by base composition

and

by secondary

structure; an important

aspect of the binding to

gene

J2

mRNA

is that

the regulatory region has an

extended

sequence lacking secondary

structure.

Using

the known equilibrium

constants,

we can

plot

the binding of

p32

to its target sites

as

a function of

protein

concentration.

iii.,i-jtir

;

"::.-.ti]

shows that at concentrations

below

I0-6 M,

p32

binds

to single-stranded DNA.

At

concentrations

>10-6

M, it binds to

ge\e

32

nRNA.

At

yet greater

concentrations,

it binds

to other

nRNA sequences, with a

range of

affinities.

These

results imply that the level of.

p32

should

be autoregulated to be

<10-6

M,

which

corresponds

to

-2000

molecules

per

bacterium.

This

fits well with the measured

level of 1000

to 2000 molecules/cell.

Autogenous

Regulation

Is

Often

Used

to

Control

Synthesis

of

Macromolecu[ar

AssembLies

.

The

precursor

to

microtubutes,

free tubulin

protein,

inhibits

translation

of tubutin

mRNA.

Autogenous

regulation

is a common

type of

control among

proteins that

are

incorporated

into macromolecular

assemblies.

The assem-

bled

particle itself may

be

unsuitable

as

a reg-

ulator because

it is too

large,

too numerous,

or

too

restricted

in its

location.

The

need

for syn-

thesis of

its components,

though,

may

be

reflected in the

pool

of free

precursor subunits.

If the assembly

pathway is blocked

for any

rea-

son,

free subunits

accumulate

and

shut off

the

unnecessary

synthesis

of

further

components.

Eukaryotic

cells

have

a common

system

in

which

autogenous

regulation

of

this tlpe

occurs.

Tubulin

is the

monomer

from

which

micro-

tubules, a

major

filamentous

system

of

all

eukaryotic

cells,

are

synthesized.

The

produc-

tion of tubulin

nRNA

is controlled

by

the free

tubulin

pool.

When

this

pool

reaches

a certain

concentration,

the

production

of further

tubu-

lin mRNA

is

prevented. Again,

the

principle is

the same:

tubulin

sequestered

into

its macromol-

ecular assembly

plays no

part

in

regulation,

but

the

level of the

free

precursor

pool

determines

whether

further

monomers

are

added

to it.

The target

site

for

regulation

is a short

sequence

at the

start

of the

coding

region.

We

do

not know

yet

what

role this

sequence

plays,

but two

models

are

illustrated

in

Ih*{"1ftil

.irl-+-l}.

Tirbulin may

bind

directly

to the

mRNA,

or

it

12.25

Autogenous Regutation

Is 0ften

Used

to

Control

Synthesis

of

Macromotecutar

Assembties

327

rs ro

erlner

^

o

--oo

O6

o

f

)tein

Tubulin

is

assembled

into

microtubules

nffif

Free tubulin

binds to

either

i-i*i":,1i i.i.+t

Tubutin

is assembted

into microtubuLes

when it

is

synthesized. Accumutation

of excess free

tubu-

[in induces

instabiLity

jn

the tubu[in

mRNA

by acting at

a

site at

the start

of the reading frame

jn

mRNA

or at the

corresponding

position

in

the nascent

protein.

may

bind to

the nascent

polypeptide

represent-

ing

this region.

Whichever

model

applies,

excess

tubulin

causes

tubulin

nRNA

that is located

on

polysomes

to

be degraded,

so the

consequence

of

the reaction

is

to make

the tubulin

mRNA

unstable.

Autogenous

control is

an intrinsically

self-

Iimiting

system,

by

contrast

with the

extrinsic

control

that

we discussed previously.

A repres-

sor

protein's

ability

to bind

an

operator may

be

controlled

by the level

of an

extraneous

small

molecule,

which

activates

or inhibits

its activ-

ity. In

the

case of autogenous

regulation,

though,

the

critical

parameter

is

the concentration

of

the

protein

itself.

Sum

mary

Transcription

is regulated

by the

interaction

b etwe

e n tr an

s

-

acling

f a ctors

and cli

-

a cting

sites.

A trans-acing

factor

is

the

product

of a

regula-

tor gene.

It is

usually

protein

but

also

can be

RNA.

It

diffuses in

the

cell, and

as a result

it can

act

on any

appropriate

target gene.

A cis-acting

site in

DNA

(or

RNA)

is a

sequence

that func-

CHAPTER

12

The

0peron

tions by

being recognized

in situ.It has

no cod-

ing function

and can regulate

only those

sequences with

which it is

physically

contigu-

ous. Bacterial

genes

coding for

proteins

whose

functions

are related,

such as successive

enzymes

in

a

pathway,

may

be organized

in a

cluster that

is transcribed

into a

polycistronic

mRNA from

a

single

promoter.

Control

of this

promoter

reg-

ulates expression

of the

entire

pathway.

The

unit

of

regulation,

which contains

structural

genes

and crs-acting

elements,

is

called the

operon.

Initiation

of transcription

is regulated

by

interactions

that occur in

the vicinity

of the

pro-

moter. The

ability

of

RNA

polymerase

to initi-

ate at the

promoter

is

prevented

or

activated

by

other

proteins.

Genes

that are

active unless

they

are turned

off are said to

be under negative

con-

trol.

Genes that are

active only

when

specifi-

cally

turned on are

said to

be under

positive

control.

The type

of control

can be

determined

by the dominance

relationships

between

wild

type

and mutants

that are

constitutive/dere-

pressed

(permanently

on) or uninducible/super-

repressed

(permanently

off).

A repressor protein prevents

RNA

poly-

merase

either from

binding to

the

promoter

or

from

activating

transcription.

The

repressor

binds to

a target

sequence, the

operator,

that

usually

is located

around

or upstream

of the

startpoint.

Operator

sequences

are short

and

often are

palindromic.

The

repressor

is

often a

homomultimer

whose

svmmetrv

reflects

that

of

its target.

The

ability of the repressor prorein

to bind

to its

operator is

regulated

by a

small molecule.

An inducer

prevents

a repressor

from

binding;

a corepressor

activates

it. Binding

of the

inducer

or corepressor

to its

site

produces

a change

in

rhe

strucrure

of the DNA-binding

site of the

repressor.

This allosteric

reaction

occurs in

both

free

repressor

proteins

and directly

in

repres-

sor

proteins

already

bound to

DNA.

The lactose

pathway

operates

by induction,

when an

inducer

B-galactoside

prevents

the

repressor

from

binding

its operator;

transcrip-

tion and

translation

of the lacZ

gene

then

produce

B-galactosidase,

the enzyme

that

metabolizes

p-galactosides.

The

tryptophan

pathway

operates

by repression;

the corepres-

sor

(tryptophan)

activates

the repressor pro-

tein,

so that it

binds to

the operator

and

prevents

expression

of

the

genes

that

code

for

the

enzymes

that

biosynthesize

tryptophan.

A

repressor

can

control

multiple

targets

that have

copies

of an operator

consensus

sequence.

328

A

protein

with a high

affinity lor a

partic-

ular target sequence in DNA has a lower affin-

ity for all DNA.

The ratio

defines the specificity

of the

protein.

There are many more nonspe-

cific sites

(any

DNA sequence) than specific

target sites

in a

genome;

as a

result,

a

DNA-

binding

protein

such as a repressor or RNA

polymerase

is

"stored"

on

DNA.

(It

is likely

that none, or very

little,

is free.) The specificity

for

the

target sequence must be

great

enough

to counterbalance the excess of

nonspecific

sites over specific

sites. The

balance

for bacte-

rial

proteins

is adjusted so that the amount of

protein

and

its specificity allow specific recog-

nition of the target in

"on"

conditions, but

allow almost

complete release of the target

in

"off"

conditions.

References

Introduction

Resea rc

h

Jacob,

F. and

Monod,

J.

(

196 I

).

Genetic

regulatory

mechanisms

in

the synthesis

of

proteins.

J. Mol. Biol ),318-)89.

Regutation Can

Be Negative

or

Positive

Review

Miller, J.

and Reznikoff, W, eds

(1980).

The

Operon,

2nd ed., Woodbury, NY: Cold

Spring

Harbor

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The lac Genes Are Controtled

by a Repressor

Reviews

Barkley,

M. D. and Bourgeois, S.

(1978).

Repressor

recognition of operator

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Tfte

Operon,

e ds. Miller, J. and

Reznikoff,

W.

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York: Cold Spring

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Beckwith,

J.

(1978).

lac:

Ihe

genetic

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Operon,

eds. Miller, J. and

Reznikoff, W. New

York: Cold

Spring Harbor

Laboratory, I I-30.

Beyreuther,

I(.

(1978).

Chemical structure

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functional

organization of the

lac repressor

from E. coli.ln

The Operon, eds. Miller, J. and

Reznikoff, W. New

York: Cold Spring

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Miller, J.

H.

(I978).

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gene

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operon

control and

its

use as

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genetic

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York: Cold Spring

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l-88.

Weber,

I(. and Geisler,

N.

(1978).

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the understanding

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Miller, J.

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6.

Wilson,

C. J., Zahn,

H., Swint-I(ruse,

L., and

Matthews,

K. S.

(2006).

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lactose

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allosteric

behavior

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protein folding. Cell.

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Jacob,

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Monod, J.

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l96l

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synthesis

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The Reoressor

Monomer

Has Several

Domains

Resea

rch

Friedman,

A. M., Fischmann,

T. O.,

and Steitz,

T. A.

(

1995). Crystal

structure

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core tetramer

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its

implications

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Iooping. Science

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Lewis, M. et al

(

1996).

Crystal

structure

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lac-

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DNA and

inducer.

Science

271,

1247-1254.

Mutant

PhenotYPes

Corretate

with

the

Domain

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Pace, H. C.,

I(ercher,

M.

A., Lu,

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P.,

Miller, J.

H., Chang,

G.,

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M'

(1997)r.

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genetic map in

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ICeina,

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Cruz, C.,

Ehret,

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Miller, J.

H.

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XIV. Analysis

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CHAPTER

12 The

0peron

Lewin Benjamin (ed.) Genes IX

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