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the transposon,

separating

its

strands

and ter-

minating

at

its

ends.

Replication is

probably

accomplished

by

host-coded functions.

At this

juncture,

the structure

has become a cointe-

grate, possessing

direct

repeats of the transpo-

son at the

junctions

between

the replicons

(as

can be seen

by tracing

the

path

around

the

cointegrate).

i:"ri.tiiil-

ii

!. l5 Nonrepticative

transposition

results

when

a crossover

structure

is

reteased

by

nicking.

This

inserts

the

transposon

into

the target

DNA,

ftanked by

the direct

repeats ofthe

target.

and

the donor

is teft

with a doubLe-

strand

break.

i:i-li-ilii

:.ii,ii: Both

strands

of

Tn10 are

cteaved

sequen-

tiaLty,

and then

the

transposon

isjoined

to the

njcked

tar-

get

site.

replication,

the transposon

at

the

new

site

will

contain

information

from

only

one

of the

par-

ent

Tnl0 strands.

If. however,

transposition

takes

place

by

physical movement

of the

existing

trans-

poson,

the

mismatches

will

be

conserved

at the

new site.

This

proved

to be

the

case.

21..8

Nonrepticative

Transposition

Proceeds

by

Breakage

and

Reunion

533

Nonrep[icative

Transposition

Proceeds

by

Breakage and Reunion

Nonrepticative

transposition

resutts

jf

a crossover

structure

is nicked on the unbroken

pair

of donor

strands

and the target

strands on either side

of the

transposon

are ligated.

Two

pathways

for nonreplicative

transposition

differ according

to whether

the first

pair

of

transposon

strands are

joined

to the target

before

the second

pair

are

cut

(Tn5).

or whether

a[[ four

strands are

cut before

joining

to the target

(Tn10).

The crossover

structure

can also be used

in

non-

replicative

transposition.

The

principle

of non-

replicative

transposition

by

this mechanism

is

that a breakage

and

reunion reaction

allows the

target to

be reconstructed

with the

insertion of

the

transposon;

the donor

remains broken.

No

cointegrate

is formed.

:-ir,.ii-:i: ,,

i-

:::

shows the cleavage

events

that

generate nonreplicative

transposition

of

phage

Mu. Once

the unbroken

donor strands

have

been

nicked,

the target strands

on either

side

of the

transposon

can be

ligated. The single-

stranded

regions

generated

by

the staggered

cuts

must be

filled

in

by

repair synthesis.

The

product

of this

reaction is a

target replicon

in

which

the transposon

has been inserted

between

repeats of

the sequence

created by

the original

single-strand

nicks.

The donor replicon

has a

double-strand

break across the

site where

the

transposon

was originally

located.

Nonreplicative

transposition

can

also occur

by an

alternative

pathway

in which

nicks are

made in target

DNA, but

a double-strand

break

is made

on either

side of

the transposon,

releas-

ing

it entirely

from

flanking donor sequences

(as

envisaged

in

Figure 21.7).

This

"cut

and

paste"

pathway is used by

Tnl0, as

illustrated

in

;;;l--;1'r::.iir.

A neat experiment

to

prove

that

Tnl0 trans-

poses

nonreplicatively

made use of an

artificially

constructed

heteroduplex

of Tnl0 that

contained

single base

mismatches.

If

transposition

involves

Tn

is

joined

to target

r

: i.ij

*i

.'l't..:

.:

Cteavage

of Tn 5 from

flanking

DNA involves

nicking,

interstrand

reaction,

and hairpjn

cleavaqe.

i:!r.:

:

;

+-:

::

Lr'i

. l: :r

Each

subunit

of the Tn

5

transDosase

nas

one end

of the

transposon

located in

its

active site

ano

also makes

contact

at a

different

site

with

the otner

end

of

the transposon.

The

basic

difference

in Figure

2l.l6

from

the

model

of Figure

21.15

is

that

both

strands

of

Tn10

are cleaved

before

any

connection

is

made

to the

target

site. The

first

step

in the

reaction

is

recognition

of the

transposon

ends

by

the

transposase,

forming

a

proteinaceous

structure

within

which

the reaction

occurs.

At

each

end

of

the transposon,

the

strands

are

cleaved

in

a

specific

order:

the transferred

strand

(the

one to

be

connected

to

the target

site) is

cleaved

first,

followed

by

the

other

strand.

(This

is

the same

order

as

in the

Mu

transposition

of Figure

2 I . l4

and

Figure

21.15.)

Tn5

also

transposes

by

nonreplicative

trans-

position,

and ;'i*l.it:tf ,l

i . ]. tr

shows

the

interesting

cleavage

reaction

that

separates

the

transposon

CHAPTER

21

Transposons

from

the flanking

sequences.

First

one DNA

strand is

nicked. The

3'-OH

end

that is

released

then

attacks

the other

strand

of

DNA.

This

releases

the flanking

sequence

and

joins

the

two strands

of

the transposon

in

a hairpin.

An

activated

water

molecule

then

attacks

the

hair-

to

generate

free

ends for

each

strand

of

the

transposon.

In

the next

step,

the cleaved

donor

DNA

is released,

and the

transposon

is

joined

to

the

nicked

ends at

the target

site.

The

transposon

and

the target

site remain

constrained

in the

proteinaceous

structure

created

by

the

trans-

posase (and

other

proteins).

The

double-strand

cleavage

at each

end

of the

transposon pre-

cludes

any replicative-type

transposition

and

forces

the reaction

to

proceed

by nonreplica-

tive transposition,

thus

giving

the

same

out-

come

as in Figure

21.14,

but with

the individual

cleavage

and

joining

steps

occurring

in

a dif-

ferent

order.

The

Tn 5

and Tn I

0 transposases

both

func-

tion

as dimers.

Each

subunit

in

the

dimer

has

an active

site

that successively

catalyzes

the dou-

ble-strand

breakage

of the

two

strands

at one

end

of the

transposon,

and

then

catalyzes

stag-

gered

cleavage

of the

target

site.

{:i{llJfti

;ii.'lil

illustrates

the

structure

of the

Tn5

transposase

bound

to the

cleaved

transposon.

Each

end

of

the

transposon

is

located

in the

active

site of

one subunit.

One end

of the

subunit

also

con-

tacts

the other

end

of the

transposon.

This

con-

trols

the

geometry

of the

transposition

reaction.

Each

of the

active

sites

will cleave

one strand

of

the target

DNA.

It is the geometry

of

the com-

plex

that determines

the distance

between

these

sites

on the

two target

strands

(nine

base

pairs

in

the case

of Tn5).

TnA

Transposition

Requires

Transposase

and

Resolvase

.

Repticative

transposition

of TnA

requires

a

transposase

to form

the cointegrate

structure

and

a resotvase

to retease

the

two replicons.

r

The

action

of

the resolvase

resembtes

[ambda

Int

protein

and betongs

to the

general

famity

of

topoisomerase-tike

site-specifi

c recombination

reactions.

which

pass

through

an intermediate

in

which

the

protein

is

covalently

bound

to the DNA.

Replicative

transposition

is

the

only

mode

of

mobility

of the TnA

family,

which

consists

of

534

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by a sector of

the new

phenotlpe

residing

within

the tissue

of the original

phenotype.

The size of

the sector depends on the

number

of divisions

in the

lineage

giving

rise to it, so the size of the

area of the

new

phenotype

is determined by the

timing of the change

in

genotype.

The earlier

its

occurrence

in the cell lineage, the

greater

the

number of descendants and thus the size of

patch

in the mature tissue.

This is

seen

most vividly in

the

variation in kemel color, when

patches

of one

color appear

within another color.

Insertion of a controlling element

may affect

the activity

of adjacent

genes.

Deletions, dupli-

cations,

inversions, and translocations

all occur

at the sites where controlling elements

are

pres-

ent. Chromosome

breakage is a common con-

sequence of

the

presence

of some elements.

A

unique

feature of

the maize

system

is that the

activities

of the controlling elements

are regu-

lated during

development. The elements trans-

pose

and

promote genetic

rearrangements at

characteristic

times and frequencies during

plant

development.

The characteristic behavior of controlling

elements

in maize is typified by the Ds ele-

ment, which

was originally identified by

its

ability

to

provide

a site

for chromosome break-

age.

The consequences are

illustrated in

r:i,-

.::.:i

.

. :.

Consider a heterozygote

in which

Ds lies on

one homolog between the

cen-

tromere

and a series of dominant

markers. The

other

homolog

lacks Ds and has recessive

mark-

ers

(C

bz,

andwx). Breakage at

Ds

generates

an

acentric

fragment carrying the dominant

markers. As a

result

of

its lack of a centromere,

this fragment

is lost at

mitosis. Thus the descen-

dant

cells

have only the recessive

markers car-

ried by the

intact chromosome.

This

gives

the

type

of situation

whose results are depicted

in

Figure

2I.22.

:'

:.!.:.i'ri

,

,.,

shows that breakage

at Ds leads

to the formation

of two unusual chromosomes.

These are

generated

byjoining

the broken ends

of the

products

of

replication. One is a U-shaped

acentric

fragment consisting of

the

joined

sister

chromatids

for the region distal to

Ds

(on

the

left

as drawn

in the figure). The other

is a U-shaped

dicentric

chromosome

comprising

the sister

chromatids

proximal

to Ds

(on

its right

in the

figure). The

latter structure leads to

the classic

breakage-fusion-bridge

cycle illustrated

in

the

figure.

Follow

the fate of the dicentric

chromo-

some

when

it attempts to segregate

on the

mitotic spindle.

Each

of

its two centromeres

pulls

toward an

opposite

pole.

The tension

: li-,iriiit.

l.

.,i

:

Clonal

anatysis

identifies

a

group

of cetls

descended

from a singte

ancestor

in

which a transposition-

mediated event

altered

the

phenotype. Timing ofthe

event

during devetopment

is indicated

by

the

number of

ceLts; tis-

sue specificity

of the

event

may be

indicated

by the

[oca-

tion ofthe cetts.

ii'i,i.iiii:

..:

ii I

A break

at

a controtling

element

causes

loss of an

acentric

fragmenU

if the

fragment

carries

the

dominant

markers

of a

heterozygote,

jts

loss

changes

the

phenotype.

The effects

of

the

dominant

markers,

CI,

Bz,

and

Wx, can be

vjsuatized

by the

cotor

of the

celts

or by

appropriate

staining.

21.11

Controttinq

Elements

in

Maize Cause

Breakage

and

Rearrangements

539

Break

in one chromosome

causes

loss of

dominant

allele

oooc

ta

It

't't

tl

ti

oooooooo

iiiiiiii

oooooooo

t/

Cells of original

genotype

r

| /

display

dominant

phenotype

V

Clone

descended

from mutant

disPlaYs

recessive

Phenotype

Centromere

Cell

PhenotYPe

@

I

ereak at

Ds

Y

Acentric

f ragment

C-------gr--4-E-c.,,

:]

Break at Ds

c-----_l

t-I-E-el

CfA

B-dI)

I

Y

First fusion-bridge

breakage

cycle

Sister

chromatid f

usion

I

Breaka9e

C-

-c-B-A-AIIB

c.,-

....:

Chromosome

with

Chromosome

with

!

duplication

of A

deletion

of A

:

Second fusion-bridge

breakage

cycle

:

trc

-----\

:

r-=

€c''

'

I

/---;---;--;---=--\

\-utrtrv__)

I

r-.-:---=:--AG_.------\

L_

r, r, rrl

v._ __)

Chromosome

with

Chromosome

with

duplication

of B

deletion

of B

some that has lost

A. In the next

cycle,

a break

occurs between

B and

C, so that

the descen-

dants are

divided into

those with

a duplication

of B

and those

with a deletion.

Successive

losses

of dominant

markers

are revealed

by

subsec-

tors

within sectors.

-,

'r'

.

'

Ds

provides

a site

to initiate

the chro-

matid

breakage-fusion-bridge

cycle. The

products

can

be

fotlowed

by

clonaI anatysis.

breaks

the

chromosome

at a random

site

between

the

centromeres.

In

the example

of

the

figure,

breakage

occurs

between

loci A

and

B, with

the

result

that

one daughter

chromo-

some

has

a duplication

of A, whereas

the other

has a

deietion.

If

A is a

dominant

marker,

the

cells

with

the duplication

will retain

a

pheno-

type,

but

cells

with the

deletion will

display

the

recessive

a

phenotype.

The

breakage-fusion-bridge

cycle

contin-

ues through

further

cell

generations,

allowing

genetic

changes

to continue

in

the

descendants.

For

example,

consider

the deletion

chromo-

CHAPTER

2L Transposons

Contro[[ing ELements

Form Families

of Transposons

.

Each family

of transposons

in maize

has

both

autonomous

and nonautonomous

controtting

e[ements.

o

Autonomous

controlting

elements

code for

proteins

that

enabte them to

transoose.

r

Nonautonomous

controtting

elements have

mutations

that

eliminate their

capacity

to catalyze

transposition,

but they can transpose

when

an

autonomous

element

provides

the necessary

protei

ns.

r

Autonomous

controtling

elements have

changes

of

phase,

when

their

properties

alter

as a resutt

of

changes in

the state of methytation.

The

maize

genome

contains

several

families

of

controlling

elements. The

numbers,

types,

and

locations

of the

elements

are characteristic

for

each individual

maize

strain.

They

may

occupy

a

significant

part

of the

genome.

The

members

of each family

are divided

into

two

classes:

.

Autonomous

controlling

elements

have

the ability

to excise

and

transpose.

As

a result

of the continuing

activity

of

an

autonomous

element,

its

insertion

at any locus

creates

an

unstable

or

"mutable"

allele.

Loss

of

the auto-

nomous

element

itself,

or

of its ability

to

transpose,

converts

a

mutable

allele

to

a stable

allele.

.

Nonautonomous

controlling

ele-

ments

are

stable;

they do

not transpose

or suffer

other

spontaneous

changes

in

condition.

They

become

unstable

only

when

an autonomous

member

of the

same

family is

present

elsewhere

in the

genome.

When

complemented

it

trans

by

an autonomous

element,

a

nonau-

tonomous

element

displays

the

usual

range

of activities

associated

with

autonomous

elements,

including

the

ability

to transpose

to new

sites.

Nonau-

tonomous

elements

are

derived

frorn

autonomous

elements

by loss

of trans-

acting functions

needed

for

transposition.

Lewin Benjamin (ed.) Genes IX

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