Analysis

of

the

Tribolium

homeotic

complex

insights

into

mechanisms

constraining

insect

Hox

clusters

The

remarkable

conservation

of

Hox

clusters

is

an

accepted

but

little

understood

principle

of

biology

Some

organizational

constraints

have

been

identified

for

vertebrate

Hox

clusters

but

most

of

these

are

thought

to

be

recent

innovations

that

may

not

apply

to

other

organisms

Ironically

many

model

organisms

have

disrupted

Hox

clusters

and

may

not

be

well-suited

for

studies

of

structural

constraints

In

contrast

the

red

flour

beetle

Tribolium

castaneum

which

has

a

long

history

in

Hox

gene

research

is

thought

to

have

a

more

ancestral-type

Hox

cluster

organization

Here

we

demonstrate

that

the

Tribolium

homeotic

complex

-LRB-

HOMC

-RRB-

is

indeed

intact

with

the

individual

Hox

genes

in

the

expected

colinear

arrangement

and

transcribed

from

the

same

strand

There

is

no

evidence

that

the

cluster

has

been

invaded

by

non-Hox

protein-coding

genes

although

expressed

sequence

tag

and

genome

tiling

data

suggest

that

noncoding

transcripts

are

prevalent

Finally

our

analysis

of

several

mutations

affecting

the

Tribolium

HOMC

suggests

that

intermingling

of

enhancer

elements

with

neighboring

transcription

units

may

constrain

the

structure

of

at

least

one

region

of

the

Tribolium

cluster

This

work

lays

a

foundation

for

future

studies

of

the

Tribolium

HOMC

that

may

provide

insights

into

the

reasons

for

Hox

cluster

conservation

Introduction

Hox

clusters

arose

near

the

origins

of

the

animal

kingdom

-LRB-

Larroux

et

al.

Ryan

et

al.

-RRB-

The

last

common

ancestor

of

the

protostomes

and

deuterostomes

is

thought

to

have

had

a

cluster

of

at

least

seven

genes

characterized

by

a

common

transcriptional

orientation

and

by

colinearity

in

the

order

of

the

genes

and

their

expression

domains

along

the

anterior

posterior

axis

-LRB-

reviewed

in

Garcia-Fernandez

-RRB-

In

various

metazoan

lineages

Hox

clusters

have

gained

or

lost

genes

by

duplication

and

deletion

but

often

have

maintained

their

chromosomal

order

transcriptional

orientation

and

both

spatial

and

temporal

colinearity

of

expression

patterns

-LRB-

reviewed

in

Ferrier

and

Minguillon

-RRB-

suggesting

that

Hox

cluster

organization

has

been

subject

to

strong

constraints

during

evolution

Classical

model

systems

such

as

Drosophila

and

Caenorhabditis

elegans

have

provided

many

important

insights

into

the

developmental

functions

of

Hox

genes

but

do

not

provide

particularly

good

examples

of

Hox

cluster

conservation

The

Hox

cluster

of

Drosophila

melanogaster

is

split

into

two

parts

-LRB-

the

Antennapedia

-LRB-

ANTC

-RRB-

and

bithorax

-LRB-

BXC

-RRB-

complexes

-RRB-

shows

changes

in

transcriptional

orientation

of

some

genes

and

includes

interspersed

genes

of

independent

origin

as

well

as

Hox-derived

genes

that

have

evolved

novel

developmental

roles

-LRB-

reviewed

in

Ferrier

and

Minguillon

-RRB-

These

alterations

suggest

that

the

constraints

keeping

the

Hox

cluster

intact

may

have

been

lost

in

the

lineage

leading

to

Drosophila

Additional

Hox

cluster

rearrangements

-LRB-

breaks

microinversions

and

gene

transpositions

-RRB-

have

been

found

in

other

Drosophila

species

-LRB-

Negre

et

al.

Negre

and

Ruiz

Von

Allmen

et

al.

-RRB-

as

well

as

in

the

silk

moth

Bombyx

mori

-LRB-

Yasukochi

et

al.

-RRB-

The

Hox

genes

of

C.

elegans

-LRB-

reviewed

in

Aboobaker

and

Blaxter

-RRB-

and

the

tunicate

Oikopleura

dioica

-LRB-

Seo

et

al.

-RRB-

have

undergone

even

more

extreme

loss

and

rearrangement

such

that

of

their

remaining

Hox

genes

are

clustered

In

most

cases

the

Hox

genes

of

these

organisms

still

show

spatial

but

not

temporal

colinearity

Rapid

development

seems

to

be

the

common

denominator

among

most

of

these

organisms

perhaps

making

temporal

colinearity

of

Hox

genes

unnecessary

or

even

undesirable

-LRB-

Ferrier

and

Holland

Ferrier

and

Minguillon

Negre

et

al.

-RRB-

While

studies

of

disrupted

Hox

clusters

have

provided

some

insights

into

Hox

cluster

maintenance

a

more

complete

understanding

will

require

analysis

of

organisms

where

they

are

still

intact

Studies

of

vertebrate

Hox

clusters

have

uncovered

several

potential

mechanisms

that

may

promote

temporal

colinearity

and

therefore

constrain

the

organization

of

these

clusters

-LRB-

reviewed

in

Kmita

and

Duboule

-RRB-

These

include

progressive

changes

in

chromatin

state

along

the

length

of

the

cluster

varying

affinity

of

regulatory

elements

to

a

gradient

of

signal

and

the

presence

of

global

enhancer

elements

outside

the

cluster

that

regulate

multiple

genes

within

the

cluster

However

it

is

not

clear

whether

these

mechanisms

apply

to

other

organisms

Duboule

-LRB-

-RRB-

has

suggested

that

the

modern

vertebrate

Hox

clusters

are

actually

more

organized

than

the

ancestral

cluster

Some

of

the

mechanisms

constraining

the

organization

of

vertebrate

Hox

clusters

likely

evolved

concomitant

with

the

co-option

of

Hox

genes

for

functions

such

as

limb

development

-LRB-

Duboule

Kmita

and

Duboule

-RRB-

and

therefore

may

not

be

applicable

to

other

lineages

Based

on

this

model

we

might

expect

to

gain

a

better

understanding

of

the

ancestral

constraints

on

Hox

clusters

by

studying

a

less

organized

but

still

intact

cluster

Such

clusters

have

been

described

in

organisms

as

diverse

as

the

cephalochordate

amphioxus

-LRB-

Garcia-Fernandez

and

Holland

Minguillon

et

al.

-RRB-

sea

urchins

-LRB-

Cameron

et

al.

-RRB-

and

the

insects

Apis

-LRB-

Honey

Bee

Genome

Sequencing

Consortium

Dearden

et

al.

-RRB-

and

Anopheles

-LRB-

Holt

et

al.

Negre

and

Ruiz

-RRB-

Evidence

also

suggests

that

the

red

flour

beetle

Tribolium

castaneum

has

an

intact

Hox

cluster

Conventional

cloning

and

sequencing

of

the

portion

of

the

cluster

corresponding

to

the

Drosophila

Antennapedia

complex

has

shown

that

this

region

of

the

homeotic

complex

-LRB-

HOMC

-RRB-

is

intact

in

Tribolium

-LRB-

Brown

et

al.

-RRB-

Genetic

mapping

also

suggests

that

the

integrity

of

the

Tribolium

Hox

cluster

has

been

maintained

-LRB-

Beeman

-RRB-

Moreover

the

genetic

methodologies

possible

with

Tribolium

as

well

as

the

application

of

RNAi

have

provided

a

comprehensive

description

of

the

full

repertoire

of

Hox

genes

and

their

functions

-LRB-

e.g.

Beeman

et

al.

Beeman

et

al.

Brown

et

al.

Shippy

et

al.

Shippy

et

al.

Stuart

et

al.

Stuart

et

al.

Tomoyasu

et

al.

-RRB-

The

Tribolium

genome

has

recently

been

sequenced

giving

us

the

opportunity

to

explore

the

structure

of

its

Hox

cluster

in

detail

Here

we

present

an

analysis

of

several

Hox

mutations

along

with

the

transcriptional

profile

of

the

cluster

during

embryonic

development

We

discuss

these

results

with

respect

to

potential

mechanisms

of

Hox

cluster

organization

and

constraint

Materials

and

methods

Sequence

and

transposable

element

analysis

Sequence

analysis

was

performed

using

Vector

NTI

Advance

-LRB-

Invitrogen

-RRB-

Basic

Local

Alignment

Search

Tools

-LRB-

BLASTs

-RRB-

against

Tribolium

genome

sequence

-LRB-

-RRB-

were

performed

at

Tcastaneum

or

and

subsequent

analysis

was

performed

using

Genboree

-LRB-

-RRB-

or

NCBI

Map

Viewer

-LRB-

-RRB-

The

entire

HOMC

sequence

was

used

as

a

BLASTn

query

against

a

collection

of

expressed

sequence

tags

-LRB-

ESTs

-RRB-

provided

by

Dr.

Yoonseong

Park

-LRB-

Department

of

Entomology

Kansas

State

University

Manhattan

KS

USA

-RRB-

Transposable

elements

were

identified

and

classified

using

Censor

to

search

the

arthropod

subset

of

Repbase

-LRB-

Kohany

et

al.

-RRB-

Array

design

and

probe

synthesis

Sequence

for

the

Tribolium

HOMC

was

taken

from

the

Baylor

HSGC

assembly

The

tiled

region

consists

of

bases

from

-LRB-

to

-RRB-

stretching

between

the

two

non-Hox

genes

flanking

the

complex

NimbelGen

designed

mer

oligos

covering

this

region

at

two

densities

-LRB-

-RRB-

one

feature

per

bp

and

-LRB-

-RRB-

one

feature

per

bp

An

additional

region

spanning

kb

on

either

side

of

the

putative

homolog

of

was

tiled

at

a

higher

density

of

one

feature

every

bp

Visualization

and

scaling

of

tiling

data

was

performed

using

Integrated

Genome

Browser

-LRB-

Affymetrix

-RRB-

Tribolium

embryos

grown

at

°C

in

standard

media

were

collected

by

sieving

dechorionated

for

min

in

bleach

and

homogenized

in

μl

of

Trizol

using

a

teflon

pestle

Total

RNA

was

then

extracted

using

the

standard

Trizol

protocol

-LRB-

Invitrogen

-RRB-

dsDNA

was

prepared

from

μg

of

total

RNA

with

random

hexamers

according

to

Kapranov

et

al.

-LRB-

-RRB-

with

the

following

modifications

Primers

were

annealed

using

a

ramp

to

°C

and

the

first

strand

reaction

was

not

subdivided

for

second

strand

synthesis

The

resulting

cDNA

was

used

as

template

by

NimbelGen

for

labeling

and

hybridization

-LRB-

Squazzo

et

al.

-RRB-

Fluorescent

in

situ

hybridization

Probe

labeling

embryo

fixation

and

RNA

FISH

were

performed

according

to

Kosman

et

al.

-LRB-

-RRB-

with

the

following

modifications

Tribolium

embryos

were

dechorionated

in

bleach

for

min

and

agitated

for

min

to

h.

Embryos

were

devitellinized

by

alternating

min

vortexing

with

min

shaking

for

min

after

the

addition

of

cold

methanol

followed

by

passage

through

an

syringe

three

to

five

times

Primers

used

for

making

the

and

probes

are

as

follows

AGATAAGATATAATGAGGTGTAGAGTTG

TGATTAACATGGACGGCTTCATTAG

CATCCTATGCACATGCGTTC

CGTTTTAATGGGTGCATCGT

Dig-labeled

RNA

probes

were

detected

using

sheepαDIG

-LRB-

Roche

-RRB-

primary

and

donkeyαsheep

Alexa

Fluor

-LRB-

Molecular

Probes

-RRB-

secondary

antibodies

Genetics

Beetles

were

cultured

at

°C

on

whole

wheat

flour

supplemented

with

brewer

's

yeast

as

described

by

Beeman

et

al.

-LRB-

-RRB-

Strains

used

were

and

-LRB-

wild

type

-RRB-

Ey

and

Eyeless

-LRB-

Ey

Beeman

et

al.

-RRB-

and

AbdominalExtra

sclerite

-LRB-

AEs

Beeman

et

al.

-RRB-

are

dominantly

marked

balancer

chromosomes

that

suppress

crossing

over

within

the

HOMC

Cuticle

preparations

were

performed

as

described

by

Shippy

et

al.

-LRB-

-RRB-

For

documentation

cuticles

were

placed

in

lactic

acid/ethanol

on

a

depression

slide

and

covered

with

a

coverslip

Images

were

captured

at

several

focal

planes

using

a

Nikon

digital

camera

and

combined

into

a

single

image

using

Auto-Montage

software

-LRB-

Syncrosopy

-RRB-

RNAi

Parental

RNAi

for

ptl/Tc-Antp

was

performed

by

injection

of

dsRNA

into

the

abdomens

of

female

pupae

Eggs

were

collected

from

injected

females

at

intervals

aged

to

hatching

and

subjected

to

cuticle

preparation

Analysis

of

breakpoints

Eggs

were

collected

after

overnight

incubation

at

°C

and

allowed

to

develop

for

days

Genomic

DNA

was

isolated

from

individual

homozygous

larvae

as

described

by

Gloor

et

al.

-LRB-

-RRB-

Polymerase

chain

reaction

-LRB-

PCR

-RRB-

surveys

of

the

HOMC

were

used

to

identify

likely

breakpoint

positions

and

fragments

spanning

these

putative

breakpoints

were

amplified

using

universal

PCR

-LRB-

Beeman

and

Stauth

Sarkar

et

al.

-RRB-

PCR

products

were

cloned

and

sequenced

at

the

Kansas

State

University

DNA

Sequencing

Center

The

resulting

sequences

were

compared

to

the

Tribolium

genome

sequence

to

characterize

the

breakpoints

GenBank

accession

numbers

for

these

sequences

are

as

follows

A

-LRB-

-RRB-

and

B

-LRB-

-RRB-

Analysis

of

breakpoints

Tribolium

genomic

DNA

was

isolated

from

Ey

AEs/Ey

and

pupae

as

described

by

Brown

et

al.

-LRB-

-RRB-

with

the

exception

that

DNA

was

not

purified

on

a

CsCl

gradient

Digested

DNAs

were

separated

on

a

agarose

gel

by

field

inversion

gel

electrophoresis

and

transferred

to

GeneScreen

nylon

membrane

-LRB-

NEN

Life

Sciences

-RRB-

To

look

for

restriction

fragment

length

polymorphisms

associated

with

the

blot

was

probed

with

a

kb

HindIII

fragment

containing

the

end

of

the

mxp/Tc-pb

coding

region

Inverse

PCR

-LRB-

Ochman

et

al.

-RRB-

was

used

to

clone

breakpoints

associated

with

Ey

genomic

DNA

was

digested

with

a

restriction

enzyme

-LRB-

EcoRI

HindIII

and

RsaI

for

breakpoint

fragments

A

B

and

C

respectively

-RRB-

After

circularization

of

the

fragments

two

rounds

of

PCR

were

performed

with

primers

designed

from

known

sequence

Resulting

fragments

were

cloned

using

the

TOPO-TA

Cloning

Kit

-LRB-

Invitrogen

-RRB-

sequenced

and

submitted

to

GenBank

under

the

following

accession

numbers

A

-LRB-

-RRB-

B

-LRB-

-RRB-

C

-LRB-

-RRB-

Sequences

were

compared

to

the

Tribolium

genome

sequence

to

determine

the

location

of

breakpoints

Analysis

of

transposon

insertion

site

The

piggyBac-insertion

site

was

amplified

by

vectorette

PCR

as

described

by

Lorenzen

et

al.

-LRB-

-RRB-

The

resulting

product

was

sequenced

by

Elim

Biopharmaceuticals

Inc.

-LRB-

Hayward

CA

USA

-RRB-

and

the

sequence

was

submitted

to

GenBank

as

accession

number

Results

The

Tribolium

Hox

cluster

has

retained

an

ancestral

organization

Several

bacterial

artificial

chromosome

-LRB-

BAC

-RRB-

clones

encompassing

the

ANTC-like

region

of

the

Tribolium

Hox

cluster

were

previously

sequenced

and

annotated

-LRB-

Brown

et

al.

-RRB-

Using

the

newly

assembled

Tribolium

genome

sequence

we

have

performed

a

similar

analysis

of

the

BXC-like

portion

of

the

cluster

and

find

that

this

region

contains

the

Tribolium

orthologs

of

Ultrabithorax

-LRB-

Ubx

Bennett

et

al.

-RRB-

abdominal-A

-LRB-

abd-A

Shippy

et

al.

-RRB-

and

Abdominal-B

-LRB-

Abd-B

-RRB-

As

in

Drosophila

the

transcription

units

in

this

part

of

the

complex

are

larger

than

those

of

the

ANTC-like

portion

due

to

the

presence

of

longer

introns

As

expected

from

previous

molecular

and

genetic

studies

-LRB-

Beeman

Brown

et

al.

-RRB-

all

of

the

Tribolium

Hox

genes

map

to

a

single

cluster

on

This

cluster

spans

approximately

Kb

within

a

single

scaffold

of

the

assembled

genome

sequence

A

few

small

sequencing

gaps

are

present

in

the

assembly

but

more

than

half

can

be

filled

by

other

available

sequences

-LRB-

i.e.

the

three

BAC

clones

previously

sequenced

for

the

ANTC-like

portion

of

the

cluster

and

four

BACs

from

the

BXC-like

region

sequenced

for

verification

of

the

shotgun

genome

assembly

Tribolium

Genome

Consortium

-RRB-

The

total

length

of

the

filled

gaps

is

approximately

bp

-LRB-

mismatches

in

the

sequence

flanking

the

gaps

lead

to

some

ambiguity

-RRB-

which

is

only

slightly

longer

than

the

estimated

total

length

of

these

gaps

-LRB-

bp

-RRB-

Thus

estimation

of

sequencing

gaps

in

the

HOMC

region

appears

to

be

quite

accurate

Two

of

these

gaps

are

immediately

adjacent

to

transposable

element

insertion

sites

and

may

result

from

difficulties

in

assembling

repetitive

DNA

These

two

sites

account

for

about

bp

of

the

total

gaps

in

the

HOMC

In

two

other

cases

gaps

in

the

genome

assembly

are

associated

with

tandem

duplications

that

are

not

present

in

the

BAC

assemblies

an

approximately

duplication

between

Tc-Deformed

-LRB-

Tc-Dfd

-RRB-

and

and

an

approximately

duplication

between

prothoraxless/Tc-Antennapedia

-LRB-

ptl/Tc-Antp

-RRB-

and

Tc-fushi

tarazu

-LRB-

Tc-ftz

-RRB-

We

designed

primers

to

amplify

across

the

regions

in

question

using

genomic

DNA

-LRB-

the

same

inbred

strain

that

was

used

for

the

Tribolium

genome

sequence

-RRB-

In

both

cases

the

size

of

the

resulting

fragment

is

consistent

with

that

predicted

from

the

BAC

sequence

-LRB-

data

not

shown

-RRB-

suggesting

that

these

gaps

and

duplications

are

artifacts

of

the

genome

assembly

process

It

is

important

to

note

that

these

artifacts

affect

only

a

small

fraction

of

the

HOMC

sequence

but

they

underscore

the

increased

quality

of

finished

versus

draft

sequences

The

single

Tribolium

Hox

cluster

contains

orthologs

of

all

eight

Drosophila

Hox

genes

as

well

as

orthologs

of

the

Hox-derived

genes

fushi

tarazu

and

zerknüllt

-LRB-

zen

Tribolium

Genome

Consortium

and

Fig.

-RRB-

-LRB-

In

the

case

of

zen

Tribolium

has

two

paralogs

apparently

resulting

from

a

recent

duplication

in

the

beetle

lineage

independent

of

the

zen

duplication

that

occurred

in

the

Drosophila

lineage

-LRB-

Brown

et

al.

-RRB-

-RRB-

These

genes

are

arranged

in

the

same

order

on

the

chromosome

as

their

counterparts

in

other

insects

As

in

Apis

-LRB-

Dearden

et

al.

Negre

and

Ruiz

-RRB-

but

in

contrast

to

Drosophila

and

Anopheles

-LRB-

Negre

and

Ruiz

-RRB-

the

Hox

and

Hox-derived

genes

in

the

Tribolium

cluster

are

all

oriented

in

the

same

direction

-LRB-

Fig.

-RRB-

In

addition

the

two

miRNA

genes

-LRB-

and

-RRB-

that

have

been

described

in

other

insect

Hox

clusters

are

found

at

conserved

positions

in

the

Tribolium

HOMC

-LRB-

Tanzer

et

al.

and

Fig.

-RRB-

Fig.

transcription

across

the

complete

Tribolium

Hox

complex

The

tiling

array

consists

of

bp

probes

that

estimate

degree

of

transcription

Relative

intensities

for

each

probe

are

represented

as

peaks

correlated

with

a

consensus

annotation

of

the

Tribolium

Hox

complex

-LRB-

below

-RRB-

Peak

height

shown

as

Percentile

Probe

Intensity

-LRB-

PPI

-RRB-

corresponds

to

the

level

of

transcription

for

a

particular

probe

The

nucleotide

position

for

each

segment

is

displayed

in

the

upper

left

and

right

corners

of

the

panel

-LRB-

numbers

correspond

with

linkage

group

release

-RRB-

New

ESTs

-LRB-

cyan

-RRB-

are

displayed

in

the

annotation

track

along

with

transposable

elements

-LRB-

gray

-RRB-

For

annotated

genes

and

ESTs

the

arrow

indicates

the

direction

of

transcription

Red

arrows

indicate

the

location

of

two

RNA-FISH

probes

The

ANTC

and

BXC

clusters

of

Drosophila

melanogaster

contain

a

number

of

non-Hox

protein-coding

genes

In

contrast

there

is

no

evidence

for

non-Hox

protein-coding

genes

in

the

Tribolium

HOMC

-LRB-

The

Tribolium

Genome

Consortium

-RRB-

Here

we

corroborate

those

findings

by

using

several

methods

to

address

whether

unrelated

genes

might

be

interspersed

among

the

Tribolium

Hox

genes

First

we

searched

the

Tribolium

genome

for

orthologs

of

genes

that

are

located

within

the

D.

melanogaster

clusters

and

determined

that

of

these

genes

are

located

within

the

Tribolium

HOMC

Second

we

analyzed

predicted

proteins

within

the

region

to

determine

whether

any

of

them

have

recognizable

orthologs

in

other

species

Other

than

the

Hox

and

Hox-derived

genes

we

found

no

evolutionarily

conserved

proteins

among

either

the

GLEAN

predictions

or

the

GNOMON

ab

initio

predictions

that

map

to

the

Hox

cluster

Third

we

searched

a

collection

of

Tribolium

ESTs

for

expressed

sequences

within

the

HOMC

By

this

approach

we

identified

three

non-Hox

EST

clusters

that

appear

to

represent

noncoding

transcripts

as

well

as

evidence

for

a

mariner

transposase

gene

-LRB-

see

below

-RRB-

but

no

other

protein-coding

genes

were

found

Finally

we

analyzed

the

embryonically

transcribed

sequences

identified

by

a

tiling

array

to

determine

if

any

were

likely

to

encode

proteins

Again

we

found

no

evidence

of

non-Hox-related

protein-coding

genes

other

than

those

within

transposable

elements

Although

there

are

caveats

to

these

analyses

-LRB-

e.g.

gene

prediction

methods

are

imperfect

the

tiling

array

represents

only

the

embryonic

transcriptome

and

EST

coverage

is

incomplete

-RRB-

our

results

strongly

suggest

that

the

protein-coding

genes

in

the

Tribolium

Hox

complex

-LRB-

excluding

genes

within

transposable

elements

-RRB-

are

all

either

Hox

or

Hox-derived

genes

Comparison

of

transposable

element

density

in

the

Hox

complexes

of

various

animals

has

led

to

the

suggestion

that

higher

abundance

of

transposable

elements

in

Hox

clusters

is

correlated

with

loss

of

structural

integrity

Mammalian

Hox

complexes

have

a

reduced

number

of

transposons

compared

to

other

regions

of

the

genome

-LRB-

Ferrier

and

Minguillon

-RRB-

Moreover

when

transposons

are

present

they

seem

to

be

preferentially

inserted

into

nontranscribed

regions

of

the

clusters

-LRB-

Mainguy

et

al.

-RRB-

In

contrast

transposons

occur

fairly

frequently

in

the

split

Drosophila

clusters

-LRB-

Fried

et

al.

-RRB-

Though

the

prediction

of

three

transposable

elements

in

the

Tribolium

Hox

complex

-LRB-

Fig.

-RRB-

may

be

an

underestimate

the

same

method

predicts

fivefold

more

in

the

Drosophila

Hox

clusters

Additionally

only

three

transposable

elements

-LRB-

all

mariners

-RRB-

have

been

found

in

the

larger

but

intact

Apis

Hox

complex

-LRB-

Dearden

et

al.

-RRB-

These

numbers

are

consistent

with

the

apparent

inverse

correlation

between

transposon

number

and

the

level

of

Hox

cluster

organization

Taken

together

these

observations

suggest

that

with

respect

to

gene

content

order

and

orientation

the

Tribolium

Hox

cluster

closely

resembles

the

putative

ancestral

Hox

cluster

-LRB-

Garcia-Fernandez

-RRB-

Thus

the

constraints

preserving

the

integrity

of

the

Hox

cluster

may

still

be

in

force

in

Tribolium

To

determine

whether

these

constraints

extend

outside

the

Tribolium

Hox

cluster

we

examined

synteny

beyond

the

cluster

itself

As

previously

described

Tc-chaoptic

partially

overlaps

the

UTR

of

Tc-labial

-LRB-

Tc-lab

-RRB-

on

the

opposite

strand

-LRB-

Nie

et

al.

-RRB-

Working

outward

the

first

gene

on

the

same

strand

as

the

Hox

genes

is

a

dolichyl

glycosyltransferase

orthologous

to

D.

melanogaster

Beyond

the

Tc-Abd-B

locus

is

a

cluster

of

putative

serine

carboxypeptidase

genes

-LRB-

and

-RRB-

and

the

ortholog

of

D.

melanogaster

-LRB-

-RRB-

We

identified

the

orthologs

of

these

genes

in

D.

melanogaster

Anopheles

gambiae

and

Apis

mellifera

and

determined

their

map

positions

of

these

genes

map

near

the

Hox

cluster

in

any

of

the

other

insects

Likewise

orthologs

of

the

genes

adjacent

to

lab

and

Abd-B

in

D.

melanogaster

do

not

map

near

the

Hox

clusters

of

the

other

three

insects

These

results

suggest

that

the

constraints

preserving

the

Hox

cluster

act

only

on

the

Hox

genes

themselves

and

not

the

surrounding

region

The

Tribolium

Hox

cluster

produces

numerous

noncoding

transcripts

We

developed

a

tiling

microarray

covering

the

Tribolium

Hox

complex

to

identify

the

transcription

units

active

during

a

broad

window

of

Tribolium

embryonic

development

Tiling

array

signal

intensity

profiles

were

compared

with

previously

described

Hox

cDNA

structures

Though

the

tiling

density

is

not

fine-scaled

enough

to

effectively

resolve

intron

exon

boundaries

there

is

a

near-perfect

correlation

between

tiling

array-predicted

transcription

and

the

position

of

exons

in

the

well-characterized

Hox

genes

The

only

caveat

is

that

the

exons

of

maxillopedia/Tc-proboscipedia

-LRB-

mxp/Tc-pb

-RRB-

and

Tc-Abd-B

exhibit

weaker

signals

than

the

other

exons

of

these

genes

The

most

likely

explanation

is

that

the

exon

is

present

only

in

a

small

subset

of

the

transcripts

derived

from

the

gene

-LRB-

i.e.

a

minor

spliceoform

-RRB-

During

the

first

days

of

development

numerous

regions

of

the

Hox

complex

including

intergenic

and

intronic

noncoding

regions

are

actively

transcribed

-LRB-

Fig.

-RRB-

Interestingly

neither

of

the

two

most

likely

noncoding

candidates

the

previously

described

miRNAs

is

robustly

identified

on

the

tiling

array

Transcription

at

the

locus

is

not

detected

and

transcription

at

the

locus

is

weak

It

may

be

that

is

not

expressed

during

the

stages

examined

whereas

in

situ

hybridization

assays

show

that

is

strongly

expressed

during

part

of

the

developmental

window

examined

-LRB-

Fig.

-RRB-

Fig.

pattern

of

two

HOMC

noncoding

transcripts

in

Tribolium

embryos

Probe

positions

are

shown

in

Fig.

a

Expression

pattern

from

a

probe

located

kb

of

the

start

of

ptl/Tc-Antp

b

The

expression

pattern

of

the

Tribolium

homolog

of

the

miRNA

-LRB-

-RRB-

The

most

intense

hybridization

signals

are

detected

in

the

central

kb

of

the

Hox

complex

encompassing

the

ptl/Tc-Antp

and

Ultrathorax/Tc-Ultrabithorax

-LRB-

Utx/Tc-Ubx

-RRB-

genes

Strikingly

transcription

in

this

region

is

almost

equally

intense

for

coding

and

noncoding

loci

and

for

both

the

introns

and

exons

of

the

protein-coding

genes

There

are

hundreds

of

discrete

regions

-LRB-

bp

or

longer

-RRB-

where

signal

intensity

is

many

times

greater

than

for

verified

Hox

gene

exons

It

is

not

possible

to

determine

from

the

single

time

point

we

have

analyzed

if

any

of

these

discrete

regions

are

part

of

larger

transcripts

To

verify

that

the

observed

signals

in

the

tiling

array

represent

authentic

transcription

RNA

fluorescent

in

situ

hybridization

-LRB-

FISH

-RRB-

was

performed

with

a

representative

region

between

ptl/Tc-Antp

and

Utx/Tc-Ubx

-LRB-

in

Fig.

-RRB-

This

region

is

expressed

in

a

Hox-like

pattern

with

distinct

anterior

and

posterior

borders

in

the

posterior

region

of

the

elongating

germ

band

-LRB-

Fig.

-RRB-

Interestingly

signal

is

detected

primarily

in

two

spots

per

nucleus

presumably

at

the

sites

of

nascent

transcription

This

suggests

either

rapid

degradation

or

processing

of

a

primary

transcript

as

would

be

seen

for

a

pri-miRNA

or

an

intron

In

our

search

for

additional

genes

within

the

Tribolium

Hox

cluster

we

identified

three

ESTs

that

appear

to

represent

noncoding

transcripts

One

seems

to

be

a

chimeric

artifact

arising

from

the

fusion

of

a

precursor

and

part

of

a

rRNA

gene

The

second

represented

by

two

independent

cDNAs

maps

between

ptl/Tc-Antp

and

Utx/Tc-Ubx

while

the

third

is

located

within

the

first

intron

of

Utx/Tc-Ubx

The

last

two

are

transcribed

from

the

strand

opposite

the

Hox

genes

and

are

correlated

with

regions

of

strong

signal

in

the

tiling

array

analysis

The

mutation

affects

regulation

of

both

mxp/Tc-pb

and

Cx/Tc-Scr

Because

complex

regulatory

regions

may

act

as

a

constraining

force

keeping

Hox

clusters

intact

we

analyzed

the

mutation

which

was

shown

to

have

unusual

effects

on

the

expression

of

mxp/Tc-pb

-LRB-

Shippy

et

al.

-RRB-

homozygotes

lack

most

if

not

all

normal

mxp/Tc-pb

expression

but

both

heterozygotes

and

homozygotes

display

strong

ectopic

mxp/Tc-pb

expression

in

a

pattern

reminiscent

of

Cephalothorax/Tc-Sex

combs

reduced

-LRB-

Cx/Tc-Scr

-RRB-

expression

-LRB-

albeit

with

an

apparent

posterior

shift

of

some

domains

-RRB-

This

ectopic

expression

is

sufficient

to

rescue

some

aspects

of

mxp/Tc-pb

function

so

is

not

an

mxp/Tc-pb

-LRB-

Shippy

et

al.

-RRB-

Interestingly

we

find

that

fails

to

complement

a

allele

of

Cx/Tc-Scr

-LRB-

and

in

fact

appears

to

be

for

Cx/Tc-Scr

-RRB-

but

complements

alleles

of

Tc-Dfd

and

ptl/Tc-Antp

-LRB-

data

not

shown

-RRB-

To

better

understand

this

complex

mutation

we

characterized

the

breakpoints

associated

with

is

associated

with

a

chromosomal

rearrangement

involving

at

least

four

breakpoints

Although

we

have

not

ruled

out

the

presence

of

additional

breakpoints

the

simplest

interpretation

of

our

data

is

that

a

fragment

of

the

HOMC

-LRB-

including

Tc-Dfd

Cx/Tc-Scr

and

Tc-ftz

-RRB-

has

been

removed

from

the

HOMC

and

inserted

between

a

fragment

of

and

a

non-HOMC

fragment

of

-LRB-

Fig.

-RRB-

This

scenario

is

consistent

with

previously

reported

pseudo-linkage

of

and

associated

with

-LRB-

Beeman

et

al.

-RRB-

and

provides

an

explanation

for

the

mid-embryonic

lethality

of

the

homozygotes

-LRB-

Shippy

et

al.

-RRB-

That

is

non-HOMC

breakpoints

interrupt

both

the

TFIIA-L

ortholog

which

is

a

component

of

the

transcriptional

machinery

-LRB-

Yokomori

et

al.

-RRB-

and

a

homolog

of

groucho

which

is

an

important

transcriptional

corepressor

in

Drosophila

-LRB-

Jimenez

et

al.

Paroush

et

al.

-RRB-

We

conclude

that

the

breakpoint

between

Tc-ftz

and

ptl/Tc-Antp

is

likely

to

account

for

the

loss

of

Cx/Tc-Scr

function

associated

with

probably

by

separating

the

Cx/Tc-Scr

transcription

unit

from

some

or

all

of

its

regulatory

units

The

rearrangement

probably

juxtaposes

these

regulatory

elements

with

the

mxp/Tc-pb

transcription

unit

providing

a

likely

explanation

for

the

Cx/Tc-Scr-like

expression

of

mxp/Tc-pb

in

embryos

Fig.

of

the

HOMC

In

these

schematic

diagrams

the

positions

of

cloned

breakpoint

fragments

are

underlined

Wild-type

chromosomal

position

-LRB-

not

to

scale

-RRB-

on

-LRB-

purple

-RRB-

is

indicated

by

a

gradient

of

color

to

illustrate

the

effects

of

inversions

a

In

the

rearrangement

an

approximately

fragment

of

the

HOMC

has

been

transposed

between

fragments

of

and

b

The

mutation

is

a

large

inversion

that

splits

the

Hox

cluster

into

two

parts

Small

fragments

at

each

end

of

the

inversion

appear

to

have

been

deleted

including

part

of

the

ptl/Tc-Antp

locus

Cx/Tc-Scr

regulatory

elements

map

near

or

within

ptl/Tc-Antp

Beeman

et

al.

-LRB-

-RRB-

observed

that

mutations

in

Cx/Tc-Scr

and

ptl/Tc-Antp

often

partially

fail

to

complement

one

another

Because

this

is

precisely

the

type

of

genetic

interaction

that

would

be

predicted

if

the

separation

of

Hox

genes

has

deleterious

effects

we

decided

to

analyze

an

allele

of

ptl/Tc-Antp

that

shows

such

effects

The

mutation

which

results

in

transformation

of

the

larval

legs

toward

antennae

as

well

as

reductions

of

some

labial

and

thoracic

tissue

has

been

proposed

to

be

a

allele

of

ptl/Tc-Antp

-LRB-

Beeman

et

al.

-RRB-

However

adults

transheterozygous

for

have

a

very

different

phenotype

from

that

produced

by

larval

ptl/Tc-Antp

RNAi

-LRB-

Tomoyasu

et

al.

-RRB-

and

instead

resemble

adults

in

which

both

ptl/Tc-Antp

and

Cx/Tc-Scr

have

been

knocked

down

-LRB-

Tomoyasu

personal

communication

-RRB-

This

result

raises

the

possibility

that

the

mutation

affects

the

function

of

both

genes

To

address

this

issue

we

performed

parental

RNAi

with

ptl/Tc-Antp

and

found

that

the

resulting

larvae

-LRB-

Fig.

-RRB-

show

a

phenotype

almost

identical

to

that

of

homozygotes

-LRB-

Fig.

-RRB-

suggesting

that

the

mutation

primarily

affects

the

function

of

ptl/Tc-Antp

during

embryonic

development

However

there

is

a

subtle

difference

between

the

and

ptl/Tc-Antp

RNAi

phenotypes

in

the

positioning

of

the

appendages

which

are

located

near

the

ventral

midline

in

the

RNAi

larvae

but

more

laterally

in

the

mutants

This

difference

is

likely

attributable

to

partial

loss

of

Cx/Tc-Scr

function

in

because

Cx/Scr

is

responsible

for

the

midline

position

of

the

labial

appendages

in

Tribolium

-LRB-

Shippy

et

al.

-RRB-

and

other

insects

-LRB-

Hughes

and

Kaufman

Pattatucci

et

al.

Rogers

et

al.

-RRB-

Thus

appears

to

be

not

only

a

allele

of

ptl/Tc-Antp

but

also

a

hypomorphic

allele

of

Cx/Tc-Scr

Fig.

and

enhancer

trap

phenotypes

of

ptl/Tc-Antp

mutations

The

antennae

-LRB-

ant

-RRB-

and

the

labial

-LRB-

lab

-RRB-

and

thoracic

-LRB-

-RRB-

segments

are

denoted

where

relevant

a

e

Cuticle

preps

displaying

the

phenotypes

of

wild-type

-LRB-

a

-RRB-

ptl/Tc-Antp

RNAi

-LRB-

b

-RRB-

-LRB-

c

-RRB-

-LRB-

d

-RRB-

and

-LRB-

e

-RRB-

first

instar

larvae

Enhancer

trap-driven

EGFP

expression

in

a

embryo

-LRB-

f

-RRB-

appears

in

a

very

similar

pattern

to

Cx/Tc-Scr

expression

-LRB-

purple

-RRB-

in

a

wild-type

embryo

-LRB-

g

-RRB-

A

larva

-LRB-

h

-RRB-

and

pupa

-LRB-

i

-RRB-

also

display

EGFP

enhancer

trap

expression

in

parts

of

the

labial

and

first

thoracic

segments

To

understand

why

the

mutation

affects

both

ptl/Tc-Antp

and

Cx/Tc-Scr

we

characterized

its

mutant

lesion

-LRB-

s

-RRB-

We

found

that

is

associated

with

an

inversion

of

about

Mb

with

breakpoints

in

ptl/Tc-Antp

and

a

distant

region

of

-LRB-

Fig.

-RRB-

In

addition

there

are

small

deletions

at

each

end

of

the

inversion

-LRB-

approximately

kb

of

the

ptl/Tc-Antp

transcription

unit

including

all

of

exon

and

approximately

kb

at

the

other

end

-RRB-

Consistent

with

this

conclusion

we

find

that

can

act

as

a

crossover

suppressor

for

reducing

recombination

between

Reindeer

-LRB-

a

mutation

near

one

end

of

-RRB-

and

the

HOMC

from

its

normal

value

of

cM

-LRB-

Beeman

et

al.

-RRB-

to

approximately

cM

These

results

suggest

that

breakpoints

within

the

ptl/Tc-Antp

gene

affect

the

function

of

both

ptl/Tc-Antp

and

Cx/Tc-Scr

probably

by

disrupting

the

function

of

Cx/Tc-Scr

regulatory

elements

-LRB-

see

Discussion

-RRB-

Additional

evidence

for

the

presence

of

Cx/Tc-Scr

regulatory

elements

in

the

vicinity

of

ptl/Tc-Antp

comes

from

a

piggyBac-insertion

line

recovered

during

an

insertional

mutagenesis

project

The

line

carries

a

homozygous

lethal

insertion

in

the

last

intron

of

ptl

-LRB-

Fig.

-RRB-

Crosses

between

heterozygotes

produce

a

class

of

embryos

-LRB-

putative

homozygotes

-RRB-

with

the

and

legs

partially

transformed

toward

antennae

-LRB-

Fig.

-RRB-

a

phenotype

consistent

with

partial

loss

of

Ptl/Tc-Antp

function

However

individuals

carrying

the

insertion

display

an

embryonic

enhancer

trap

expression

pattern

-LRB-

Fig.

-RRB-

very

similar

to

the

expression

pattern

of

Cx/Tc-Scr

-LRB-

Curtis

et

al.

Fig.

-RRB-

despite

the

fact

that

the

insertion

site

is

about

kb

upstream

of

Cx/Tc-Scr

larvae

and

pupae

also

show

weak

enhancer

trap

patterns

consistent

with

predicted

Cx/Tc-Scr

domains

-LRB-

Fig.

i

-RRB-

As

expected

from

the

phenotype

of

homozygotes

fails

to

complement

as

assayed

by

adult

viability

and

crosses

of

to

heterozygotes

produce

embryos

with

a

phenotype

similar

to

but

slightly

weaker

than

that

of

homozygotes

-LRB-

Fig.

-RRB-

In

contrast

fully

complements

both

the

embryonic

phenotype

and

the

adult

viability

of

a

allele

of

Cx/Tc-Scr

-LRB-

Shippy

et

al.

-RRB-

indicating

that

Cx/Tc-Scr

function

is

not

compromised

by

the

insertion

-LRB-

data

not

shown

-RRB-

These

results

suggest

that

is

a

hypomorphic

allele

of

ptl/Tc-Antp

and

it

will

hereafter

be

referred

to

as

Together

with

the

data

from

the

and

mutations

the

Cx/Tc-Scr-like

enhancer

trap

phenotype

of

provides

strong

evidence

that

Cx/Tc-Scr

regulatory

elements

are

located

near

and

probably

within

ptl/Tc-Antp

Although

additional

experiments

will

be

necessary

to

pinpoint

the

location

of

regulatory

elements

and

verify

this

conclusion

it

is

intriguing

to

think

that

overlap

of

regulatory

elements

of

one

Hox

gene

with

the

transcription

unit

of

another

Hox

gene

might

be

an

important

mechanism

of

Hox

cluster

constraint

Fig.

of

Cx/Tc-Scr

regulatory

elements

with

the

ptl/Tc-Antp

locus

The

gene

structure

of

ptl/Tc-Antp

-LRB-

coding

sequence

is

shaded

gray

-RRB-

and

the

positions

of

mutant

lesions

are

shown

in

the

diagram

The

inferred

positions

of

Cx/Tc-Scr

regulatory

elements

in

the

ptl/Tc-Antp

region

are

indicated

below

the

diagram

Discussion

The

Hox

clusters

of

several

insects

have

now

been

completely

sequenced

While

breaks

in

the

cluster

seem

to

have

occurred

several

times

in

the

Drosophila

lineage

the

clusters

of

Apis

Anopheles

and

Tribolium

are

intact

This

suggests

that

many

insect

clusters

are

still

subject

to

constraints

to

maintain

their

organization

Of

the

insects

with

intact

clusters

Tribolium

is

by

far

the

most

genetically

tractable

and

has

a

strong

history

of

Hox

gene

studies

thus

offering

the

best

system

for

understanding

the

constraints

acting

on

an

intact

insect

Hox

cluster

Below

we

discuss

insights

provided

by

our

analysis

of

the

Tribolium

HOMC

into

mechanisms

that

might

be

responsible

for

Hox

cluster

integrity

Temporal

colinearity

Among

organisms

for

which

both

Hox

cluster

sequence

and

expression

data

are

available

the

presence

of

an

intact

cluster

appears

to

be

correlated

with

temporal

colinearity

of

Hox

gene

expression

while

disrupted

clusters

are

associated

with

lack

of

temporal

colinearity

-LRB-

Monteiro

and

Ferrier

-RRB-

This

observation

has

pushed

temporal

colinearity

to

the

forefront

of

discussions

about

Hox

cluster

maintenance

However

several

questions

remain

to

be

answered

Does

temporal

colinearity

really

require

an

intact

Hox

cluster

Is

temporal

colinearity

required

for

proper

Hox

gene

function

in

organisms

with

intact

clusters

If

temporal

colinearity

is

a

key

constraint

on

Tribolium

Hox

cluster

integrity

we

might

expect

rearrangements

to

affect

the

function

of

most

or

all

Hox

genes

However

our

analysis

of

Tribolium

Hox

cluster

mutations

provides

no

evidence

for

such

global

effects

The

inversion

splits

the

complex

into

two

parts

but

all

of

the

Hox

genes

except

ptl/Tc-Antp

and

Cx/Tc-Scr

appear

to

function

normally

In

addition

the

rearrangement

results

in

the

translocation

of

several

HOMC

genes

-LRB-

Tc-zen

Tc-Dfd

Cx/Tc-Scr

and

Tc-ftz

-RRB-

to

a

new

chromosomal

location

At

least

one

of

these

genes

Tc-Dfd

is

functional

because

fully

complements

a

Tc-Dfd

allele

Likewise

the

genes

remaining

in

the

HOMC

-LRB-

with

the

exception

of

mxp/Tc-pb

-RRB-

apparently

function

normally

These

limited

effects

of

Hox

cluster

rearrangements

are

similar

to

what

has

been

reported

for

Drosophila

-LRB-

e.g.

Abbott

and

Kaufman

Pultz

et

al

-RRB-

Although

additional

experiments

will

be

required

to

determine

whether

Tribolium

Hox

genes

exhibit

temporal

colinearity

our

results

suggest

that

constraints

on

the

Tribolium

HOMC

are

more

likely

to

act

locally

Regulatory

elements

The

reported

breaks

and

transposition

sites

in

the

Hox

clusters

of

Drosophila

species

are

all

located

in

intergenic

regions

near

the

end

of

a

gene

-LRB-

Negre

et

al.

Negre

and

Ruiz

-RRB-

and

thus

are

presumably

less

likely

to

separate

a

gene

from

its

regulatory

elements

which

are

predominantly

located

of

each

Drosophila

Hox

gene

Interestingly

the

one

exception

to

this

rule

is

the

region

between

abd-A

and

Abd-B

which

contains

regulatory

regions

for

both

genes

and

is

not

split

in

any

of

the

Drosophila

species

examined

so

far

-LRB-

Negre

and

Ruiz

-RRB-

These

observations

led

to

the

conclusion

that

Drosophila

Hox

clusters

have

a

modular

organization

-LRB-

with

each

gene

and

its

regulatory

elements

representing

a

separate

module

-RRB-

and

that

Drosophila

Hox

genes

are

still

partially

clustered

simply

because

the

regulatory

regions

are

so

large

that

there

are

relatively

few

positions

where

breaks

can

occur

without

disturbing

a

module

That

is

most

of

the

remaining

linkage

in

Drosophila

Hox

clusters

-LRB-

with

the

possible

exception

of

abd-A

and

Abd-B

-RRB-

is

due

to

phylogenetic

inertia

and

given

enough

time

the

clusters

will

completely

disperse

-LRB-

Negre

and

Ruiz

-RRB-

The

question

naturally

arises

whether

phylogenetic

inertia

could

also

be

the

reason

for

the

intact

Hox

clusters

of

insects

like

Tribolium

Lewis

et

al.

-LRB-

-RRB-

suggested

that

unusual

features

of

recombination

-LRB-

Ranz

et

al.

-RRB-

may

make

drosophilids

more

tolerant

of

Hox

cluster

rearrangements

than

are

most

insects

If

this

is

the

case

intact

clusters

may

just

be

the

consequence

of

slower

rates

of

chromosomal

rearrangement

-LRB-

Negre

and

Ruiz

-RRB-

Alternatively

constraints

on

Hox

cluster

maintenance

may

still

be

functional

in

Tribolium

Our

analysis

of

mutant

breakpoints

in

the

ptl/Tc-Antp

region

indicates

that

in

at

least

one

case

Tribolium

Hox

genes

are

not

modular

That

is

at

least

some

of

the

regulatory

elements

controlling

Cx/Tc-Scr

expression

are

apparently

located

within

the

pt/Tc-Antp

gene

-LRB-

Fig.

-RRB-

Most

embryonic

enhancers

of

Cx/Tc-Scr

are

predicted

to

lie

between

the

and

breakpoints

because

the

allele

appears

to

lack

all

Cx/Tc-Scr

function

while

has

almost

normal

embryonic

Cx/Tc-Scr

function

This

conclusion

is

further

supported

by

the

expression

of

mxp/Tc-pb

in

a

Cx/Tc-Scr-like

pattern

in

mutants

presumably

due

to

juxtaposition

of

the

mxp/Tc-pb

transcription

unit

with

sequence

between

Tc-ftz

and

ptl/Tc-Antp

Adult

Cx/Tc-Scr

regulatory

elements

are

likely

located

within

or

of

ptl/Tc-Antp

because

the

rearrangement

seems

to

have

a

stronger

effect

on

the

adult

functions

of

Cx/Tc-Scr

The

presence

of

Cx/Tc-Scr

regulatory

elements

near

or

within

ptl/Tc-Antp

is

supported

by

the

observation

that

a

piggyBac

insertion

within

the

ptl/Tc-Antp

transcription

unit

-LRB-

-RRB-

shows

an

enhancer

trap

phenotype

that

appears

to

be

driven

by

Cx/Tc-Scr

regulatory

elements

In

Drosophila

elements

which

drive

Scr-like

expression

patterns

have

been

found

in

the

region

between

ftz

and

Antp

-LRB-

Gorman

and

Kaufman

-RRB-

However

these

elements

are

apparently

redundant

because

breakpoints

in

this

region

only

slightly

reduce

Scr

expression

levels

within

its

normal

domain

Moreover

a

deficiency

which

removes

most

if

not

all

of

the

Antp

transcription

unit

has

no

effect

on

embryonic

Scr

expression

Thus

it

is

possible

that

the

modularity

of

the

Drosophila

Hox

cluster

is

a

recent

innovation

resulting

from

changes

in

the

position

of

cis-regulatory

elements

This

newly

acquired

modularity

might

have

allowed

breaks

in

the

Hox

cluster

in

the

Drosophila

lineage

In

contrast

overlap

of

regulatory

elements

with

neighboring

genes

might

still

act

as

a

constraint

on

the

integrity

of

the

Hox

cluster

in

Tribolium

Noncoding

transcripts

Although

noncoding

transcripts

within

Hox

clusters

have

been

recognized

for

many

years

-LRB-

Cumberledge

et

al.

Lipshitz

et

al.

Sanchez-Herrero

and

Akam

-RRB-

there

has

recently

been

renewed

interest

in

these

enigmatic

RNAs

At

least

two

of

these

ncRNAs

and

can

have

homeotic

function

and

attenuate

the

actions

of

protein-coding

Hox

genes

-LRB-

Hornstein

et

al.

Ronshaugen

et

al.

-RRB-

In

addition

to

the

miRNAs

numerous

long

noncoding

RNAs

have

been

identified

within

the

Hox

clusters

of

both

flies

and

mammals

-LRB-

Bae

et

al.

Rinn

et

al.

-RRB-

ncRNAs

are

implicated

in

a

vast

array

of

processes

including

regulation

of

transcription

translation

epigenetic

control

of

chromatin

mono-allelic

expression

dosage

compensation

and

silencing

-LRB-

reviewed

in

Mattick

and

Makunin

-RRB-

In

the

Drosophila

BXC

these

transcripts

have

been

implicated

in

the

control

of

Hox

gene

expression

although

there

is

some

controversy

as

to

whether

they

promote

-LRB-

Sanchez-Elsner

et

al.

-RRB-

or

repress

-LRB-

Petruk

et

al.

-RRB-

Hox

gene

transcription

Mainguy

et

al.

-LRB-

-RRB-

found

evidence

for

extensive

noncoding

transcription

in

the

mammalian

Hox

clusters

and

proposed

that

polycistronic

and

antisense

transcription

might

play

a

role

in

keeping

Hox

genes

clustered

Our

transcriptional

profiling

data

demonstrates

that

Tribolium

also

shows

considerable

noncoding

transcription

in

the

Hox

complex

While

the

tiling

array

has

provided

a

revealing

snapshot

of

transcription

levels

during

embryonic

development

much

additional

work

will

be

necessary

to

characterize

the

actual

transcripts

For

example

the

high

levels

of

transcription

in

the

ptl/Tc-Antp

and

Utx/Tc-Ubx

regions

could

be

the

result

of

several

individual

transcripts

or

one

long

transcript

Interestingly

dicistronic

transcripts

spanning

the

Antp

and

Ubx

orthologs

have

been

reported

in

crustaceans

-LRB-

Shiga

et

al.

-RRB-

and

centipedes

-LRB-

Brena

et

al.

-RRB-

If

such

transcripts

have

an

important

function

they

might

constrain

linkage

in

at

least

some

parts

of

the

Hox

cluster

The

data

available

thus

far

indicate

that

noncoding

transcripts

are

prevalent

in

both

intact

and

broken

Hox

clusters

However

it

is

not

clear

whether

these

transcripts

perform

the

same

functions

in

all

organisms

Perhaps

noncoding

RNAs

play

a

different

or

more

critical

role

in

organisms

with

intact

clusters

Future

studies

in

this

area

are

likely

to

provide

important

insights

into

Hox

gene

function

and

possibly

into

Hox

cluster

conservation

Conclusions

The

results

presented

here

provide

a

foundation

for

further

studies

of

the

constraints

acting

on

Hox

clusters

While

it

is

important

to

keep

in

mind

that

multiple

factors

may

have

contributed

to

the

maintenance

of

Hox

clusters

during

evolution

-LRB-

Kmita

and

Duboule

-RRB-

the

intact

structure

of

the

Tribolium

Hox

cluster

and

the

suite

of

tools

now

available

for

this

insect

makes

it

an

ideal

candidate

for

such

research

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O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B I O O O O O O O O O B O O O O O O O O O

tribolium
homeotic
hox cluster
tiling
insect


Expression of alternansucrase in potato plants Alternan , which consists of alternating α - -LRB- 1 → 3 -RRB- / α - -LRB- 1 → 6 -RRB- - linked glucosyl residues , was produced in potato tubers by expressing a mature alternansucrase -LRB- Asr -RRB- gene from Leuconostoc mesenteroides NRRL B-1355 in potato . Detection of alternan was performed by enzyme-linked immunosorbent assay in tuber juices , revealing a concentration between 0.3 and 1.2 mg g-1 fresh wt . The Asr transcript levels correlated well with alternan accumulation in tuber juices . It appeared that the expression of sucrose-regulated starch-synthesizing genes -LRB- ADP-glucose pyrophosphorylase subunit S and granule-bound starch synthase I -RRB- was down-regulated . Despite this , the physico-chemical properties of the transgenic starches were unaltered . These results are compared to those obtained with other transgenic potato plants producing mutan -LSB- α - -LRB- 1 → 3 -RRB- - linked glucosyl residues -RSB- and dextran -LSB- α - -LRB- 1 → 6 -RRB- - linked glucosyl residues -RSB- . Introduction Production of novel polymers in plants by genetic modification is a great opportunity to obtain plants with unique properties that can not be generated by conventional breeding -LRB- Kok-Jacon et al. 2003 -RRB- . In addition , modifications of native polymers in planta could also generate crops with added nutritional , environmental or commercial value . For instance , production of biodegradable plastics in crops such as flax offers new perspectives for the replacement of oil-derived plastics -LRB- Wróbel et al. 2004 -RRB- . Another example is the production of a freeze-thaw-stable potato starch exhibiting novel physicochemical properties , thereby increasing the number of industrial applications -LRB- Jobling et al. 2002 -RRB- . Alternan is a unique polymer which is produced by three Leuconostoc mesenteroides strains : NRRL B-1355 , NRRL B-1498 and NRRL B-1501 -LRB- Jeanes et al. 1954 -RRB- . Alternan synthesized by L. mesenteroides NRRL B-1355 is mediated by the alternansucrase ASR -LRB- EC 2.4.1.140 -RRB- which is a large glucansucrase of 2,057 amino-acids -LRB- Argüello-Morales et al. 2000 -RRB- . Its C-terminal domain -LRB- also referred to as glucan-binding domain or GBD -RRB- exhibits short repeats specific for ASR , which could contribute to its distinct features -LRB- Janeček et al. 2000 -RRB- . The resulting polymer has a unique structure with alternating α - -LRB- 1 → 3 -RRB- / α - -LRB- 1 → 6 -RRB- - linked glucose residues , present for 46 % and 54 % , respectively . Due to this structure , alternan is a highly soluble and low viscous polymer , which is resistant to microbial and mammalian enzymes making it suitable for the production of ingredients for functional foods such as prebiotics -LRB- Côté 1992 -RRB- . Also , novel industrial applications were investigated by hydrolyzing native alternan polymers with isolates of Penicillium bacterial strains , creating potential replacers of commercial gum arabic -LRB- Leathers et al. 2002 ; 2003 -RRB- . Furthermore , ASR is an attractive enzyme because of its efficiency in bond formation , which is higher than that of the dextransucrase -LRB- DSRS -RRB- -LRB- Richard et al. 2003 -RRB- . In addition , mutated ASR enzymes showed a high efficiency in glucosylating acceptor molecules -LRB- cellobiose , α-alkylglucosides -RRB- in comparison to native ASR and DSRS enzymes , which might enable novel industrial applications -LRB- Argüello-Morales et al. 2001 ; Richard et al. 2003 ; Luz Sanz et al. 2006 -RRB- . In this work , we describe the production of alternan in potato tubers by expressing ASR . Modification of starch structure was envisaged with ASR , because of its high acceptor reaction efficiency . The effect of ASR on starch biosynthesis was studied at the microscopical , molecular and biochemical level , and compared to the effects of the dextransucrase -LRB- DSRS -RRB- and mutansucrase -LRB- GTFI -RRB- , producing less soluble polymers , such as dextran and mutan that are mainly composed of α - -LRB- 1 → 6 -RRB- and α - -LRB- 1 → 3 -RRB- - linked glucose residues , respectively -LRB- Kok-Jacon et al. 2005a , b -RRB- . Materials and methods Construction of binary plant expression vector containing the Asr gene An expression cassette containing the patatin promoter -LRB- Wenzler et al. 1989 -RRB- , the chloroplastic ferredoxin signal peptide -LRB- FD -RRB- from Silene pratensis -LRB- Pilon et al. 1995 -RRB- fused to the NOS terminator was cloned into the pBluescript SK -LRB- pBS SK -RRB- plasmid , resulting in pPF that was used as starting material for cloning the alternansucrase -LRB- Asr -RRB- gene . A mature Asr gene from L. mesenteroides NRRL B-1355 -LRB- Argüello-Morales et al. 2000 ; AJ250173 -RRB- was ligated in frame between the signal peptide FD and the NOS terminator . The mature Asr gene was amplified by PCR , with a forward primer containing a SmaI restriction site -LRB- 5 ′ - CATCAGGGCCCCGGGGATACAAAT-3 ′ -RRB- and a reverse primer containing a NruI restriction site -LRB- 5 ′ - CTCCTTTCGCGAATCCTTCCCTTA-3 ′ -RRB- using the proofreading Pfu turbo DNA polymerase -LRB- 2.5 units/μl ; Stratagene , UK -RRB- and cloned into the SmaI/NruI restriction sites of pPF , resulting in pPFAsr . FD and the fused Asr gene were completely sequenced in one direction by Baseclear -LRB- The Netherlands -RRB- to verify the correctness of the construct . pPFAsr was digested with SacI and SalI and subsequently ligated into a pBIN20 binary vector -LRB- Hennegan and Danna 1998 -RRB- , resulting in pPFA -LRB- Fig. 1 -RRB- . Fig. 1Schematic representation of pPFA binary vector used for potato plant transformation Transformation and regeneration of potato plants pPFA was transformed into Agrobacterium tumefaciens strain LBA 4404 using electroporation -LRB- Takken et al. 2000 -RRB- . Internodal stem segments from the tetraploid potato genotype -LRB- cv . Kardal -LRB- KD -RRB- -RRB- were used for Agrobacterium-mediated transformation , which was performed as described by Kok-Jacon et al. -LRB- 2005a -RRB- . Starch isolation Potato tubers were peeled and homogenized in a Sanamat Rotor -LRB- Spangenberg , The Netherlands -RRB- . The resulting homogenate was allowed to settle overnight at 4 °C and the potato juice was decanted and stored at − 20 °C for characterization of soluble alternan . The starch pellet was washed three times with water , air-dried at room temperature for at least three days and stored at room temperature . Immunological detection of alternans in tuber juices and gelatinized starches Presence of alternans was investigated with enzyme-linked immunosorbent assay -LRB- ELISA -RRB- as described by Kok-Jacon et al. -LRB- 2005a -RRB- , using monoclonal anti-α - -LRB- 1 → 6 -RRB- dextran antibodies -LRB- 45.21.1 -LRB- groove-type ; IgA/Kappa -RRB- and 16.4.12 EBI -LRB- cavity-type ; IgA/Kappa -RRB- -RRB- -LRB- Wang et al. 2002 -RRB- with tuber juices and gelatinized starches . The monoclonal anti-α - -LRB- 1 → 6 -RRB- dextran antibodies detect structures containing both internal and terminal epitopes of α - -LRB- 1 → 6 -RRB- dextran which can be applicable for the detection of α - -LRB- 1 → 6 -RRB- linked glucose residues present in alternan -LRB- Sharon et al. 1982 ; Dr Denong Wang , personal communication -RRB- . Expression analysis of Asr and genes involved in starch biosynthesis using semi-quantitative and real-time quantitative RT-PCR analysis RNA was isolated from 3 g -LRB- fresh weight -RRB- of potato tuber material from selected transgenic lines according to Kuipers et al. -LRB- 1994 -RRB- . Semi-quantitative and real-time quantitative RT-PCR 's were performed as described by Kok-Jacon et al. -LRB- 2005a -RRB- . AsrRT primers , 5 ′ - ACCGGTTCCATCAACTAATAAT-3 ′ and 5 ′ - GACATCTCGGAAGGATCCC-3 ′ -LRB- Tm = 55 °C , 35 cycles -RRB- were based on the Asr gene sequence -LRB- Argüello-Morales et al. 2000 -RRB- . RNA sample from Karnico potato tubers expressing a sense/antisense GBSSI cDNA inverted-repeat construct referred to as RVT34-77 -LRB- Heilersig 2005 -RRB- was used as a positive control , because its GBSSI expression level was completely down-regulated . Determination of morphological and physicochemical starch properties Analysis of starch granule morphology was performed by light microscopy and scanning electron microscopy -LRB- SEM -RRB- as described by Kok-Jacon et al. -LRB- 2005a -RRB- . Median values of the granule size distribution -LRB- d50 -RRB- , gelatinization analysis , amylose content , starch content , chain length distributions -LRB- HPSEC , HPAEC -RRB- were determined as described by Kok-Jacon et al. -LRB- 2005a -RRB- . Results Detection of alternan in transgenic potato juices To enable plastidic protein targeting , the mature Asr gene was fused to the ferredoxin -LRB- FD -RRB- signal peptide -LRB- Gerrits et al. 2001 -RRB- . The resulting gene fusion was inserted between the patatin promoter -LRB- Fig. 1 -RRB- allowing high-tuber expression -LRB- Wenzler et al. 1989 -RRB- and the Nos terminator sequence . At the FD ▲ Asr fusion , two mutations were present because a SmaI restriction site was engineered at this position -LRB- VTAM ↓ ATYKVTLITK ▲ ADT became VTAM ↓ ATYKVTLITP ▲ GDT , in which ↓ represents the splice site for amyloplast entry and ▲ the gene fusion -RRB- . Furthermore , differences from the published ASR sequence -LRB- Argüello-Morales et al. 2000 -RRB- were found at three positions -LRB- Y208H , D221G and G1092S -RRB- , but these did not affect conserved residues . After Agrobacterium-mediated plant transformation , thirty independent transgenic potato clones were obtained using the Kardal -LRB- KD -RRB- genotype . Five plants of each transgenic clone were grown in the greenhouse from which the tubers were pooled for further characterization . KDAxx referred to the transformed potato plant serie in which A represents the Asr gene and xx the clone number . The untransformed genotype is referred to as KD-UT . Detection of alternan was performed by analyzing tuber juices of the transformants with ELISA using anti-dextran antibodies -LRB- Wang et al. 2002 -RRB- . Alternan was detected in 4 out of 29 tubers -LRB- about 14 % -RRB- in a concentration ranging from 0.3 to 1.2 mg g − 1 fresh wt -LRB- Fig. 2 -RRB- in the transformants KDA16 , KDA19 , KDA27 and KDA13 . As expected , no alternan was found in KD-UT plants . According to the tuber juice results , the KDA transformants were divided in three classes : -LRB- − -RRB- , -LRB- + -RRB- and -LRB- + + -RRB- , representing no , intermediate -LRB- ≤ 1 mg g − 1 FW -RRB- and high -LRB- > 1 mg g − 1 FW -RRB- levels of alternan , respectively . All the transformants containing alternan and two from the -LRB- − -RRB- class were selected for further characterization : KDA13 -LRB- + + -RRB- , KDA16 -LRB- + -RRB- , KDA19 -LRB- + -RRB- , KDA27 -LRB- + -RRB- , KDA1 -LRB- − -RRB- and KDA24 -LRB- − -RRB- . RNA was isolated from potato tubers and subjected to RT-PCR analysis . The expression levels were determined for the Asr and Ubi3 genes , of which the latter is used as a control because of its constitutive expression -LRB- Garbarino and Belknap 1994 -RRB- -LRB- Fig. 3 -RRB- . Heterologous Asr gene expression was detected in the expressers KDA13 , KDA16 , KDA19 , KDA27 . No Asr mRNA was detected in the -LRB- − -RRB- class transformants and in the KD-UT plants . The Asr expression levels correlated well with the ELISA results described above . Fig. 2Detection of alternans accumulated in potato juices by ELISA using anti-dextrans antibodies . Based on the alternan concentration -LSB- in mg g − 1 fresh wt -LRB- FW -RRB- -RSB- , three categories of transformants were made , where -LRB- − -RRB- , -LRB- + -RRB- and -LRB- + + -RRB- represent no , intermediate and high alternan accumulation , respectively . Transgenic clones indicated with grey bars were selected for further characterizationFig . 3RT-PCR analysis of the selected KDA transformants and KD-UT tuber RNA . The upper panel shows the PCR products using the primers designed on the Asr sequence . The lower panel shows the PCR products using the primers designed on the Ubi3 sequence that served as an internal control . pPFAsr plasmid : positive control Alternan accumulation does not interfere with plant , tuber and starch morphologies Asr expressing plants -LRB- green parts and tubers -RRB- did not exhibit any morphological changes in comparison to KD-UT plants -LRB- data not shown -RRB- . In addition , starch morphology of Asr expressing plants was quite similar to that of KD-UT . With SEM , a rough surface was present on some of the -LRB- + + -RRB- class transformant granules -LRB- Fig. 4B , F -RRB- , but was considered as not significant when compared to dextran - -LRB- Fig. 4C , G -RRB- and mutan - -LRB- Fig. 4D , H -RRB- accumulating plants . In general , starch granules from the -LRB- + -RRB- and -LRB- − -RRB- class transformants were similar to those of the KD-UT -LRB- data not shown -RRB- . Starch granules comparable to those illustrated in Fig. 4 -LRB- F -RRB- were scored by analyzing a population of 100 granules in triplicate for each selected transformant -LRB- data not shown -RRB- . KDA13 , belonging to the -LRB- + + -RRB- class transformant , exhibited -LRB- 12 % ± 1.0 -RRB- of altered starch granules , followed by the -LRB- + -RRB- class transformant -LSB- KDA19 -LRB- 9.3 % ± 0.6 -RRB- ; KDA27 -LRB- 8.3 % ± 0.6 -RRB- -RSB- . For the -LRB- − -RRB- class transformant and KD-UT , the frequency of altered granules was lower , which was around the 7 % . Fig. 4SEM analysis of starch granules -LRB- × 350 : upper panel -RRB- and -LRB- × 1,000 : lower panel -RRB- from KD-UT -LRB- A , E -RRB- compared to that of selected transformants producing foreign polymers with decreasing water-solubility -LRB- KDA13 that produces alternan -LRB- B and F ; + + : highly soluble -LRB- S -RRB- -RRB- , KDD30 that produces dextran -LRB- C and G ; + : soluble -LRB- L -RRB- -RRB- and KDIC15 that produces mutan -LRB- D and H ; − : insoluble -LRB- I -RRB- -RRB- . Degrees of polymer solubility were defined according to Robyt -LRB- 1996 -RRB- in which class S = more soluble referring to glucans precipitated by 40 -- 44 % -LRB- v/v -RRB- ethanol , L = less soluble referring to glucans precipitated by 34 -- 37 % ethanol and I = water-insoluble The physicochemical properties and starch content of KDA transformants remain unchanged Median granule size -LRB- d50 -RRB- , gelatinization characteristics -LRB- T0 and ΔH -RRB- , amylose and starch content measurements were performed on selected transformants -LRB- Table 1 -RRB- . From these results , it can be seen that no consistent changes were detected for the different classes of transformants . Furthermore , chain length distribution experiments -LRB- HPSEC and HPAEC -RRB- were also done , particularly because ASR exhibits a high acceptor reaction efficiency . After complete debranching of starch with isoamylase , no consistent changes were found with HPSEC and HPAEC in comparison to KD-UT starches -LRB- data not shown -RRB- . In addition , debranched starches , which were further treated with α-amylase , were analyzed with HPAEC in order to detect the presence of novel structural elements on starch molecules such as alternating α - -LRB- 1 → 3 -RRB- / α - -LRB- 1 → 6 -RRB- linkages . Again , no consistent changes were detected with HPAEC in comparison to KD-UT starches -LRB- data not shown -RRB- . Table 1Summary of granule size -LRB- d50 -RRB- , gelatinization characteristics -LRB- To , ΔH -RRB- , amylose and starch content measurements of starches from the selected transformants and KD-UT . Data -LRB- ± SD -RRB- are the average of two or three independent measurementsTransformantsd50 -LRB- μm -RRB- * T0 -LRB- °C -RRB- † ΔH -LRB- kJ/g -RRB- ‡ Amylose content -LRB- % -RRB- Starch content -LRB- mg/g FW -RRB- KD-UT26.5 -LRB- ± 0.3 -RRB- 67.9 -LRB- ± 0.1 -RRB- 14.5 -LRB- ± 0.1 -RRB- 22.3 -LRB- ± 0.2 -RRB- 214.8 -LRB- ± 117.5 -RRB- KDA1 -LRB- − -RRB- 24.4 -LRB- ± 0.2 -RRB- 68.1 -LRB- ± 0.1 -RRB- 17.0 -LRB- ± 0.1 -RRB- 22.2 -LRB- ± 0.2 -RRB- 103.4 -LRB- ± 66.3 -RRB- KDA24 -LRB- − -RRB- 25.0 -LRB- ± 0.2 -RRB- 68.0 -LRB- ± 0.1 -RRB- 16.3 -LRB- ± 1.2 -RRB- 21.3 -LRB- ± 0.4 -RRB- 86.7 -LRB- ± 41.9 -RRB- KDA16 -LRB- + -RRB- 24.9 -LRB- ± 0.3 -RRB- 67.9 -LRB- ± 0.2 -RRB- 16.4 -LRB- ± 1.3 -RRB- 22.2 -LRB- ± 0.1 -RRB- 140.0 -LRB- ± 88.2 -RRB- KDA19 -LRB- + -RRB- 27.9 -LRB- ± 0.2 -RRB- 67.7 -LRB- ± 0.0 -RRB- 15.2 -LRB- ± 0.1 -RRB- 23.0 -LRB- ± 0.2 -RRB- 137.1 -LRB- ± 38.2 -RRB- KDA27 -LRB- + -RRB- 22.8 -LRB- ± 0.7 -RRB- 67.7 -LRB- ± 0.2 -RRB- 16.2 -LRB- ± 0.5 -RRB- 22.2 -LRB- ± 0.4 -RRB- 289.3 -LRB- ± 39.7 -RRB- KDA13 -LRB- + + -RRB- 24.0 -LRB- ± 0.1 -RRB- 67.8 -LRB- ± 0.1 -RRB- 16.0 -LRB- ± 0.7 -RRB- 22.2 -LRB- ± 0.5 -RRB- 107.2 -LRB- ± 49.4 -RRB- * Median value of the granule size distribution † Temperature of onset of starch gelatinization ‡ Enthalpy released Expression levels of AGPase and GBSSI genes are down-regulated in the -LRB- + -RRB- and -LRB- + + -RRB- KDA class The expression levels of key genes involved in starch biosynthesis such as sucrose synthase -LRB- SuSy -RRB- , ADP-glucose pyrophosphorylase subunit S -LRB- AGPase -RRB- , starch synthase III -LRB- SSIII -RRB- , starch branching enzyme I -LRB- SBEI -RRB- and granule-bound starch synthase I -LRB- GBSSI -RRB- were monitored by real-time quantitative RT-PCR -LRB- Fig. 5 -RRB- . All these genes seemed to be down-regulated , particularly the AGPase and GBSSI genes . In most cases , the extent of AGPase and GBSSI down-regulation corresponded well with the amount of alternan that was accumulated in the potato tubers . However , AGPase down-regulation did not correlate with a reduction in starch content for the -LRB- + + -RRB- transformants -LRB- 107.2 ± 49.4 mg g − 1 FW -RRB- when compared to KD-UT -LRB- 214.8 ± 117.5 mg g − 1 FW -RRB- . Concerning GBSSI , the down-regulation was about 20 times less than for the transformant RVT34-77 in which GBSSI is completely inhibited . Typically , no reduction in amylose content was observed for the KDA transformants -LRB- Table 1 -RRB- , irrespective of their GBSSI messenger RNA level . Thus , the observed reduction in GBSSI expression for the -LRB- + -RRB- and -LRB- + + -RRB- KDA classes were significant within the selected transformants , but relatively small with respect to the RVT34-77 transformant . Fig. 5Real-time quantitative RT-PCR analysis of KDA24 -LRB- − -RRB- , KDA27 -LRB- + -RRB- and KDA13 -LRB- + + -RRB- transformants and KD-UT tuber RNA using the following specific primers : SuSy , sucrose synthase ; AGPase , ADP-glucose pyrophosphorylase subunit S ; SSIII , starch synthase III ; SBEI , starch branching enzyme I ; GBSSI , granule-bound starch synthase I. RNA levels for each gene were expressed relative to the amount of Ubi3 RNA , as described in materials and methods . RNA sample from Karnico potato tubers expressing a sense/antisense GBSSI cDNA construct exhibiting a complete GBSSI down-regulation -LRB- RVT34-77 -RRB- , was used as a positive control Discussion This report is the first study on the production of alternan in potato tubers . Their presence in potato juices was demonstrated by ELISA using anti-dextran antibodies . Expression of ASR did not interfere with plant growth and development , and tuber and starch yield penalties were not observed . These results were similar to those obtained with the dextransucrase -LRB- DSRS -RRB- expression -LRB- Kok-Jacon et al. 2005a -RRB- , but not to those obtained with the mutansucrase -LRB- GTFI -RRB- expression in which the tuber phenotype was significantly affected -LRB- Kok-Jacon et al. 2005b -RRB- . The amount of alternan accumulated in potato tubers -LRB- 1.2 mg g − 1 fresh wt -RRB- was lower than that of dextran -LRB- 1.7 mg g − 1 fresh wt -RRB- -LRB- Kok-Jacon et al. 2005a -RRB- . It might be possible that the large size of the mature ASR -LRB- 2,057 amino-acids -LRB- a.a -RRB- when compared to DSRS with only 1,527 a.a. -RRB- might reduce the efficiency with which the enzyme is transported through the amyloplast membrane . However , such explanation needs to be approached with caution because the presence of alternansucrase in the amyloplast was not directly evidenced , as no ASR antibodies were available to us . Interestingly , it has been shown that the size of ASR can be reduced -LRB- by removal 82 % -LRB- 632/767 a.a. -RRB- of the C-terminal GBD -RRB- without compromising its activity -LRB- Joucla et al. 2006 -RRB- . If the size of the protein is indeed a critical factor , than this truncated variant may be a useful tool to enhance alternan synthesis in the amyloplast . Such an approach was already employed successfully for the Streptococcus downei mutansucrase GTFI -LRB- Kok-Jacon et al. 2005b -RRB- . We have directed a mature and a GBD-truncated GTFI protein to potato amyloplasts , and found that the truncated form synthesized a larger amount of mutan , with much more pronounced effect on starch granule morphology . Although ASR is known to be efficient in catalyzing acceptor reactions -LRB- Richard et al. 2003 ; Côté and Sheng 2006 -RRB- , no evidence was found for the covalent attachment of novel , alternan-based structural elements to starch molecules . Also with dextransucrase and mutansucrase we have not been able to introduce different glycosyl linkage patterns in starch -LRB- Kok-Jacon et al. 2005a , b -RRB- . To this end , acceptor reactions of glucansucrases with starch or maltodextrins are not studied in much detail . It has been observed that the efficiency of acceptor reaction decreases with increasing length of maltodextrins -LRB- reviewed in Kok-Jacon et al. 2003 -RRB- . We had anticipated that the nascent starch polymers would be poor acceptors for the glucansucrases . However , during starch biosynthesis potential acceptors -LRB- small maltodextrins -RRB- are thought to be generated through the action of , for instance , debranching enzymes -LRB- or isoamylases -RRB- . If such a small acceptor is mutanylated , alternanylated , or dextranylated at the non-reducing end , then these novel structures might be incorporated into starch polymers through the action of certain transferases such as , for instance , branching enzyme . Apparently , this does not happen , or at a very low -LRB- undetectable -RRB- frequency , but the reason for this is unclear . Starch morphology in the ASR transformants was not significantly altered in comparison to that of dextran and mutan-accumulating plants -LRB- Fig. 4 -RRB- . This might be related to the fact that alternan is more water-soluble than dextran and mutan . An indication of the water-solubility of the three polysaccharides is given in Fig. 4 ; the more ethanol is required for precipitation , the higher the water-solubility . The water-solubility decreases in the order of alternan , dextran and mutan . We hypothesize that the co-synthesis of water-insoluble mutan and starch leads to co-crystallization of the two polymers , as a result of which the granule is packed in a less orderly fashion . This comparison should be approached with caution . For alternan and dextran , the observed differences in starch morphology may also be related to the fact that more dextran than alternan was accumulated in the potato tubers ; for mutan , we have not been able to quantify the amount accumulated in the tubers . Therefore , it can not be excluded that the observed effects are related to the amount of foreign polymer produced . Interestingly , co-synthesis of levan , a water-soluble fructosyl-based polymer , and starch resulted in a dramatically altered starch granule morphology -LRB- Gerrits 2000 -RRB- . However , it should be noted that much higher levels of levan , which were estimated to be 66 mg g − 1 fresh wt -LRB- Gerrits et al. 2001 ; Cairns 2003 -RRB- , were produced in comparison with alternan -LRB- 1.2 mg g − 1 fresh wt -RRB- or dextran -LRB- 1.7 mg g − 1 fresh wt -RRB- , and that the starch granules contained approximately 5 % of levan . This result contrasts with that of alternan - and dextran-accumulating plants in which foreign polymers were only found in the stroma . Taking together the results of potato transformants expressing glucan - or levansucrases in amyloplasts , it seems that the site of accumulation of the foreign polymer -LRB- granule or stroma -RRB- , the solubility of the foreign polymer , and the amount of foreign polymer that is actually produced are important factors in determining starch granule morphology .

O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

alternan
glucansucrase
polymer solubility
transgenic potato