The

fission

yeast

subunit

of

RNA

polymerase

II

plays

a

specialized

role

in

cell

separation

RNA

polymerase

II

is

a

complex

of

subunits

to

whose

specific

roles

are

only

partly

understood

is

essential

in

mammals

and

fission

yeast

but

not

in

budding

yeast

To

learn

more

about

the

roles

of

we

expressed

the

gene

under

the

control

of

regulatable

promoters

of

different

strength

in

fission

yeast

We

demonstrate

that

below

a

critical

level

of

transcription

affects

cellular

growth

proportional

to

its

expression

levels

cells

expressing

lower

levels

of

grew

slower

compared

to

cells

expressing

higher

levels

Lowered

expression

did

not

affect

cell

survival

under

several

stress

conditions

but

it

caused

specific

defects

in

cell

separation

similar

to

sep

mutants

Microarray

analysis

revealed

that

lowered

expression

causes

a

global

reduction

in

gene

expression

but

the

transcript

levels

of

a

distinct

subset

of

genes

were

particularly

responsive

to

changes

in

expression

These

genes

show

some

overlap

with

those

regulated

by

the

transcriptional

cascade

required

for

cell

separation

Most

notably

the

gene

expression

signature

of

cells

with

lowered

expression

was

highly

similar

to

those

of

and

mutants

and

encode

orthologs

of

metazoan

TFIIH-associated

cyclin-dependent

kinase

-LRB-

CDK

-RRB-

activating

kinase

-LRB-

-RRB-

while

and

encode

mediator

components

Our

results

suggest

that

along

with

some

other

general

transcription

factors

plays

a

specialized

role

in

a

transcriptional

pathway

that

controls

the

cell

cycle-regulated

transcription

of

a

specific

subset

of

genes

involved

in

cell

division

Introduction

RNA

polymerase

II

-LRB-

pol

II

-RRB-

transcribes

the

coding

genes

and

its

activity

is

the

main

target

for

transcriptional

control

in

all

eukaryotes

The

pol

II

enzyme

consists

of

subunits

which

are

highly

conserved

during

evolution

The

specific

functions

of

the

smaller

subunits

such

as

are

relatively

poorly

understood

and

they

can

show

differences

between

organisms

For

example

is

essential

in

mammals

and

fission

yeast

-LRB-

Schizosaccharomyces

pombe

-RRB-

but

not

in

budding

yeast

-LRB-

Saccharomyces

cerevisiae

-RRB-

Here

we

use

fission

yeast

as

a

model

to

learn

more

about

particular

roles

of

in

genome-wide

transcription

Much

of

our

current

understanding

of

the

mechanism

of

transcription

and

the

transcription

machinery

comes

from

studies

using

budding

yeast

The

distantly

related

fission

yeast

provides

a

valuable

complementary

model

system

to

dissect

the

functions

of

different

components

of

the

transcriptional

apparatus

because

its

transcription

mechanism

is

more

similar

to

that

of

mammals

in

some

respects

For

example

the

initiation

of

transcription

in

both

mammalian

cells

and

S.

pombe

occur

bp

downstream

of

the

TATA

box

whereas

in

S.

cerevisiae

this

distance

can

vary

between

and

bp

-LRB-

Li

et

al.

-RRB-

Moreover

transcriptional

initiation

from

mammalian

promoters

introduced

into

S.

pombe

occurs

at

the

same

site

as

in

mammalian

cells

-LRB-

Toyama

and

Okayama

-RRB-

As

in

other

eukaryotes

S.

pombe

pol

II

is

a

multi-subunit

enzyme

containing

the

subunits

to

The

four

core

subunits

and

are

homologous

to

the

β

β

α

and

α

subunits

respectively

of

the

eubacterial

RNA

polymerase

Five

subunits

and

are

shared

by

the

three

eukaryotic

RNA

polymerases

I

II

and

III

whereas

and

are

subunits

specific

to

pol

II

-LRB-

Mitsuzawa

and

Ishihama

-RRB-

The

functions

of

most

of

these

smaller

subunits

are

not

well

characterized

and

form

a

conserved

heterodimer

complex

in

archaebacteria

yeast

plants

and

humans

-LRB-

Choder

-RRB-

Crystallographic

studies

of

pol

II

and

biochemical

data

provide

evidence

for

a

role

of

the

heterodimer

in

transcription

initiation

and

RNA

binding

in

S.

cerevisiae

-LRB-

Armache

et

al.

Bushnell

and

Kornberg

Edwards

et

al.

Orlicky

et

al.

-RRB-

S.

pombe

interacts

with

an

RNA-binding

protein

implying

a

role

in

the

coupling

of

RNA

processing

to

transcription

and

it

associates

with

dehydrogenase

and

actin

-LRB-

Mitsuzawa

et

al.

-RRB-

The

in

vivo

functions

of

have

been

characterized

in

S.

cerevisiae

and

several

lines

of

evidence

suggest

that

it

plays

an

important

role

in

the

response

and

survival

to

stress

-LRB-

reviewed

by

Choder

-RRB-

S.

cerevisiae

is

also

important

for

activated

transcription

from

a

subset

of

promoters

and

in

carbon

and

energy

metabolism

at

moderate

temperatures

-LRB-

Pillai

et

al.

-RRB-

Recent

data

show

that

it

is

required

for

the

decay

of

a

class

of

mRNAs

whose

products

are

involved

in

protein

synthesis

and

it

interacts

with

subunits

of

the

mRNA

decay

complex

that

enhances

decapping

-LRB-

Lotan

et

al.

-RRB-

We

are

only

beginning

to

understand

the

role

of

the

subunit

in

S.

pombe

it

binds

to

the

TFIIF-interacting

carboxyl-terminal

domain

-LRB-

CTD

-RRB-

phosphatase

and

it

has

been

proposed

to

play

a

role

in

the

assembly

of

the

pol

II

complex

thereby

promoting

CTD

phosphorylation

for

the

reutilization

of

pol

II

in

a

new

cycle

of

transcription

-LRB-

Kimura

et

al.

-RRB-

S.

pombe

exhibits

features

that

resemble

its

budding

yeast

counterpart

while

others

place

it

closer

to

its

orthologs

in

multicellular

eukaryotes

It

is

essential

for

viability

whereas

in

S.

cerevisiae

it

is

not

required

for

cell

growth

under

optimal

conditions

-LRB-

Sakurai

et

al.

Choder

-RRB-

S.

pombe

contains

amino

acids

placing

it

closer

in

size

to

its

counterparts

in

humans

-LRB-

amino

acids

-RRB-

and

plants

-LRB-

amino

acids

-RRB-

than

to

S.

cerevisiae

-LRB-

amino

acids

-RRB-

Stoichiometric

amounts

of

associate

with

pol

II

in

S.

pombe

and

higher

eukaryotes

while

the

fraction

of

pol

II

molecules

containing

in

optimally

growing

S.

cerevisiae

cells

is

only

about

Finally

is

more

tightly

associated

with

S.

pombe

pol

II

than

with

S.

cerevisiae

pol

II

-LRB-

Sakurai

et

al.

-RRB-

Here

we

provide

evidence

that

influences

the

growth

and

gene

expression

of

S.

pombe

cells

in

a

dose-dependent

fashion

under

optimal

conditions

We

show

that

lower

levels

of

expression

do

not

affect

the

survival

of

cells

under

several

stress

conditions

Our

results

imply

a

particularly

important

role

for

in

the

expression

and

regulation

of

a

subset

of

genes

involved

in

cell

separation

The

defects

in

cell

growth

and

separation

as

well

as

the

gene

expression

signatures

associated

with

low

levels

of

expression

are

similar

to

the

phenotypes

and

expression

signatures

of

a

range

of

transcriptional

regulatory

mutants

with

defects

in

cell

separation

Materials

and

methods

Strains

and

molecular

genetic

methods

The

S.

pombe

strains

used

in

this

study

are

listed

in

Table

was

the

parental

haploid

strain

used

to

generate

the

strains

expressing

under

the

and

promoters

-LRB-

Basi

et

al.

-RRB-

The

strains

were

constructed

by

PCR

using

the

constructs

as

described

-LRB-

Bähler

et

al.

-RRB-

and

the

kanamycin-resistant

transformants

were

selected

on

KsNoT

media

-LRB-

Linder

et

al.

-RRB-

Recombinant

DNA

methods

were

performed

as

described

-LRB-

Sambrook

et

al.

-RRB-

and

standard

methodology

and

media

for

the

manipulation

of

S.

pombe

were

used

-LRB-

Moreno

et

al.

-RRB-

Table

used

in

this

studyStrainRelevant

h

Leupold

-LRB-

-RRB-

h

This

h

This

h

This

study

Growth

experiments

Pre-cultures

for

the

growth

curves

were

made

by

inoculating

the

respective

strains

in

Edinburgh

Minimal

Medium

-LRB-

EMM

-RRB-

without

thiamine

or

with

μM

thiamine

and

growing

the

cultures

for

h

at

°C

with

shaking

Subsequently

the

pre-culture

was

diluted

into

ml

of

fresh

EMM

medium

at

an

and

allowed

to

grow

at

°C

with

shaking

Samples

were

removed

at

various

timepoints

thereafter

for

measuring

ODs

at

nm

microscopic

examination

and

microarray

hybridization

experiments

The

low

OD

measurements

in

cells

expressing

under

the

and

promoters

is

not

caused

by

the

phenotype

because

mutants

with

similar

phenotypes

such

as

show

strong

increases

in

OD

during

growth

-LRB-

unpublished

data

-RRB-

Quantitative

RT-PCR

experiments

To

measure

expression

the

strains

expressing

under

the

three

promoters

were

grown

for

h

in

EMM

medium

at

°C

-LRB-

promoter

on

-RRB-

or

cultivated

for

a

further

h

in

the

presence

of

μM

thiamine

-LRB-

promoter

off

-RRB-

RNA

was

isolated

by

phenol

extraction

purified

using

the

RNeasy

kit

-LRB-

Qiagen

-RRB-

and

treated

with

RNase-free

DNase

-LRB-

Roche

-RRB-

The

reverse

transcription

reactions

were

performed

using

Superscript

III

-LRB-

Invitrogen

-RRB-

Real-time

PCR

was

performed

using

the

Brilliant

SYBR

Green

QPCR

Core

Reagent

Kit

-LRB-

Stratagene

-RRB-

on

an

QPCR

system

-LRB-

Stratagene

-RRB-

with

primers

specific

for

or

-LRB-

control

-RRB-

-LRB-

sequences

available

on

request

-RRB-

Arbitrary

expression

units

were

calculated

using

a

standard

curve

made

from

serial

dilutions

of

fission

yeast

genomic

DNA

Stress

response

assays

Serial

dilutions

-LRB-

-RRB-

of

strains

generated

in

our

study

were

spotted

on

EMM

plates

with

or

without

μM

thiamine

The

plates

were

supplemented

with

mM

hydrogen

peroxide

or

different

concentrations

of

sorbitol

-LRB-

and

M

-RRB-

to

test

the

response

to

oxidative

and

osmotic

stress

respectively

These

plates

were

incubated

at

°C

for

days

and

photographed

The

effect

of

temperature

stress

was

tested

by

incubating

the

strains

at

°C

for

days

Microscopy

Unfixed

cells

were

observed

at

intervals

for

h

by

light

microscopy

using

a

Zeiss

Axioscope

fluorescence

microscope

set

up

for

differential

interference

contrast

-LRB-

DIC

-RRB-

with

a

objective

and

Axiovision

digital

imaging

system

To

stain

the

nuclei

cells

were

spread

onto

microscopic

glass

slides

Subsequently

the

cells

were

fixed

by

heating

at

°C

for

min

and

DAPI

-LRB-

-RRB-

was

added

at

a

final

concentration

of

μg/ml

To

view

the

division

septa

calcofluor

was

added

to

cultures

at

a

final

concentration

of

mg/ml

and

the

cultures

were

incubated

in

the

dark

at

room

temperature

for

min

The

cells

were

washed

twice

with

phosphate

buffer

saline

and

μl

of

cell

suspension

was

spotted

onto

microscopic

glass

slides

DAPI

and

calcofluor-treated

cells

were

visualized

under

a

Zeiss

Axioscope

microscope

as

above

Microarray

experiments

To

compare

the

gene

expression

profiles

as

a

function

of

expression

cells

were

grown

in

the

presence

of

thiamine

to

of

and

ml

of

culture

was

centrifuged

The

remaining

culture

was

washed

four

to

five

times

with

EMM

and

subsequently

grown

in

EMM

medium

without

thiamine

for

h

and

centrifuged

To

identify

transcripts

affected

by

lowered

expression

the

desired

strains

were

grown

in

the

presence

of

thiamine

for

and

h

and

harvested

A

wild-type

parental

strain

grown

in

the

presence

of

thiamine

served

as

a

reference

Although

thiamine

leads

only

to

minimal

expression

changes

-LRB-

Jenkins

et

al.

-RRB-

this

experimental

design

corrects

for

effects

caused

by

the

presence

of

thiamine

Pellets

were

frozen

in

liquid

nitrogen

and

kept

at

°C

until

further

processing

RNA

was

isolated

by

phenol

extraction

and

purified

by

the

RNeasy

kit

-LRB-

Qiagen

-RRB-

cDNA

probes

were

prepared

with

Superscript

-LRB-

Invitrogen

-RRB-

and

labeled

with

or

Detailed

methods

were

as

described

-LRB-

Lyne

et

al.

-RRB-

Microarrays

were

scanned

with

a

Genepix

scanner

and

analyzed

with

Genepix

software

-LRB-

Axon

Instruments

-RRB-

Data

filtering

normalization

and

quality

control

were

performed

as

described

-LRB-

Lyne

et

al.

-RRB-

The

data

are

based

on

two

to

three

independent

biological

repeats

with

dye

swaps

To

quantify

global

effects

on

gene

expression

we

repeated

microarray

analysis

spiked

with

external

control

RNAs

The

expression

data

were

normalized

using

Bacillus

subtilis

spikes

added

in

equal

quantities

to

the

RNA

samples

before

labeling

-LRB-

Lee

et

al.

-RRB-

Each

microarray

contains

control

elements

for

the

spikes

which

are

spread

across

the

complete

grid

of

the

array

in

a

spatially

even

manner

Clustering

and

visualization

were

done

with

GeneSpring

Gene

annotations

were

taken

from

the

S.

pombe

GeneDB

database

-LRB-

-RRB-

Overlaps

between

different

gene

lists

were

calculated

using

the

hypergeometric

distribution

All

normalized

data

sets

are

available

from

our

website

Results

Dosage-dependent

effect

of

on

cell

growth

is

an

essential

subunit

of

S.

pombe

pol

II

To

elucidate

its

in

vivo

function

-LRB-

s

-RRB-

we

constructed

haploid

strains

expressing

from

its

normal

chromosomal

location

using

the

thiamine-regulated

promoter

Three

different

versions

of

this

promoter

were

used

the

wild-type

promoter

has

the

highest

level

of

promoter

activity

and

maintains

substantial

basal

levels

of

expression

even

under

repressed

conditions

the

two

derivatives

-LRB-

medium

strength

-RRB-

and

-LRB-

weakest

strength

-RRB-

have

reduced

levels

of

activity

due

to

mutations

in

the

TATA

box

-LRB-

Basi

et

al.

-RRB-

We

performed

quantitative

PCR

of

cells

expressing

under

the

three

promoters

both

in

the

absence

-LRB-

promoter

on

-RRB-

or

presence

-LRB-

promoter

off

-RRB-

of

thiamine

to

verify

changing

levels

of

mRNA

-LRB-

Fig.

-RRB-

Expression

levels

of

showed

a

range

of

more

than

in

the

different

conditions

and

the

relative

expression

levels

were

as

expected

for

the

three

promoters

of

different

strength

The

levels

were

especially

low

when

expressed

from

the

and

promoters

in

the

presence

of

thiamine

The

gene

is

highly

expressed

and

was

used

as

a

control

gene

Although

it

shows

much

less

variation

in

expression

levels

than

there

are

some

changes

in

accordance

with

levels

-LRB-

Fig.

-RRB-

This

probably

reflects

effects

on

global

transcription

as

a

function

of

expression

levels

-LRB-

see

below

-RRB-

Fig.

levels

of

mRNA

when

transcribed

from

different

promoter

constructs

Cells

expressing

from

the

or

promoter

were

grown

for

h

in

EMM

medium

at

°C

-LRB-

promoter

on

gray

bars

-RRB-

or

cultivated

for

a

further

h

in

the

presence

of

thiamine

-LRB-

promoter

off

white

bars

-RRB-

-LRB-

left

-RRB-

and

-LRB-

right

control

-RRB-

mRNA

levels

from

the

same

cells

were

measured

by

quantitative

RT-PCR

The

PCR

was

performed

on

reverse

transcription

reactions

which

were

carried

out

in

the

presence

-LRB-

RT

-RRB-

or

absence

-LRB-

RT

-RRB-

of

reverse

transcriptase

To

analyze

effects

of

expression

levels

the

three

strains

were

grown

at

°C

either

in

the

absence

or

presence

of

thiamine

and

cell

growth

was

monitored

In

the

absence

of

thiamine

all

strains

grew

similar

to

each

other

and

to

the

wild-type

strain

-LRB-

Fig.

-RRB-

This

shows

that

expressed

from

the

weak

promoter

under

induced

conditions

was

sufficient

to

sustain

normal

cell

growth

When

these

strains

were

grown

in

the

presence

of

thiamine

however

there

was

a

direct

correlation

between

residual

promoter

activity

and

growth

rate

cells

expressing

from

the

promoter

showed

the

same

growth

rate

as

wild-type

cells

while

cells

expressing

from

the

promoter

and

even

more

so

from

the

promoter

showed

reduced

growth

rates

-LRB-

Fig.

-RRB-

Thus

in

the

presence

of

thiamine

the

lowered

levels

become

limiting

for

cell

growth

and

residual

growth

directly

reflects

basal

expression

levels

from

the

different

promoter

constructs

Fig.

of

on

cell

growth

Wild-type

cells

-LRB-

filled

circle

-RRB-

and

cells

expressing

from

either

the

-LRB-

filled

square

-RRB-

-LRB-

filled

triangle

-RRB-

or

-LRB-

crosses

-RRB-

promoter

were

grown

in

EMM

medium

at

°C

was

measured

at

the

indicated

times

a

In

the

absence

of

thiamine

all

strains

grew

at

similar

rates

b

In

the

presence

of

thiamine

the

growth

rates

were

dependent

on

the

residual

levels

of

being

expressed

from

the

different

promoters

Effect

of

on

cell

growth

under

stress

conditions

is

required

for

stress

response

in

S.

cerevisiae

-LRB-

Choder

-RRB-

We

therefore

determined

if

played

a

similar

role

under

stress

conditions

in

S.

pombe

Cells

expressing

varying

levels

of

were

subjected

to

temperature

stress

-LRB-

°C

-RRB-

osmotic

stress

-LRB-

M

sorbitol

-RRB-

or

oxidative

stress

-LRB-

mM

hydrogen

peroxide

-RRB-

We

found

that

neither

in

the

presence

nor

absence

of

thiamine

the

cells

exhibited

any

increased

stress

sensitivity

compared

to

control

cells

when

grown

under

the

different

stress

conditions

-LRB-

data

not

shown

-RRB-

Similar

results

were

obtained

when

cells

were

grown

in

up

to

M

sorbitol

No

significant

growth

of

either

the

wild-type

or

expressing

cells

was

observed

in

the

presence

or

absence

of

thiamine

at

and

M

concentrations

of

sorbitol

These

observations

indicate

that

low

levels

of

do

not

affect

the

survival

of

cells

under

any

of

the

tested

stress

conditions

Repression

of

transcription

causes

defects

in

cell

separation

We

next

examined

whether

different

levels

of

expression

would

influence

specific

cellular

processes

Cells

expressing

from

any

of

the

three

promoters

in

the

absence

of

thiamine

did

not

lead

to

any

obvious

phenotypes

even

after

h

of

growth

in

medium

without

thiamine

-LRB-

Fig.

-RRB-

Fig.

affects

cell

separation

and

morphology

Cells

expressing

from

either

the

or

promoter

were

analyzed

by

microscopy

a

Cells

grown

in

EMM

in

the

absence

of

thiamine

-LRB-

promoter

on

-RRB-

at

°C

appear

like

normal

wild-type

cells

b

When

grown

in

the

presence

of

thiamine

-LRB-

promoter

off

-RRB-

cells

expressing

from

the

promoter

appear

normal

while

those

expressing

under

the

promoter

display

defects

in

cell

morphology

and

septation

Multiple

phenotypes

including

elongated

cells

with

single

or

multiple

septa

and

growth

by

branching

are

evident

c

Cells

expressing

under

the

promoter

were

analyzed

by

fluorescence

microscopy

in

the

presence

of

thiamine

DAPI

staining

revealed

single

nuclei

in

each

cell

compartment

while

calcofluor

staining

showed

septa

-LRB-

some

of

them

aberrant

-RRB-

separating

the

cell

compartments

d

Control

cells

and

mutants

showing

similar

phenotypes

as

cells

expressing

under

the

promoter

cells

From

left

to

right

wild-type

cells

deletion

mutants

deletion

mutants

and

double

deletion

mutants

In

thiamine-containing

medium

however

cells

expressing

from

the

weak

promoter

became

more

elongated

than

the

wild-type

cells

after

h

of

growth

-LRB-

Fig.

-RRB-

Moreover

after

continued

incubation

in

the

same

medium

for

another

h

-LRB-

h

with

thiamine

-RRB-

cells

also

showed

defects

in

cell

separation

which

became

more

evident

after

an

additional

h

of

incubation

-LRB-

h

with

thiamine

-RRB-

-LRB-

Fig.

-RRB-

Most

cells

were

elongated

with

either

a

single

or

multiple

septa

and

some

cells

also

displayed

bent

morphologies

and/or

growth

by

branching

Cells

expressing

from

the

medium

strength

promoter

displayed

no

obvious

cell

separation

defects

until

h

of

growth

in

the

presence

of

thiamine

Similar

defects

as

described

above

however

became

apparent

after

h

of

growth

in

thiamine

-LRB-

data

not

shown

-RRB-

In

contrast

cells

expressing

from

the

strong

promoter

did

not

exhibit

any

aberrations

in

morphology

or

division

after

growth

in

thiamine-containing

medium

for

h

-LRB-

Fig.

-RRB-

and

continued

to

look

like

wild-type

cells

even

at

h

of

growth

in

thiamine-containing

medium

-LRB-

data

not

shown

-RRB-

Thus

the

basal

level

of

expression

from

the

strongest

promoter

is

sufficient

to

prevent

the

cell

separation

defects

seen

with

the

weaker

promoters

We

investigated

these

phenotypes

in

more

detail

for

cells

expressing

under

the

promoter

by

staining

the

division

septa

and

cell

nuclei

-LRB-

Fig.

-RRB-

Several

nuclei

were

present

separated

from

each

other

by

either

a

single

septum

or

multiple

aberrant

septa

Some

cells

also

contained

large

deposits

of

septal

material

at

random

positions

-LRB-

Fig.

-RRB-

We

conclude

that

lowered

expression

levels

lead

to

filamentous

growth

with

multiple

cells

remaining

attached

to

each

other

Similar

phenotypes

to

those

of

cells

expressing

under

the

promoter

have

observed

in

several

different

mutants

-LRB-

see

Discussion

-RRB-

for

example

deletions

in

two

transcription

factor

genes

and

also

lead

to

defects

in

cell

separation

-LRB-

Fig.

Bähler

-RRB-

These

findings

raise

the

possibility

that

plays

a

specific

role

in

cell

separation

after

cytokinesis

Low

levels

of

transcripts

most

strongly

affect

expression

of

genes

involved

in

septation

We

used

S.

pombe

whole-genome

microarrays

to

investigate

the

effects

on

gene

expression

caused

by

low

levels

of

Wild-type

cells

and

cells

expressing

under

the

different

regulatable

promoters

were

grown

in

the

presence

and

absence

of

thiamine

followed

by

RNA

extraction

Cells

expressing

under

the

promoter

did

not

show

any

significant

differences

in

gene

expression

profiles

when

compared

with

wild-type

cells

either

in

the

presence

or

absence

of

thiamine

-LRB-

data

not

shown

-RRB-

consistent

with

our

earlier

observations

that

these

cells

show

normal

growth

and

phenotypes

If

was

expressed

under

the

weakest

promoter

in

the

presence

of

thiamine

however

gene

expression

changed

substantially

As

down-regulation

of

is

expected

to

have

a

global

effect

on

transcription

we

used

external

spikes

for

normalization

-LRB-

Materials

and

methods

-RRB-

This

revealed

that

mean

transcript

levels

were

lower

compared

to

wild-type

cells

grown

under

the

same

conditions

-LRB-

Fig.

-RRB-

The

expression

levels

of

were

two

to

threefold

lower

under

these

conditions

compared

to

the

levels

under

its

own

promoter

Thus

lowered

expression

leads

to

reduced

overall

transcript

levels

Fig.

on

global

and

specific

gene

expression

as

a

function

of

levels

a

Microarray

experiments

of

cells

expressing

under

the

weakest

promoter

The

histograms

show

the

distribution

of

gene

expression

ratios

of

cells

grown

in

the

presence

of

thiamine

for

and

h

relative

to

wild-type

cells

grown

in

the

presence

of

thiamine

Genes

in

both

histograms

are

colored

by

their

relative

expression

levels

at

h

as

indicated

at

upper

right

External

spikes

have

been

used

for

normalization

revealing

a

global

down-regulation

of

transcript

levels

by

-LRB-

h

-RRB-

to

-LRB-

h

-RRB-

on

average

when

expression

is

lowered

The

averages

of

two

independent

repeats

for

each

timepoint

are

shown

b

Overlap

between

the

genes

downregulated

when

is

expressed

under

the

weakest

promoter

in

the

presence

of

thiamine

and

genes

downregulated

in

both

and

mutants

-LRB-

Lee

et

al.

-RRB-

The

number

in

brackets

represents

the

overlap

expected

by

chance

given

the

sizes

of

the

gene

sets

considered

and

the

total

number

of

genes

The

overlap

is

highly

significant

as

calculated

using

the

hypergeometric

distribution

-LRB-

P

-RRB-

c

Hierarchical

cluster

analysis

of

genes

that

are

either

upregulated

or

threefold

downregulated

when

is

expressed

under

the

weakest

promoter

in

presence

of

thiamine

Horizontal

rows

represent

the

profiles

of

hierarchically

clustered

genes

Columns

represent

experiments

of

different

mutant

strains

and

different

time-points

for

the

same

strain

The

transcript

levels

for

each

strain

-LRB-

indicated

at

bottom

-RRB-

relative

to

levels

in

wild-type

cells

are

color-coded

as

indicated

at

right

and

missing

data

are

shown

in

gray

The

microarray

data

from

and

mutants

are

from

Lee

et

al.

-LRB-

-RRB-

and

the

data

from

and

mutants

are

from

Rustici

et

al.

-LRB-

-RRB-

On

top

of

this

genome-wide

down-regulation

of

transcription

transcripts

were

at

least

upregulated

in

three

of

the

four

samples

tested

while

genes

were

at

least

threefold

downregulated

in

three

of

the

four

samples

tested

upon

lowering

expression

-LRB-

Supplementary

Tables

and

-RRB-

These

genes

were

categorized

according

to

their

functions

-LRB-

Supplementary

Table

-RRB-

The

majority

of

the

genes

whose

expression

levels

were

affected

by

encoded

proteins

involved

in

metabolism

and

transport

Many

of

the

upregulated

transcripts

in

cells

with

lowered

expression

levels

overlapped

with

genes

upregulated

during

different

environmental

stresses

-LRB-

Chen

et

al.

-RRB-

and

nitrogen

starvation

-LRB-

Mata

et

al.

-RRB-

-LRB-

P

to

-RRB-

This

probably

reflects

an

indirect

effect

caused

by

the

defects

in

these

cells

A

substantial

number

of

the

downregulated

genes

in

the

category

of

cell

cycle

and

cell

division

played

specific

roles

in

cytokinesis

and

cell

separation

For

example

the

two

genes

and

encode

β-glucanase

and

α-glucanase

respectively

degrades

the

primary

division

septum

between

the

new

ends

of

daughter

cells

and

hydrolyzes

the

old

cell

wall

surrounding

the

septum

leading

to

full

separation

of

daughter

cells

-LRB-

Martín-Cuadrado

et

al.

Dekker

et

al.

-RRB-

The

genes

and

have

also

been

shown

to

be

involved

in

cell

separation

-LRB-

Alonso-Nunez

et

al.

-RRB-

and

encodes

a

protein

involved

in

formation

of

contractile

ring

during

cytokinesis

-LRB-

Wong

et

al.

-RRB-

Both

the

lists

of

genes

being

upregulated

and

downregulated

after

down-regulation

showed

highly

significant

overlaps

with

lists

of

genes

upregulated

or

downregulated

respectively

in

and

mutants

-LRB-

P

to

-RRB-

-LRB-

Lee

et

al.

-RRB-

whose

genes

encode

different

proteins

involved

in

transcriptional

regulation

-LRB-

see

Discussion

-RRB-

Among

the

downregulated

genes

the

most

pronounced

overlap

was

observed

with

and

mutants

-LRB-

Fig.

-RRB-

The

downregulated

genes

also

showed

some

overlap

with

genes

downregulated

in

and

mutants

-LRB-

P

-RRB-

-LRB-

Rustici

et

al.

-RRB-

which

encode

cell-cycle

transcription

factors

Figure

shows

a

cluster

analysis

of

the

genes

most

strongly

regulated

by

repression

and

their

expression

levels

in

the

various

transcriptional

mutant

backgrounds

The

and

mutants

show

highly

similar

expression

signatures

to

cells

with

compromised

expression

while

the

overlap

with

and

mutants

is

less

evident

Intriguingly

if

was

overexpressed

using

the

medium

strength

promoter

the

expression

levels

of

these

genes

were

reversed

with

downregulated

genes

becoming

upregulated

and

upregulated

genes

becoming

downregulated

-LRB-

Fig.

-RRB-

Thus

transcript

levels

of

this

gene

set

are

particularly

responsive

to

the

transcript

levels

of

Discussion

We

have

analyzed

functions

of

the

subunit

of

pol

II

in

S.

pombe

Despite

the

overall

conservation

in

structure

and

function

of

pol

II

across

species

the

subunit

presents

a

special

case

S.

pombe

is

similar

to

its

orthologs

in

higher

eukaryotes

with

respect

to

its

structure

and

essential

nature

-LRB-

Sakurai

et

al.

-RRB-

but

in

contrast

to

its

human

ortholog

it

can

form

stable

hybrid

dimers

with

S.

cerevisiae

-LRB-

Sakurai

et

al.

-RRB-

and

can

rescue

the

temperature-sensitive

phenotype

associated

with

the

lack

of

S.

cerevisiae

-LRB-

Shpakovski

et

al.

-RRB-

Thus

S.

pombe

may

have

intermediate

characteristics

useful

to

bridge

the

knowledge

from

S.

cerevisiae

and

multicellular

eukaryotes

We

used

regulatable

gene

expression

constructs

to

study

the

in

vivo

roles

of

Under

optimal

conditions

influences

the

growth

of

cells

in

a

dose-dependent

manner

when

expression

drops

below

a

certain

level

using

the

regulatable

promoters

-LRB-

Figs.

-RRB-

In

S.

cerevisiae

deletion

of

the

gene

leads

to

slow

cell

growth

under

optimal

conditions

-LRB-

Woychik

and

Young

Choder

-RRB-

while

in

S.

pombe

is

essential

-LRB-

Sakurai

et

al.

-RRB-

We

found

that

low

levels

of

expressed

from

the

weakest

promoter

were

sufficient

for

the

cells

to

survive

a

range

of

different

stress

conditions

including

temperature

stress

Accordingly

core

stress

genes

were

actually

upregulated

in

mutants

and

S.

pombe

is

therefore

unlikely

to

play

any

specialized

role

during

environmental

stress

S.

cerevisiae

is

dispensable

for

oxidative

and

osmotic

stress

-LRB-

Maillet

et

al.

-RRB-

but

unlike

in

S.

pombe

it

is

required

for

survival

of

temperature

stress

-LRB-

Woychik

and

Young

-RRB-

Fission

yeast

cells

grown

under

low

expression

of

were

elongated

and

showed

defects

in

cell

separation

as

indicated

by

the

accumulation

of

division

septa

some

of

them

highly

aberrant

-LRB-

Fig.

-RRB-

Strains

with

cell

separation

defects

similar

to

the

ones

described

here

include

mutants

in

components

of

the

exocyst

complex

-LRB-

and

-RRB-

an

anillin

homolog

-LRB-

-RRB-

septins

-LRB-

-RRB-

an

-LRB-

-RRB-

an

-LRB-

-RRB-

calcineurin

-LRB-

-RRB-

a

MAP

kinase

and

phosphatase

-LRB-

-RRB-

two

transcription

factors

-LRB-

-RRB-

and

subunits

of

the

mediator

complex

-LRB-

and

-RRB-

-LRB-

Sipiczki

et

al.

Yoshida

et

al.

Longtine

et

al.

Toda

et

al.

Sugiura

et

al.

Grallert

et

al.

Wang

et

al.

Martín-Cuadrado

et

al.

Spåhr

et

al.

Tasto

et

al.

Dekker

et

al.

-RRB-

In

S.

cerevisiae

haploid

deletion

mutants

exhibit

an

axial

budding

pattern

with

normal

yeast

cell

morphology

but

diploid

mutants

are

more

elongated

than

wild-type

cells

during

nitrogen

starvation

and

grow

in

a

unipolar

pattern

resembling

pseudohyphae

formation

-LRB-

Pillai

et

al.

-RRB-

Lowered

expression

of

causes

relatively

modest

reductions

in

global

transcription

which

probably

leads

to

the

reduced

growth

rates

under

these

conditions

-LRB-

Fig.

-RRB-

In

addition

low

transcription

results

in

substantially

lowered

expression

of

a

distinct

subset

of

genes

Among

the

strongly

affected

genes

were

and

with

known

functions

in

cell

separation

which

are

targets

of

a

transcriptional

cascade

regulated

by

the

and

transcription

factors

-LRB-

Rustici

et

al.

Alonso-Nunez

et

al.

-RRB-

Accordingly

the

genes

affected

by

lowered

expression

significantly

overlapped

with

genes

affected

in

and

mutants

Much

stronger

similarities

however

were

evident

with

the

gene

expression

signatures

of

several

strains

mutated

in

the

and

genes

which

encode

various

components

of

the

transcriptional

machinery

and

are

essential

components

of

the

mediator

complex

of

pol

II

while

and

are

components

of

a

complex

homologous

to

metazoan

a

cyclin-dependent

kinase-activating

kinase

-LRB-

CAK

-RRB-

and

a

pol

II

CTD

kinase

associated

with

transcription

factor

IIH

-LRB-

Spåhr

et

al.

Lee

et

al.

-RRB-

The

overlap

in

expression

signatures

between

these

four

mutants

and

cells

with

lowered

expression

was

evident

for

both

upregulated

and

downregulated

genes

These

differentially

regulated

genes

showed

reverse

regulation

-LRB-

upregulated

genes

became

downregulated

and

vice

versa

-RRB-

when

was

overexpressed

from

the

medium

strength

promoter

-LRB-

Fig.

-RRB-

Thus

the

expression

of

this

gene

set

is

particularly

responsive

to

expression

levels

and

also

depends

on

a

range

of

other

factors

involved

in

general

transcription

This

could

either

reflect

a

specific

transcriptional

pathway

involving

and

the

other

factors

-LRB-

either

directly

or

indirectly

via

the

control

of

a

regulator

-RRB-

or

the

affected

genes

could

be

generally

sensitive

to

transcriptional

interference

In

any

case

these

data

show

that

the

transcriptional

pathway

required

for

cell

separation

in

fission

yeast

-LRB-

Bähler

-RRB-

is

strongly

affected

when

interfering

with

expression

levels

We

speculate

that

the

complex

is

involved

in

linking

the

transcriptional

output

to

the

metabolic

status

of

the

cell

The

and

transcripts

are

down-regulated

when

cells

enter

stationary

phase

-LRB-

unpublished

observations

-RRB-

interacts

with

the

glycolytic

enzyme

dehydrogenase

-LRB-

Mitsuzawa

et

al.

-RRB-

and

global

transcription

is

sensitive

to

expression

levels

It

is

possible

that

the

transcriptional

pathway

required

for

cell

separation

is

particularly

sensitive

for

compromised

transcription

to

adjust

the

growth

mode

to

availability

of

nutrients

in

abundant

nutrients

global

transcription

is

efficient

and

growth

is

best

as

single

cells

while

in

limiting

nutrients

global

transcription

and

cell

separation

are

compromised

and

cells

grow

as

multicellular

pseudohyphae

which

may

allow

a

more

efficient

grazing

for

new

nutrients

as

growth

is

directed

In

the

first

description

of

S.

pombe

multi-cellular

pseudohyphae

similar

to

those

seen

in

mutants

have

been

observed

-LRB-

Lindner

-RRB-

the

natural

capacity

to

switch

to

multi-cellular

growth

might

have

been

lost

in

the

yeast

strains

cultured

in

the

laboratory

Taken

together

our

data

suggest

that

S.

pombe

besides

its

general

role

in

transcription

performs

a

more

specialized

function

in

regulating

a

specific

gene

expression

program

required

for

cell

separation

at

the

end

of

the

cell

cycle

It

is

possible

that

this

role

is

carried

out

by

interacting

with

other

general

factors

of

the

transcriptional

machinery

such

as

the

mediator

and

CTD

kinase

as

suggested

by

similarities

in

phenotypes

and

expression

signatures

in

our

study

Accordingly

a

comparison

of

the

X-ray

crystallographic

structure

of

the

complete

pol

II

including

the

heterodimer

with

an

electron

microscopic

structure

of

the

pol

II-mediator

complex

has

indicated

that

may

interact

with

the

mediator

-LRB-

Bushnell

and

Kornberg

-RRB-

Alternatively

the

subset

of

genes

affected

by

changes

in

expression

-LRB-

and

by

manipulating

other

general

transcription

factors

-RRB-

could

reflect

genes

that

are

especially

sensitive

to

transcriptional

stress

This

interpretation

does

not

imply

any

specific

gene

expression

program

although

it

could

be

used

by

the

cell

to

adjust

transcription

to

available

nutrients

Given

the

conservation

of

it

will

be

interesting

to

see

whether

the

human

ortholog

plays

a

similar

specialized

role

to

transcribe

particular

subsets

of

genes

Electronic

supplementary

material

Supplementary

material

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B O O O O O O O O O O O O O O O B 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 B O O O O O O O O O O B 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 B 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 B 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 B 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B 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 B 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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 B O O O O O O O B 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 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 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 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 B 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 B O O O O O O O O O O O B 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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