Identification

of

genes

potentially

involved

in

supporting

hematopoietic

stem

cell

activity

of

stromal

cell

line

Although

coculture

of

hematopoietic

stem

cells

-LRB-

HSCs

-RRB-

with

stromal

cells

is

a

useful

system

to

study

hematopoiesis

in

the

niche

little

is

known

regarding

the

precise

cellular

and

molecular

mechanisms

of

maintaining

HSCs

through

cell

cell

interactions

The

murine

preadipose

stromal

cell

line

-LRB-

-RRB-

has

been

demonstrated

to

support

HSCs

in

vitro

In

this

study

microarray

analysis

was

performed

on

cells

and

HSC-nonsupporting

subclone

cells

to

identify

genes

responsible

for

supporting

HSC

activity

Comparison

of

gene

expression

profiles

revealed

that

only

genes

were

down-regulated

by

more

than

twofold

in

subclone

cells

Of

these

down-regulated

genes

we

selected

candidate

genes

and

evaluated

for

the

maintenance

of

HSC

function

by

overexpressing

these

genes

in

subclone

cells

One

unknown

gene

-LRB-

also

named

as

-RRB-

which

is

predicted

to

encode

an

integral

membrane

protein

demonstrated

a

partial

restoration

of

the

defect

in

HSC-supporting

activity

Introduction

Hematopoietic

stem

cells

-LRB-

HSCs

-RRB-

are

capable

of

self-renewal

and

multilineage

differentiation

and

generate

all

types

of

blood

cells

throughout

the

lifetime

A

major

challenge

for

a

long

time

has

been

the

establishment

of

culture

conditions

that

can

facilitate

ex

vivo

expansion

of

HSCs

To

date

a

large

number

of

attempts

using

combinations

of

multiple

cytokines

have

resulted

in

limited

success

with

a

several-fold

increase

in

HSC

numbers

under

the

optimal

culture

conditions

-LSB-

-RSB-

A

major

problem

is

that

culture

conditions

established

thus

far

have

induced

HSC

proliferation

coupled

with

differentiation

leading

to

a

progressive

loss

of

in

vivo

long-term

repopulating

potential

-LSB-

-RSB-

Therefore

a

better

understanding

of

mechanisms

that

regulate

HSC

self-renewal

and

differentiation

is

required

to

achieve

ex

vivo

expansion

of

HSCs

In

the

adult

bone

marrow

HSCs

are

thought

to

reside

in

a

specific

microenvironment

referred

to

as

the

niche

which

is

composed

of

stromal

cells

that

play

a

vital

role

in

determining

stem

cell

fate

Indeed

several

stromal

cell

lines

have

been

established

not

only

from

bone

marrow

but

also

from

fetal

liver

and

the

aorta-gonad-mesonephros

region

and

have

been

shown

to

maintain

HSCs

in

vitro

-LSB-

-RSB-

Coculture

of

HSCs

with

stromal

cell

lines

is

a

useful

system

to

study

hematopoiesis

in

the

niche

Although

physical

contact

between

HSCs

and

stromal

cell

lines

has

been

demonstrated

to

be

essential

little

is

known

regarding

the

precise

cellular

and

molecular

mechanisms

of

maintaining

HSCs

by

stromal

cell

lines

-LRB-

-RRB-

is

a

preadipose

stromal

cell

line

derived

from

newborn

mouse

calvaria

that

can

support

long-term

hematopoiesis

in

vitro

-LSB-

-RSB-

In

addition

subclones

incapable

of

supporting

the

proliferation

of

HSCs

have

been

isolated

-LSB-

-RSB-

It

has

also

been

shown

that

the

hematopoiesis-supporting

ability

of

cells

is

not

solely

conferred

by

their

expression

of

c-kit

ligand

stem

cell

factor

-LRB-

SCF

-RRB-

In

this

study

microarray

analysis

was

performed

to

identify

genes

responsible

for

supporting

HSC

activity

of

cells

Eleven

genes

specifically

down-regulated

in

the

subclone

cells

were

selected

and

characterized

by

overexpressing

the

corresponding

cDNAs

in

the

subclone

cells

Materials

and

methods

Mice

-LRB-

-RRB-

mice

were

purchased

from

Charles

River

Laboratories

Japan

-LRB-

Yokohama

Japan

-RRB-

mice

congenic

for

the

locus

-LRB-

-RRB-

were

obtained

from

RIKEN

BRC

mice

were

obtained

from

mating

pairs

of

and

mice

All

animal

experiments

were

approved

by

the

Animal

Experiment

Committee

of

the

RIKEN

Tsukuba

Institute

Stromal

cell

culture

and

lentiviral

transduction

The

stromal

cell

line

was

obtained

from

RIKEN

BRC

Cell

Bank

and

cells

were

maintained

in

Minimum

Essential

Medium

Eagle-α

-LRB-

MEM-α

-RRB-

-LRB-

Sigma-Aldrich

St

Louis

MO

-RRB-

containing

fetal

bovine

serum

-LRB-

FBS

-RRB-

-LRB-

Sigma-Aldrich

-RRB-

at

°C

in

a

atmosphere

FANTOM

cDNA

clones

-LRB-

kindly

provided

by

Dr.

Y.

Hayashizaki

and

Dr.

J.

Kawai

RIKEN

GSC

-RRB-

corresponding

to

the

genes

used

in

this

study

were

subcloned

into

the

lentiviral

vector

plasmid

Recombinant

lentiviral

vectors

were

produced

as

described

previously

-LSB-

-RSB-

subclone

cells

were

transduced

with

lentiviral

vectors

expressing

cDNAs

at

a

multiplicity

of

infection

of

and

of

transduction

efficiency

was

confirmed

by

fluorescence-activated

cell

sorting

-LRB-

FACS

-RRB-

analysis

for

Venus

expression

Purification

of

KSL

cells

and

coculture

with

stromal

cells

lowc-Kit

lineage

marker

-LRB-

KSL

-RRB-

cells

were

purified

as

described

previously

with

minor

modifications

-LSB-

-RSB-

Briefly

bone

marrow

cells

isolated

from

to

mice

were

stained

with

a

lineage

marker

antibody

cocktail

consisting

of

biotinylated

anti-IgM

and

antibodies

-LRB-

eBioscience

San

Diego

CA

-RRB-

Lineage

marker

cells

were

depleted

using

streptavidin-coupled

Dynabeads

-LRB-

Invitrogen

Carlsbad

CA

-RRB-

The

remaining

cells

were

stained

with

fluorescein

isothiocyanate

-LRB-

FITC

-RRB-

conjugated

phycoerythrin

-LRB-

PE

-RRB-

conjugated

and

allophycocyanin

-LRB-

APC

-RRB-

conjugated

anti-c-Kit

antibodies

-LRB-

all

from

BD

Biosciences

San

Jose

CA

-RRB-

The

biotinylated

antibodies

were

developed

with

-LRB-

BD

Biosciences

-RRB-

FACS

was

performed

with

a

FACSVantage

SE

-LRB-

BD

Biosciences

-RRB-

KSL

cells

were

sorted

into

individual

wells

of

a

plate

containing

stromal

cells

-LRB-

cells/well

-RRB-

irradiated

with

Gy

and

were

cocultured

in

μL

of

MEM-α

containing

FBS

Colony-forming

cell

assay

After

days

of

coculture

KSL

HSCs

were

collected

and

plated

in

a

plate

containing

mL

of

methylcellulose

medium

-LRB-

MethoCult

GF

-RRB-

-LRB-

StemCell

Technologies

Vancouver

Canada

-RRB-

containing

ng/mL

rmSCF

ng/mL

ng/mL

and

units/mL

rhEPO

After

days

of

incubation

colonies

were

recovered

cytospun

onto

glass

slides

and

then

subjected

to

Hemacolor

-LRB-

Merck

KGaA

Darmstadt

Germany

-RRB-

staining

for

morphological

examination

Colony-forming

unit-granulocyte

erythrocyte

monocyte

megakaryocyte

-LRB-

CFU-GEMM

-RRB-

CFU-granulocyte

erythrocyte

monocyte

-LRB-

CFU-GEM

-RRB-

CFU-granulocyte

monocyte

-LRB-

CFU-GM

-RRB-

CFU-granulocyte

-LRB-

CFU-G

-RRB-

CFU-monocyte

-LRB-

CFU-M

-RRB-

and

burst

forming

unit-erythrocyte

-LRB-

BFU-E

-RRB-

were

scored

using

standard

scoring

criteria

The

total

colony

number

was

expressed

as

colony-forming

cell

-LRB-

CFC

-RRB-

Competitive

repopulation

assay

The

competitive

repopulation

assay

was

performed

by

using

the

congenic

mouse

system

as

described

previously

-LSB-

-RSB-

KSL

HSCs

after

coculture

were

mixed

with

total

bone

marrow

competitor

cells

from

mice

and

transplanted

into

lethally

-LRB-

Gy

-RRB-

irradiated

mice

At

to

weeks

after

transplantation

peripheral

blood

cells

of

the

recipient

mice

were

collected

by

retro-orbital

bleeding

After

lysis

of

red

blood

cells

with

ammonium

chloride

buffer

the

remaining

nucleated

cells

were

stained

with

FITC-conjugated

PE-conjugated

biotinylated

and

biotinylated

antibodies

followed

by

addition

of

streptavidin-PerCP

The

cells

were

stained

simultaneously

with

APC-conjugated

antibody

or

a

mixture

of

APC-conjugated

and

antibodies

FACS

analysis

was

performed

with

a

FACSCalibur

Donor

chimerism

was

determined

as

the

percentage

of

cells

When

the

percent

chimerism

was

for

all

myeloid

B-lymphoid

and

T-lymphoid

lineages

recipient

mice

were

considered

to

be

multilineage

reconstituted

Microarray

analysis

Total

RNA

was

isolated

from

stromal

cells

using

the

ISOGEN

reagent

-LRB-

Nippon

Gene

Tokyo

Japan

-RRB-

and

purified

with

the

RNeasy

MinElute

cleanup

kit

-LRB-

Qiagen

Hilden

Germany

-RRB-

Biotin-labeled

cRNA

was

prepared

from

μg

of

the

purified

total

RNA

with

a

one-cycle

cDNA

synthesis

kit

and

amplification

reagents

for

IVT

labeling

-LRB-

Affymetrix

Santa

Clara

CA

-RRB-

and

was

hybridized

to

an

Affymetrix

Gene

Chip

Mouse

Genome

array

-LRB-

Affymetrix

-RRB-

which

contains

approximately

probe

sets

for

analyzing

the

expression

levels

of

more

than

mouse

genes

After

washing

and

staining

with

the

antibody

amplification

procedure

the

microarrays

were

scanned

with

an

Affymetrix

GeneChip

Scanner

All

these

procedures

were

carried

out

according

to

the

manufacturer

's

instructions

The

expression

value

-LRB-

Signal

-RRB-

and

detection

call

-LRB-

Present

-LRB-

P

-RRB-

Absent

-LRB-

A

-RRB-

or

Marginal

-LRB-

M

-RRB-

-RRB-

for

each

probe

set

were

calculated

using

GeneChip

Operating

Software

version

-LRB-

Affymetrix

-RRB-

The

Signal

values

were

normalized

so

that

their

mean

in

each

experiment

was

in

order

to

adjust

for

minor

differences

between

the

experiments

The

change

value

-LRB-

Signal

Log

Ratio

-RRB-

and

change

call

-LRB-

Increase

Marginal

Increase

No

Change

Marginal

Decrease

or

Decrease

-RRB-

for

each

probe

set

were

calculated

by

Comparison

Analysis

of

the

software

All

experiments

were

performed

in

duplicate

using

two

independent

cell

samples

To

identify

differentially

expressed

genes

we

selected

probe

sets

that

showed

a

change

call

of

Decrease

and

a

Signal

Log

Ratio

value

of

-LRB-

more

than

twofold

down-regulation

-RRB-

or

a

change

call

of

Increase

and

a

Signal

Log

Ratio

value

of

-LRB-

more

than

twofold

up-regulation

-RRB-

in

each

of

the

two

independent

experiments

Quantitative

real-time

polymerase

chain

reaction

Total

RNA

was

isolated

from

stromal

cells

using

the

ISOGEN

reagent

and

cDNA

was

synthesized

from

μg

of

total

RNA

using

SuperScript

II

reverse

transcriptase

-LRB-

Invitrogen

-RRB-

Real-time

polymerase

chain

reaction

-LRB-

PCR

-RRB-

amplification

with

two

independent

cell

samples

was

carried

out

using

the

LightCycler

FastStart

DNA

Master

SYBR

Green

I

kit

-LRB-

Roche

Penzberg

Germany

-RRB-

according

to

the

manufacturer

's

instructions

The

primer

sets

used

in

this

study

are

listed

in

Table

Data

were

normalized

GAPDH

mRNA

levels

Gene-specific

amplification

was

confirmed

by

determining

the

melting

curves

of

the

PCR

products

and

by

a

single

band

of

the

expected

size

in

agarose

gel

electrophoresis

Table

used

in

quantitative

real-time

PCRGenePrimer

sequenceProduct

size

-LRB-

bp

-RRB-

Results

and

discussion

Subclones

of

the

cell

line

are

defective

in

supporting

HSCs

Although

subclones

and

have

been

isolated

in

terms

of

inability

to

support

long-term

hematopoiesis

in

vitro

their

HSC-supporting

capacity

has

not

been

precisely

assessed

-LSB-

-RSB-

Therefore

we

evaluated

the

maintenance

of

HSC

function

after

coculture

with

subclones

and

-LRB-

hereinafter

referred

to

as

and

respectively

-RRB-

by

in

vitro

CFC

and

in

vivo

competitive

repopulation

assays

using

KSL

cells

as

highly

purified

HSCs

-LSB-

-RSB-

Murine

bone

marrow-derived

stromal

cell

line

that

can

support

HSCs

was

also

used

as

a

control

-LSB-

-RSB-

As

shown

in

Table

the

CFC

frequency

significantly

decreased

after

days

of

coculture

with

and

cells

compared

with

cells

and

lower

levels

of

engraftment

were

observed

in

transplanted

mice

These

results

indicate

that

subclone

cells

have

a

substantial

defect

in

supporting

HSCs

even

after

short-term

coculture

Table

frequency

and

competitive

repopulation

capacityStromal

cellsCFC

frequencyNo

of

reconstituted

mice

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

A

total

of

KSL

HSCs

-LRB-

cells/well

-RRB-

were

cocultured

with

stromal

cells

for

days

and

were

then

subjected

to

the

CFC

assay

The

CFC

frequency

represents

the

colony

number

per

ten

input

KSL

HSCs

For

the

competitive

repopulation

assay

KSL

HSCs

were

cocultured

with

stromal

cells

for

days

and

then

transplanted

into

lethally

irradiated

mice

At

weeks

after

transplantation

peripheral

blood

cells

of

the

recipient

mice

were

analyzed

Data

represent

the

mean

SD

of

two

independent

experiments

P

P

versus

Student

's

t-test

Identification

of

genes

that

were

specifically

down-regulated

in

subclone

cells

We

hypothesized

that

genes

responsible

for

supporting

HSCs

would

be

down-regulated

in

subclone

cells

To

identify

genes

specifically

down-regulated

in

subclone

cells

microarray

analysis

was

performed

on

cells

and

cells

and

cells

using

an

Affymetrix

GeneChip

array

containing

approximately

probe

sets

representing

over

mouse

genes

As

expected

gene

expression

profiling

showed

a

few

differences

in

gene

expression

between

cells

and

subclone

cells

Compared

to

cells

only

genes

were

down-regulated

by

more

than

twofold

in

both

subclone

cells

Of

these

down-regulated

genes

genes

were

of

unknown

function

and

of

-LRB-

-RRB-

known

genes

encoded

membrane

and

extracellular

space

proteins

according

to

the

Gene

Ontology

cellular

component

classification

On

the

other

hand

genes

were

up-regulated

by

more

than

twofold

and

of

-LRB-

-RRB-

known

genes

encoded

membrane

and

extracellular

space

proteins

The

signal

values

and

detection

calls

for

this

analysis

are

provided

in

supplemental

data

Among

the

down-regulated

genes

eight

genes

of

known/predicted

function

including

chemokine

and

cytokine-related

genes

which

were

expected

to

effect

on

the

HSC-supporting

activity

and

three

genes

of

unknown

function

which

were

predicted

to

encode

membrane

proteins

were

selected

for

further

analysis

-LRB-

Table

-RRB-

Table

of

candidate

genes

down-regulated

in

subclone

cells

compared

with

cellsGene

symbolGene

titleRefseq

IDMicroarrayqPCRvs

cDNA

geneNM_1979860.300.460.110.190.290.230.160.131200009

cDNA

cDNA

geneNM_1337490.410.371.231.160.290.330.480.55

-LRB-

C-C

motif

-RRB-

ligand

2NM_0113330.090.130.040.060.160.070.090.04

-LRB-

C-C

motif

-RRB-

ligand

9NM_0113380.140.190.030.040.100.090.040.03

-LRB-

C-X-C

motif

-RRB-

ligand

5NM_0091410.330.230.430.380.200.130.900.60

HgfHepatocyte

growth

-LRB-

-RRB-

Interleukin

receptor

-LRB-

-RRB-

Interleukin

6NM_0311680.380.442.072.620.300.162.421.31

acid

phosphatase

type

NDNDNDNDRelative

expression

levels

in

subclone

cells

-LRB-

and

-RRB-

compared

with

and

cells

were

calculated

using

data

from

microarray

and

quantitative

real-time

PCR

-LRB-

qPCR

-RRB-

analyses

Data

represent

the

average

of

fold

changes

from

two

independent

experimentsND

not

determined

Characterization

of

candidate

genes

To

determine

the

requirement

of

candidate

genes

for

the

supportive

activity

of

cells

cells

were

transduced

with

lentiviral

vectors

containing

the

corresponding

cDNAs

In

all

experiments

the

transduction

efficiency

was

more

than

as

determined

by

FACS

analysis

for

Venus-positive

cells

KSL

HSCs

were

cocultured

with

cells

expressing

candidate

genes

for

days

and

then

subjected

to

the

CFC

assay

As

shown

in

Fig.

the

CFC

frequency

increased

when

cocultured

with

cells

overexpressing

the

candidate

genes

except

Hgf

compared

with

cells

Morphological

evaluation

of

the

colonies

revealed

that

the

number

of

CFU-GEMM

which

is

the

most

primitive

colony

type

observed

in

the

CFC

assay

increased

except

and

Of

note

although

the

overexpression

of

increased

the

number

of

colonies

to

the

same

level

as

cells

a

large

proportion

-LRB-

-RRB-

of

colonies

were

CFU-M

Fig.

of

over

expression

of

candidate

genes

on

the

CFC

frequency

A

total

of

KSL

HSCs

-LRB-

cells/well

-RRB-

were

cocultured

with

cells

cells

or

cells

expressing

the

indicated

genes

for

days

and

then

subjected

to

the

CFC

assay

Individual

colonies

were

scored

according

to

their

morphology

Data

represent

the

average

of

two

independent

experiments

We

next

performed

the

competitive

repopulation

assay

to

determine

the

effect

of

overexpressing

seven

candidate

genes

including

and

and

three

unknown

genes

As

shown

in

Table

successful

multilineage

reconstitution

was

observed

in

all

mice

transplanted

with

HSCs

cocultured

with

cells

overexpressing

or

as

well

as

with

cells

However

comparison

of

the

chimerism

showed

no

significant

differences

from

that

of

cells

suggesting

that

the

overexpression

of

neither

nor

completely

restored

the

defective

HSC-supporting

activity

of

cells

Microarray

analysis

showed

that

the

expression

of

was

approximately

twofold

up-regulated

in

and

cells

when

compared

with

cells

this

finding

was

confirmed

by

quantitative

real-time

PCR

analysis

-LRB-

Table

-RRB-

It

has

also

been

demonstrated

that

alone

or

in

combination

with

SCF

is

incapable

of

maintaining

KSL

HSCs

-LSB-

-RSB-

Although

is

involved

in

various

steps

of

hematopoiesis

and

has

been

used

for

the

ex

vivo

expansion

of

HSCs

-LSB-

-RSB-

may

not

be

crucial

for

the

maintenance

of

HSCs

governed

by

stromal

cells

Table

of

overexpression

of

candidate

genes

on

the

competitive

repopulation

capacityNo

of

reconstituted

mice

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Thirty

KSL

HSCs

were

cocultured

with

cells

cells

or

cells

expressing

the

indicated

genes

for

days

and

then

transplanted

into

lethally

irradiated

mice

At

weeks

after

transplantation

peripheral

blood

cells

of

the

recipient

mice

were

analyzed

Data

represent

the

mean

SD

P

versus

Student

's

t-test

cells

overexpressing

or

had

no

apparent

effect

on

the

HSC-supporting

activity

In

contrast

the

overexpression

of

or

resulted

in

a

certain

increase

in

the

frequency

of

reconstituted

mice

Although

these

genes

as

well

as

and

could

significantly

increase

the

CFC

frequency

the

effect

on

the

HSC-supporting

activity

assessed

by

the

competitive

repopulation

assay

was

limited

This

may

be

because

days

of

coculture

was

not

long

enough

to

detect

a

significant

difference

in

HSC-supporting

activity

It

is

of

course

well

known

that

many

growth

factors

and

cytokines

that

can

increase

the

CFC

frequency

have

little

influence

on

the

maintenance

and

proliferation

of

HSCs

Of

note

the

overexpression

of

showed

a

substantial

increase

in

the

chimerism

although

the

difference

did

not

reach

statistical

significance

-LRB-

Table

-RRB-

To

confirm

the

effect

of

expression

on

the

supportive

activity

of

cells

we

performed

the

following

experiments

First

down-regulation

of

expression

in

and

cells

compared

to

that

in

and

cells

was

confirmed

by

quantitative

real-time

PCR

analysis

-LRB-

Table

-RRB-

Next

cells

were

transduced

with

a

myc-tagged

expression

lentiviral

vector

and

its

expression

was

detected

by

Western

blotting

and

immunofluorescence

staining

with

anti-myc

antibody

Coculture

with

cells

overexpressing

myc-tagged

resulted

in

an

increase

in

the

CFC

frequency

at

a

level

similar

to

that

shown

in

Fig.

-LRB-

data

not

shown

-RRB-

The

cDNA

encodes

a

predicted

protein

of

amino

acids

which

is

rich

in

leucine

-LRB-

-RRB-

and

has

no

significant

homology

with

any

known

protein

sequences

is

also

named

as

-LRB-

transmembrane

protein

-RRB-

which

is

predicted

to

be

an

integral

membrane

protein

gene

according

to

the

Gene

Ontology

classification

Further

studies

are

required

to

determine

whether

is

involved

in

the

HSC-supporting

activity

of

cells

Thus

far

a

large

number

of

microarray

analyses

of

stromal

cell

lines

derived

from

a

variety

of

tissue

types

have

been

reported

Comparison

of

gene

expression

profiles

between

HSC-supporting

and

HSC-nonsupporting

stromal

cell

lines

has

resulted

in

the

identification

of

a

number

of

differentially

expressed

genes

-LSB-

-RSB-

In

the

present

study

we

expected

a

few

differences

in

gene

expression

by

comparing

HSC-supporting

cells

with

HSC-nonsupporting

subclone

cells

and

indeed

we

could

narrow

down

the

candidate

genes

involved

in

maintaining

HSCs

Although

one

of

the

analyzed

candidate

genes

demonstrated

a

partial

restoration

of

the

defect

in

HSC-supporting

activity

of

cells

it

is

possible

that

other

down-regulated

genes

in

subclone

cells

may

also

be

required

for

the

maintenance

of

HSCs

Continuing

studies

to

identify

genes

responsible

for

HSC-supporting

activity

of

cells

would

facilitate

the

understanding

of

the

mechanisms

that

control

stem

cell

fate

through

interaction

with

stromal

cells

Electronic

supplementary

material

Below

is

the

link

to

the

Supplemental

data

Electronic

supplementary

material

-LRB-

XLS

kb

-RRB-

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

hematopoietic stem cells
stromal cells
coculture
microarray


MHC

class

I

A

region

diversity

and

polymorphism

in

macaque

species

The

HLA-A

locus

represents

a

single

copy

gene

that

displays

abundant

allelic

polymorphism

in

the

human

population

whereas

in

contrast

a

nonhuman

primate

species

such

as

the

rhesus

macaque

-LRB-

Macaca

mulatta

-RRB-

possesses

multiple

HLA-A-like

-LRB-

Mamu-A

-RRB-

genes

which

parade

varying

degrees

of

polymorphism

The

number

and

combination

of

transcribed

Mamu-A

genes

present

per

chromosome

display

diversity

in

a

population

of

Indian

animals

At

present

it

is

not

clearly

understood

whether

these

different

A

region

configurations

are

evolutionarily

stable

entities

To

shed

light

on

this

issue

rhesus

macaques

from

a

Chinese

population

and

a

panel

of

cynomolgus

monkeys

-LRB-

Macaca

fascicularis

-RRB-

were

screened

for

various

A

region-linked

variations

Comparisons

demonstrated

that

most

A

region

configurations

are

old

entities

predating

macaque

speciation

whereas

most

allelic

variation

-LRB-

-RRB-

is

of

more

recent

origin

The

latter

situation

contrasts

the

observations

of

the

major

histocompatibility

complex

class

II

genes

in

rhesus

and

cynomolgus

macaques

which

share

a

high

number

of

identical

alleles

-LRB-

-RRB-

as

defined

by

exon

sequencing

Introduction

Major

histocompatibility

complex

-LRB-

MHC

-RRB-

represents

a

multigene

family

that

plays

a

crucial

role

in

the

generation

of

adaptive

immune

responses

in

vertebrate

species

A

key

feature

of

the

system

is

that

some

of

its

genes

display

abundant

polymorphism

at

the

population

level

In

addition

the

number

of

Mhc

class

I

or

II

genes

may

differ

significantly

between

species

as

well

as

between

individuals

of

a

species

-LRB-

Kelley

et

al.

-RRB-

MHC

polymorphisms

may

have

a

profound

impact

on

several

features

such

as

disease

susceptibility

organ

transplantation

and

reproductive

success

-LRB-

Lagaaij

et

al.

Goulder

and

Watkins

Bontrop

and

Watkins

Ziegler

et

al.

Smith

et

al.

-RRB-

The

MHC

systems

of

various

primate

species

including

humans

have

been

studied

extensively

-LRB-

Watkins

Antunes

et

al.

Bontrop

et

al.

Adams

and

Parham

de

Groot

et

al.

Lafont

et

al.

Middleton

et

al.

Marsh

et

al.

Penedo

et

al.

Abbott

et

al.

Huchard

et

al.

-RRB-

For

example

the

MHC

of

the

rhesus

macaque

-LRB-

MhcMamu

-RRB-

an

Old

World

primate

species

has

been

shown

to

share

many

similarities

with

the

human

leukocyte

antigen

-LRB-

HLA

-RRB-

system

in

humans

The

evolutionary

orthologs

of

the

HLA-DP

DQ

and

DR

genes

are

also

present

in

rhesus

monkeys

and

as

in

humans

these

loci

are

polymorphic

-LRB-

Bontrop

et

al.

-RRB-

The

number

of

Mamu-DRB

region

configurations

appears

to

be

expanded

in

comparison

with

humans

and

some

of

these

regions

seem

to

harbor

an

extended

number

of

genes

-LRB-

Doxiadis

et

al.

-RRB-

Subsequent

cDNA

studies

have

illustrated

however

that

in

humans

and

rhesus

macaques

comparable

numbers

of

DRB

genes

are

transcribed

-LRB-

de

Groot

et

al.

-RRB-

Apart

from

Mamu-E

-LRB-

Knapp

et

al.

-RRB-

F

-LRB-

Otting

and

Bontrop

-RRB-

G

-LRB-

Boyson

et

al.

Castro

et

al.

-RRB-

and

AG

-LRB-

Boyson

et

al.

-RRB-

rhesus

macaques

also

possess

a

B-like

sequence

designated

Mamu-I

which

appears

to

be

present

in

each

haplotype

-LRB-

Urvater

et

al.

-RRB-

As

observed

in

humans

these

nonclassical

genes

display

low

levels

of

polymorphism

An

ortholog

of

the

HLA-C

gene

appears

to

be

absent

in

the

rhesus

macaque

whereas

evolutionary

equivalents

of

the

classical

HLA-A

and

B

genes

have

been

described

-LRB-

Boyson

et

al.

-RRB-

The

Mamu-A

and

B

genes

have

been

subjected

to

several

rounds

of

duplication

as

was

shown

by

genomic

sequencing

-LRB-

Daza-Vamenta

et

al.

Kulski

et

al.

-RRB-

Analysis

of

a

panel

of

rhesus

macaques

mainly

originating

from

the

Indian

subcontinent

illustrated

that

the

number

and

combination

of

Mamu-A

and

B

genes

that

are

expressed

per

haplotype

may

differ

at

the

population

level

-LRB-

Otting

et

al.

-RRB-

In

the

same

study

marked

differences

in

expression

levels

were

shown

At

this

stage

it

is

not

known

whether

rhesus

macaques

originating

from

other

geographic

areas

have

unique

Mhc

class

I

alleles

and/or

region

configurations

Comparative

studies

have

illustrated

that

many

Mhc

loci

and

lineages

predate

speciation

events

The

sharing

of

Mhc

alleles

between

two

primate

species

seems

to

be

a

rare

event

and

only

a

few

cases

have

been

documented

-LRB-

Cooper

et

al.

Evans

et

al.

-RRB-

An

exception

is

provided

by

rhesus

and

cynomolgus

macaques

which

seem

to

share

a

high

number

of

Mhc

class

II

alleles

as

was

defined

by

exon

sequencing

-LRB-

Blancher

et

al.

Doxiadis

et

al.

-RRB-

Whether

this

sharing

is

because

of

introgression

or

purifying

selection

remains

to

be

elucidated

Thus

far

about

Mafa-A

sequences

have

been

published

for

the

cynomolgus

macaque

-LRB-

Uda

et

al.

Krebs

et

al.

-RRB-

The

absence

of

pedigreed

material

did

not

allow

us

to

define

Mafa-A

loci

or

region

configurations

For

that

reason

a

panel

of

pedigreed

cynomolgus

macaques

was

incorporated

in

the

present

study

Comparison

with

rhesus

macaque

Mhc

class

I

sequences

obtained

from

different

populations

enabled

us

to

draw

conclusions

on

the

evolutionary

stability

of

A

region

variations

Materials

and

methods

Animals

and

cell

lines

The

Biomedical

Primate

Research

Centre

houses

a

self-sustaining

colony

of

approximately

rhesus

macaques

mainly

of

Indian

origin

that

have

been

pedigreed

based

on

the

segregation

of

serologically

defined

MHC

allotypes

-LRB-

Bontrop

et

al.

Penedo

et

al.

Doxiadis

et

al.

-RRB-

Furthermore

a

large

collection

of

DNA

samples

as

well

as

B-cell

lines

is

available

In

this

study

B-cells

derived

from

rhesus

macaques

of

Chinese

origin

and

from

cynomolgus

monkeys

were

used

to

isolate

RNA

Most

cynomolgus

monkey

samples

originate

from

a

pedigreed

group

housed

at

the

campus

of

the

University

of

Utrecht

cDNA

cloning

and

sequencing

RNA

was

isolated

from

B

cells

-LRB-

Rneasy

kit

Qiagen

-RRB-

and

subjected

to

One-step

reverse

transcriptase

polymerase

chain

reaction

-LRB-

RT-PCR

-RRB-

as

recommended

by

the

supplier

-LRB-

Qiagen

-RRB-

The

primers

-LRB-

MAS

-RRB-

AATTCATGGCGCCCCGAACCCTCCTCCTGG

and

-LRB-

MAS

-RRB-

CTAGACCACACAAGGCGGCTGTCTCAC

were

used

which

are

specific

for

class

I

A

transcripts

in

macaques

The

final

elongation

step

was

extended

to

min

to

generate

a

dA

overhang

The

RT-PCR

products

were

cloned

using

the

InsT/Aclone

kit

-LRB-

Fermentas

-RRB-

After

transformation

to

colonies

were

picked

for

plasmid

isolations

The

separate

Mamu-A

loci

show

differences

in

expression

levels

as

indicated

by

the

numbers

of

clones

picked

from

each

animal

The

alleles

with

high

expression

levels

-LRB-

majors

-RRB-

may

be

associated

with

serotypes

and

thus

involved

in

classical

antigen

presentation

whereas

those

with

low

expression

levels

-LRB-

minors

-RRB-

are

considered

nonclassicals

and

may

exhibit

more

specialized

types

of

functions

-LRB-

Otting

et

al.

-RRB-

Sequencing

reactions

were

performed

using

the

BigDye

terminator

cycle

sequencing

kit

and

samples

were

run

on

an

automated

capillary

sequencing

system

-LRB-

ABI

Genetic

Analyzer

-RRB-

as

has

been

described

previously

-LRB-

Otting

et

al.

-RRB-

Locus-specific

PCR

reactions

To

test

the

presence

of

Mamu

and

alleles

μl

of

the

RT-PCR

samples

was

taken

for

a

single-specific-primer

PCR

-LRB-

SSP-PCR

-RRB-

reaction

in

advance

of

the

cloning

step

The

relevant

primers

-LRB-

A

-RRB-

GGGACCCGACGGGCGCCTCCAA

and

-LRB-

A

-RRB-

GGCCCTCCAGGTAGACTCTGTC

have

annealing

sites

in

exon

Amplifications

were

carried

out

starting

with

min

at

°C

followed

by

cycles

at

°C

°C

and

°C

for

min

each

The

PCR

products

were

subjected

to

direct

sequencing

and

the

reactions

were

performed

as

described

above

Phylogenetic

analysis

and

nomenclature

The

sequences

were

analyzed

with

the

Sequence

Navigator

Software

version

-LRB-

Applied

Biosystems

-RRB-

and

alleles

are

based

on

at

least

three

clones

with

identical

full-length

sequences

To

define

loci

and

lineages

alignments

of

the

sequences

were

made

using

the

MacVector

version

-LRB-

Oxford

Molecular

Group

-RRB-

followed

by

manual

adjustments

Phylogenetic

analyses

on

the

full-length

-LRB-

bp

-RRB-

sequences

were

also

performed

with

the

MacVector

software

Neighbor-joining

trees

were

constructed

with

the

Kimura

parameter

method

Bootstrap

analyses

were

performed

based

on

replications

In

total

unreported

Mamu-A

and

Mafa-A

sequences

were

submitted

to

the

European

Bioinformatics

Institute

and

European

Molecular

Biology

Laboratory

-LRB-

EBI-EMBL

-RRB-

database

Relevant

information

such

as

accession

number

and

a

reference

cell-line

are

provided

-LRB-

Table

-RRB-

Moreover

all

novel

Mamu-A

and

Mafa-A

sequences

are

named

in

accordance

with

a

generally

accepted

nomenclature

proposal

-LRB-

Klein

et

al.

Robinson

et

al.

Ellis

et

al.

-RRB-

For

example

defines

a

Mhc

allele

in

the

rhesus

macaque

which

is

encoded

by

one

of

the

class

I

loci

namely

The

first

two

digits

after

the

asterisk

define

the

lineage

whereas

the

third

and

fourth

digits

define

the

allele

number

These

allele

numbers

are

arbitrary

as

they

reflect

the

order

in

which

the

alleles

were

discovered

A

fifth

and

sixth

digit

are

used

to

mark

a

synonymous

basepair

difference

between

two

sequences

Table

of

Mamu-A

and

Mafa-A

alleles

detected

in

this

Chinese

rhesus

macaques

are

indicated

by

Ri

numbers

Nine

alleles

found

in

rhesus

macaques

of

mixed

breeding

are

also

reported

Results

and

discussion

Polymorphism

and

diversity

of

the

Mamu-A

region

comparison

of

Chinese

and

Indian

rhesus

macaques

In

a

previous

communication

five

different

Mamu-A

region

configurations

were

defined

in

a

population

of

Indian

rhesus

macaques

-LRB-

Otting

et

al.

-RRB-

These

configurations

display

diversity

with

regard

to

the

number

and

combination

of

distinct

Mamu-A

genes

present

per

chromosome

The

loci

have

been

designated

and

respectively

-LRB-

Fig.

-RRB-

As

can

be

seen

each

region

configuration

comprises

a

gene

characterized

by

high

transcription

levels

-LRB-

major

-RRB-

combined

with

one

or

two

other

Mamu-A

genes

characterized

by

lower

transcription

levels

-LRB-

minors

-RRB-

The

gene

is

probably

responsible

for

executing

the

classical

antigen

presentation

function

-LRB-

Evans

et

al.

Sidney

et

al.

Sette

et

al.

-RRB-

whereas

the

others

can

be

considered

as

nonclassicals

and

may

interact

for

instance

with

the

KIR

gene

family

present

on

NK

cells

Two

complete

MHC

haplotypes

in

the

rhesus

macaque

are

sequenced

-LRB-

Daza-Vamenta

et

al.

Kulski

et

al.

-RRB-

and

they

represent

two-region

configurations

One

haplotype

contains

the

combination

whereas

in

the

other

the

pair

is

observed

Both

region

configurations

are

present

in

the

animals

of

Indian

origin

-LRB-

Fig.

-RRB-

Fig.

representation

of

different

Mamu-A

region

configurations

observed

in

Indian

rhesus

macaques

The

exact

order

and

physical

distances

of

the

loci

on

the

genome

are

still

unknown

The

relative

levels

of

polymorphism

and

transcription

of

the

loci

are

indicated

Two

Mamu-A

region

configurations

-LRB-

and

-RRB-

are

confirmed

by

the

sequencing

of

the

complete

rhesus

MHC

region

-LRB-

Daza-Vamenta

et

al.

Kulski

et

al.

-RRB-

Within

the

present

panel

of

Chinese

animals

different

full-length

Mamu-A

cDNAs

were

detected

that

can

be

grouped

into

various

loci

and

lineages

-LRB-

Fig.

-RRB-

A

complete

listing

of

the

alleles

detected

in

the

Chinese

macaques

is

provided

as

electronic

supplementary

material

-LRB-

Table

-RRB-

Because

of

codominant

expression

rhesus

macaques

can

be

heterozygous

for

the

gene

and

thus

can

express

up

to

two

allotypes

Nine

animals

were

found

to

express

three

different

sequences

These

animals

shared

one

of

these

sequences

in

separated

clades

in

phylogenetic

tree

-LRB-

Fig.

and

-RRB-

The

triplets

of

the

three

animals

are

indicated

by

asterisks

The

clustering

of

the

three

alleles

obtained

from

one

animal

in

clades

distinct

from

and

indicates

that

one

allele

belongs

to

a

separate

locus

The

newly

detected

loci

are

named

according

to

the

generally

accepted

nomenclature

system

-LRB-

Klein

et

al.

Robinson

et

al.

Ellis

et

al.

-RRB-

Representatives

of

two

loci

and

are

also

observed

in

cynomolgus

macaques

that

express

more

than

two

sequences

Fig.

tree

of

Mamu-A

and

Mafa-A

gene/alleles

detected

in

this

study

The

locus

is

represented

by

four

alleles

only

The

tree

is

based

on

full-length

sequences

although

analyses

on

only

exon

and

showed

no

significant

differences

The

nomenclature

of

the

new

alleles

is

based

on

a

more

extended

tree

-LRB-

not

shown

-RRB-

containing

all

the

Mamu-A

and

Mafa-A

alleles

now

available

Asterisk

three

alleles

of

animal

of

which

one

represents

the

locus

Double

asterisks

three

alleles

of

animal

of

which

one

represents

the

locus

Triple

asterisks

three

alleles

of

animal

of

which

one

represents

the

locus

Thirty-three

new

alleles

were

detected

in

the

Chinese

rhesus

macaques

Although

some

of

the

alleles

in

this

population

group

into

lineages

that

are

also

present

in

Indian

animals

sharing

of

alleles

for

the

locus

is

rare

and

was

observed

only

once

The

allele

is

detected

in

both

populations

whereas

the

and

for

alleles

are

highly

similar

and

differ

for

one

synonymous

basepair

substitution

Hence

it

is

concluded

that

most

allelic

polymorphism

observed

for

the

locus

was

probably

generated

after

the

rhesus

macaque

populations

were

separated

Sequence

comparisons

illustrated

that

most

of

the

variations

map

at

the

contact

residues

of

the

peptide-binding

site

-LRB-

data

not

shown

-RRB-

Thus

polymorphism

at

the

highly

divergent

locus

characterized

by

high

expression

levels

must

have

resulted

from

positive

Darwinian

selection

-LRB-

Hughes

and

Nei

Borghans

et

al.

-RRB-

The

gene

displays

differential

haplotype

distribution

in

the

Indian

population

of

rhesus

macaques

-LRB-

Fig.

-RRB-

This

also

appears

to

be

the

case

for

Chinese

animals

as

cDNAs

were

detected

in

out

of

of

them

In

this

panel

alleles

that

differ

from

the

ones

earlier

detected

in

Indian

animals

were

defined

In

the

Indian

population

low

expression

levels

characterize

the

gene

which

appears

also

to

be

the

case

in

Chinese

animals

Five

Chinese

macaques

possess

the

sequence

which

is

always

observed

in

combination

with

In

concordance

with

the

Indian

animals

the

number

of

clones

detected

also

reflects

low

expression

levels

A

full-length

cDNA

was

detected

in

only

three

animals

In

contrast

to

the

allele

in

the

Indian

macaque

the

Chinese

allele

has

a

stopcodon

at

the

last

triplet

of

exon

encoding

the

transmembrane

part

of

the

class

I

protein

This

suggests

that

the

corresponding

gene

product

may

settle

in

the

membrane

but

the

signal

transduction

is

impaired

As

was

observed

earlier

in

Indian

animals

this

locus

is

characterized

by

extremely

low

expression

levels

-LRB-

Fig.

-RRB-

For

that

reason

the

presence

of

cDNAs

in

the

other

animals

was

tested

with

SSP

on

the

RT-PCR

samples

and

out

of

Chinese

animals

appeared

to

be

positive

for

this

locus

The

and

genes

are

also

characterized

by

low

expression

levels

and

are

considered

to

represent

minors

In

contrast

to

the

previously

studied

Indian

population

the

Chinese

animals

were

selected

randomly

from

a

large

population

Because

of

the

lack

of

pedigree

data

and

segregation

profiles

it

was

not

possible

to

firmly

establish

the

segregation

of

different

Mamu-A

genes

present

per

chromosome

Haplotypes

can

only

be

deduced

based

on

sharing

of

sequences

between

animals

and

such

haplotypes

are

listed

in

Table

At

this

stage

it

is

impossible

to

determine

in

which

region

configurations

the

and

genes

are

present

Table

Mamu-A

haplotypes

deduced

by

sharing

of

alleles

grouped

into

two-region

configurations

Mafa-A

region

polymorphism

and

diversity

in

cynomolgus

monkeys

Sixty-two

alleles

were

detected

in

the

cynomolgus

macaques

analyzed

As

observed

in

the

rhesus

macaques

most

animals

had

two

polymorphic

sequences

in

combination

with

orthologs

of

the

and/or

genes

In

total

alleles

were

detected

illustrating

the

high

level

of

polymorphism

of

this

locus

These

alleles

were

compared

with

sequences

reported

earlier

by

two

other

research

groups

-LRB-

Uda

et

al.

Krebs

et

al.

-RRB-

that

have

used

different

nomenclature

systems

Only

four

alleles

and

detected

in

our

panel

are

identical

to

the

earlier

described

sequences

A

A

A

and

A

A

total

of

out

of

animals

have

at

least

one

sequence

and

alleles

could

be

distinguished

-LRB-

Table

-RRB-

The

oligomorphic

locus

is

also

present

in

the

pigtailed

macaque

-LRB-

Macaca

nemestrina

-RRB-

as

was

recently

described

-LRB-

Pratt

et

al.

Lafont

et

al.

-RRB-

In

these

studies

of

the

animals

possess

the

gene

and

lower

transcription

levels

were

also

observed

in

comparison

to

the

other

Mane-A

alleles

The

gene

is

observed

in

only

two

animals

whereas

the

locus

is

detected

in

individuals

Two

alleles

were

found

that

differ

in

only

one

basepair

-LRB-

nonsynonymous

-RRB-

substitution

-LRB-

Fig.

-RRB-

As

in

the

Chinese

rhesus

macaques

the

alleles

have

a

stopcodon

at

the

end

of

exon

The

and

alleles

were

detected

in

respectively

eight

and

four

animals

and

display

low

levels

of

polymorphism

Phylogenetic

analyses

illustrate

that

apart

from

loci

rhesus

and

cynomolgus

monkeys

also

share

lineages

-LRB-

Fig.

-RRB-

As

found

in

rhesus

macaques

the

alleles

are

characterized

by

high

transcription

levels

whereas

the

other

loci

display

moderate

or

low

expression

levels

Because

most

of

the

cynomolgus

macaques

are

pedigreed

the

combination

of

sequences

that

are

inherited

on

one

chromosome

could

be

defined

As

an

example

the

kinship

tree

of

one

breeding

group

is

provided

-LRB-

Fig.

-RRB-

The

haplotypes

are

grouped

based

on

the

combination

of

loci

-LRB-

Table

-RRB-

Seven

combinations

of

loci

or

region

configurations

are

recognized

-LRB-

Fig.

-RRB-

and

four

of

them

-LRB-

two

to

five

-RRB-

are

also

observed

in

the

Indian

rhesus

macaques

-LRB-

Fig.

-RRB-

which

indicates

that

they

predate

speciation

segregates

in

combination

with

and

but

not

exclusively

The

exact

region

configurations

of

containing

haplotypes

are

not

yet

known

Fig.

of

a

cynomolgus

macaque

family

showing

segregation

of

Mafa-A

alleles

The

animals

analyzed

are

indicated

by

shading

A

question

mark

indicates

that

the

sire

has

not

been

identifiedTable

haplotypes

as

defined

by

segregation

grouped

into

seven-region

configurations

region

configuration

appears

to

lack

the

gene

At

this

stage

it

is

unknown

whether

the

locus

is

really

absent

or

was

missed

because

of

PCR

failure

for

example

because

of

a

mutation

in

a

primer

site.Fig

representation

of

different

Mafa-A

region

configurations

in

cynomolgus

macaques

Four

configurations

-LRB-

-RRB-

are

shared

with

the

Indian

rhesus

macaques

-LRB-

Fig.

-RRB-

One

region

configuration

lacks

the

gene

It

is

not

yet

known

whether

the

locus

is

really

absent

or

missed

because

of

PCR

failure

Should

a

gene

be

found

in

extended

studies

than

this

region

configuration

has

to

be

deleted

from

the

list

Sharing

of

and

sequences

between

different

macaque

species

A

comparison

of

all

available

Mamu-A

to

Mafa-A

sequences

showed

that

six

full-length

cDNA

transcripts

are

shared

between

both

species

of

macaque

This

is

the

case

for

five

pairs

and

one

pair

-LRB-

Table

-RRB-

One

of

these

cynomolgus

monkey

alleles

is

identical

to

an

Indian

rhesus

allele

whereas

the

other

five

are

shared

with

animals

of

Chinese

origin

This

observation

was

unexpected

as

the

two

rhesus

macaque

populations

investigated

share

only

one

allele

The

fact

that

cynomolgus

macaques

share

more

alleles

with

Chinese

rhesus

macaques

than

with

Indian

individuals

may

be

explained

by

the

fact

that

there

is

an

overlap

in

the

geographic

areas

inhabited

by

both

species

in

Indochina

-LRB-

eastern

Asia

-RRB-

It

is

known

that

rhesus

macaques

and

cynomolgus

monkeys

can

interbreed

and

produce

offspring

-LRB-

Tosi

et

al.

-RRB-

Sharing

of

alleles

was

not

observed

for

the

minors

controlled

by

the

loci

Table

Mhc

class

I

sequences

detected

in

the

two

species

of

macaqueMacaca

mulattaMacaca

by

Krebs

et

al.

bPublished

by

Otting

et

al.

Different

modes

of

selection

operating

on

the

Mhc

class

I

and

II

genes

in

macaques

Although

Mamu-A

and

Mafa-A

sequences

are

interspersed

in

the

phylogenetic

tree

the

vast

majority

of

the

alleles

are

species

unique

and

for

rhesus

macaque

most

of

them

appear

to

be

population

specific

This

is

in

sharp

contrast

to

the

situation

observed

for

the

macaque

class

II

region

where

sharing

for

different

exon

sequences

at

the

Mhc-DR

but

especially

the

DQ

and

DP

genes

is

far

more

common

-LRB-

Blancher

et

al.

Doxiadis

et

al.

-RRB-

About

half

of

the

and

and

one

third

of

the

Mafa-DRB

sequences

are

identical

to

rhesus

orthologs

As

animals

of

the

same

populations

have

been

used

to

study

Mhc

class

I

and

II

sequences

the

present

results

exclude

the

possibility

that

this

high

sharing

of

Mhc

class

II

sequences

is

because

of

introgression

A

more

likely

explanation

is

that

in

macaques

the

exon

sequences

of

the

Mhc

class

II

genes

which

encode

the

peptide-binding

site

have

been

subjected

to

purifying

selection

As

a

consequence

many

Mhc

class

II

alleles

in

these

two

macaque

species

predate

speciation

processes

A

databank

search

indicated

that

the

phenomenon

also

extends

to

other

macaque

species

-LRB-

Robinson

et

al.

-RRB-

The

Mhc

class

I

alleles

in

macaques

are

largely

unique

illustrating

that

selection

has

favored

diversity

Mhc

class

I

proteins

are

involved

in

the

presentation

of

intracellular

pathogens

such

as

viruses

and

parasites

that

are

known

to

evolve

at

high

mutation

rates

Recognition

of

Mhc

class

I

molecules

combined

with

a

foreign

peptide

may

result

in

the

lysis

of

an

infected

cell

The

Mhc

class

II

molecules

select

peptides

originating

from

extracellular

pathogens

such

as

bacteria

and

fungi

for

binding

Mhc

class

II-mediated

activation

may

result

in

antibody

production

and/or

providing

help

to

cytotoxic

T

cells

The

present

results

illustrate

that

probably

because

of

coevolution

with

intracellular

pathogens

macaques

have

generated

a

highly

complex

and

divergent

Mhc

class

I

repertoire

The

fact

that

Mhc

class

I

alleles

evolve

quickly

even

within

a

species

has

been

documented

in

humans

for

instance

-LRB-

Belich

et

al.

Watkins

et

al.

-RRB-

The

high

degree

of

sharing

of

Mhc

class

II

sequences

seems

to

be

unique

for

macaques

and

has

not

been

observed

in

any

other

group

of

vertebrate

species

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

Table

Overview

of

Mamu-A

genes/alleles

in

the

panel

of

Chinese

rhesus

macaques

Sequences

represented

by

two

ciphers

-LRB-

for

example

A

-RRB-

are

not

confirmed

and

are

not

submitted

to

the

to

the

EBI-EMBL

database

-LRB-

PDF

KB

-RRB-

O O O O O O O O O O O O O O O O O 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 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 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 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 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 O O O O O O O O O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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