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The
Human
Marina
By
Interleukin-la
of Chromosome
Nicolas
Lafage,
Maroc,
Patrice
Yves
Interleukin-la
distinct,
cytokines
that
reactions,
that
play
and
Il-i
a
described
progenitor
a key
tissue
is
Dubreuil,
Is Located
on the
2 at Band
q13
Ren#{233}
de Waal
Carcassonne,
and
role
in inflammation,
repair.
identical
to
immunologic
Recently.
it has been
hematopoietin
1,
as a hematopoietic
cells in synergy
growth
with
factor
other
shown
which
was
acting on early
hematopoietic
C
I
NTERLEUKIN-1
(IL-l)
represents
a family
of polypeptide hormones
that
plays
a crucial
role in the body’s
response
to microbial
invasion,
inflammation,
immunologic
reactions,
and
have been
tric points
tissue
repair.’2
The
reported
in protein
(pi) ranging
from
at p1 5.0 and 7.0.
of two distinct,
nant species
the isolation
encoding
proteins
with
now referred
to as IL-la
biologic
activities
of IL-l
species
with different
4.0 to 8.0, with two
isoelecpredomi-
Cloning
and sequencing
but distantly
related,
growth
hybridization
that the IL-lf3 gene was located
some
2 at region
q 1 3-q2 1 . To
of the iL-la
location
situ
From
on
the Department
Cancer
Center
Ia Recherche
Centre
gene
hybridization
and
Medicale
de Recherche,
Dardilly,
June
Supported
by grantsfrom
du Rhbne
6, 1988;
Contre
Address
and
I 19 de l’Institut
(INSERM).
Submitted
reported
Cell
Marseille,
yet.
Using
in
cells
with
a
Biology,
Nationalde
France;
and
September
the Ligue
et de
Unicet,
des Bouches
le Cancer.
reprint
requests
I 19 and Regional
Cancer
to Marina
Center,
Lafage,
MD,
27, Bd LeI Roure,
INSERM
13009
U.
Marseile,
France.
The publication
charge
payment.
“advertisement”
indicate
costs
ofthis
article
This
article
must
in accordance
with
were defrayed
therefore
18 U.S.C.
be
section
in part
by page
hereby
1734
marked
solely
to
this fact.
© 1 989 by Grune
& Stratton,
probe,
arm
chromosomal
we report
that
ofchromosome
region
as the
IL-lfl
AND
the
IL-la
2 at band
gene
q13,
is
in the
gene.
METHODS
probes.
hybridization
IL-la
The
human
and
Northern
blot
preparations.
Normal
human
male
prometaphase
preboiled
ribonuclease
A (Sigma,
St Louis) in 2 x sodium
saline citrate
(SSC)
(0.3 mol/L
sodium
chloride-O.03
mol/L
trisodium
citrate, pH7) for 60 minutes at 37#{176}C,
rinsed three times in
2 x SSC, ethanol dehydrated,
and air dried. Chromosomal
DNA
was denatured
by immersion
of the slides in 70% deionized
formamide/2 x SSC for two minutes at 70#{176}C,
followed by quick rinsing in
cold (4#{176}C)
2 x SSC and dehydration
in ethanol.
In situ hybridization
was performed
according
to the procedure
described
by Mattei
et al with slight
modifications.”
The DNA
probe
was labeled by nick translation
using [3HJ dCTP and [3H]
dTTP (Amersham,
Buckinghamshire,
UK) to a specific activity of
6 x l0 cpm/g
and separated
from unincorporated
nucleotides
on a
Sephadex
G-50 column
in 2 x SSC. Ethanol
precipitation
was
performed
with a 2,000-fold
excess of sonicated
salmon sperm DNA.
The radiolabeled
DNA probe was resuspended
at a final concentration of 100 ng/mL
in hybridization
buffer
(50% deionized
formamide, 2 x SSC, 1 x Denhardt’s
solution,
10% dextran sulfate, 0.1%
sodium dodecyl sulfate (SDS), 40 mmol/L
sodium phosphate,
final
pH 7).
1. 1988.
Departementale
cDNA
ig/mL
France.
accepted
IL-la
on the long
Inc.
spreads were obtained
from amethopterin-synchronized
peripheral
blood lymphocyte
cultures.’#{176}At the release period, 5-bromodeoxyuridine (l0
mol/L) was used to ensure good quality posthybridization chromosomal
banding. Slide preparations
were treated with 100
Regional
Ia Sante
P#{233}busque,
cDNA probe used for both in
analysis was a 1.7 kb Hind III
fragment
(nucleotides
236-2033)
excised
from the recombinant
plasmid
pCD-IL-ia
provided
by Dr N. Arai (DNA-X
Research
Institute, Palo Alto, CA).9
The human IL-1$ cDNA probe used for Northern
blot analysis
was a 0.8 kb Pst I fragment
isolated
from the IL-Ifl-SP6
plasmid
provided
by C. Vaquero
(INSERM
U.152,
Paris),
which corresponds
to the complete
coding sequence
of human IL-I/3.4
of chromothe precise
prometaphase
& Stratton,
located
Chromosome
has not been
ofHematology
Unite
Grune
human
situ
revealed
on the long arm
our knowledge,
human
by
DNA
led to
genes
chromosomes
Marie-Jos#{234}phe
MATERIALS
IL-i
activity.34
These
proteins
are
(p1 5.0, 17 Kd) and IL-i/3
(p1 7.0,
factors.57
on human
Mabefijt,
Arm
Mannoni
1989
same
17 Kd). Recently,
it has been shown that IL-la
is identical
to
hemopoietin
1 , which is described
as a hematopoietic
growth
factor,
acting
on early progenitor
cells in synergy
with other
hematopoietic
In situ
Patrice
Long
growth
factors.
In this report we discuss our use of in situ
hybridization
on human prometaphase
cells with a human
IL-i a eDNA probe to localize the human IL-i a gene on the
proximal
part of the long arm of chromosome
2 at band
q13. in the same chromosomal
region as the IL-lfl gene.
and interleukin-1$
(lI-18)
are two
but distantly
related,
polypeptidic
(IL-la)
biochemically
Gene
Inc.
0006-4971/89/7301-0038$3.00/0
Lt)
I
z
:j:s
iuEE
1j1j1
thbu
(I1t
U-
C
U-
20
Fig 1 .
grains
=
over
of silver
all
somes in 86 prometaphases
z
ciiiixui
‘0
104
Distribution
scored
II
l2
:
:.n..
j
IS
14
IS
lb
7
5
9
20
(lii’
71
aiiiiciii
22
X
Blood,
ta#{243}
in situ
V
hybridization
with
chromo-
after
the
laprobe.
Vol 73, No 1 (January),
1989:
pp 104-107
II-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
LOCALIZATION
OF HUMAN
IL-la
GENE
AT 2Q13
105
at 4#{176}C
for 27 days. Autoradiographs
were then developed.
errors due to slipping of silver grains during the banding
procedure,
chromosomes
were first stained
with buffered
Giemsa
solution. Only complete,
well-spread
prometaphases
with at least one
chromosome-associated
silver grain were photographed.
R-banding
was then performed
by a modification
of the fluorochrome-photolysis-Giemsa
(FPG)
method,’2
and the same prometaphases
were
rephotographed,
thus allowing the identification
of the chromosomal
bands. Chi-square
(x2) analysis,
which tests the hypothesis
that
labeling is random on all chromosomes,
was used. The expected
number of grains was based on the relative
DNA content of each
chromosome,
according
to Mendelsohn
et al.’3
and stored
..
To avoid
..
..
.
..
.
Northern
blot
analysis.
Total
RNA
human cell lines (5637, HL6O, and
isothiocyanate
as described.’4
RNA
.
.
.
.
.
..
2
Fig 2.
Localization
of the human
Il-la
ization.
Diagram
of G-banded
chromosome
distribution
of labeled
sites.
gene by in situ hybrid2 showing
the detailed
The DNA probe was denatured
by incubation
at 70#{176}C
for 15
minutes,
quickly cooled in ice, applied to the chromosome
preparations, and hybridized
for 17 hours at 42#{176}C.
Slides were washed in
successive
changes
to remove
nonspecifically
bound DNA:
three
times in 50% formamide/2
x SSC for ten minutes at 39#{176}C,
three
times in 2 x SSC for ten minutes
at 39#{176}C,
and three times in 2 x
SSC for ten minutes
at room temperature.
Finally, slides were rinsed
in 0. 1 x SSC for one hour and 30 minutes
at room temperature
and
for 30 minutes at 4#{176}C.
After dehydration
in ethanol, the slides were
coated with NTB2
photographic
emulsion
(Kodak,
Rochester,
NY)
Fig 3.
Three
partial
representative
chromosome
spreads
showing
the specific
location
of
IL-i a on chromosome
2. Top: Arrowheads
indicate
silver
grains
on Giemsa-stained
chromosomes.
after
autoradiography.
Bottom:
Chromosomes
with silver grains (arrowheads)
were subsequently
identified
by R-banding.
was
prepared
from
three
by lysis in guanidine
were resuspended
in
diethylpyrocarbonate-treated
H20.
Aliquots
(25 tg) of total
RNA
were loaded in horizontal
1% agarose gels containing
10% formaldehyde, submitted to electrophoresis,
and transferred
to nylon filters.’3
The probes were labeled in vitro by random
priming.’6
Prehybridization was performed
in 50% deionized formamide,
1 x Denhardt’s
solution,
0.1% SDS, 5 x sodium saline phosphate
EDTA (SSPE)
buffer (0.05 mol/L sodium phosphate
containing
0.9 mol/L
NaCI
and 0.4 mol/L
ethylene
diamine
tetraacetic
acid (EDTA),
pH 7.7),
and 200 g/mL denatured
calf thymus DNA. Blots were hybridized
at 42#{176}C
for 24 hours in the same buffer additionally
containing
I x
106 cpm/mL
denatured
probe. Blots were washed
at room temperature twice in 2 x SSC, 0.1% SDS for 30 minutes and once in I x
SSC,
0.1% SDS for 30 minutes.
They were then exposed to X-ray
films at -80#{176}C
with intensifying
screens.
RESULTS
Results
of in situ
probe
are shown
cells were analyzed
AND
hybridization
GCT)
pellets
DISCUSSION
with
the
in Figs 1 and 2. Eighty-six
for silver grains
associated
IL-la
cDNA
prometaphase
with chromo-
somes.
A total of 1 96 silver grains
was scored
(ie, an average
of 2.3 grains
per cell), and 47 grains
(24% of the total grains)
were
found
to be associated
with
chromosome
2 (x2,
p < l0).
Of the 47 silver grains
associated
with chromosome 2, 28 grains
(60%)
mapped
to region
2q1, the proximal
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
LAFAGE ET AL
106
Fig 4.
Northern
blot analysis of IL-la and
lI-l$
expression
in human cell lines. Duplicate
Northern
blots.
prepared
as described
in Materials
and Methods. were hybridized with the IL-la (left
panel) and lI-ift (right panel) probes. The different
lanes contained: marker DNA fragments (lambdaHind Ill and 4xl 74 replicative form-Hae III. lane M).
RNA extracted from 5637 (lane 1). HI 60 (lane 2).
stimulated
HL 60
(3 x 10
mol/I
TPA
for
two
hours. lane 3), and GCT cells (lane 4).
region
ofthe
precisely,
long
at the 2q 1 3 band
significant
cluster
regions
gene
arm
ofchromosome
the maximal
(Fig
number
2
of silver
(x2.
p
i0).
<
grains
was
More
I). Consequently,
we assigned
2 at band
the human
it did
IL-ia
q 13.
the
long
arm
of chromosome
The human
IL-lf. gene has been localized
on chromosome
2 at region
q13-q21,8
and we report
that the IL-la
gene is
located
in the same chromosomal
region.
These two genes are
chromosomal
region as the
these two genes are related4
distantly
the
related,
reveals
and
a comparison
45% homology.4
However,
of their
the
highest
used
in these
experiments.
sequences
of the IL-la
In addition,
probe
and the
found
41%
that
they
patches
scattered
one should expect
and
the
share
IL-lfl
hybridization
(see
confirm
the specificity
blot
human
homology,
all over the coding
no cross-hybridization
situ
Northern
selected
sequences
percentage
is found in the 5’ portions
of the cDNAs,
1-235)
absent
from the IL-la
cDNA
homology
(nucleotides
probe
coding
gene
using
Materials
of the
sequences.
between
transcript4
cells (Fig
the
and
to
same
cell-surface
possibility
for
I .8 kb IL-lfl
a gene
duplication
2 at
our
the two
event.4
band
ql3,
in the
same
IL-l
It has
data
additionally
molecules
recently
were
been
support
produced
shown
by
that
the
genes encoding
two other
cytokines,
GM-CSF
and IL-3,
previously
localized
on the same chromosomal
region,’9’2#{176}are
in fact tandemly
arrayed,
separated
by only 9 kb.2’ Whether
the
IL-la
remains
and
IL-1f3
genes
are
close
at the
molecular
level
to be determined.
ACKNOWLEDGMENT
in
Methods).
To further
probe,
we performed
from
detected
the
IL-ifl
gene. Keeping
in mind that
and that their products
share the
receptor,’8
that
Therefore,
the IL-ia
conditions
analysis
on RNAs
extracted
cell lines.
The IL-la
probe
expected
2.2 kb IL-la
giant cell tumor
(GCT)
a portion
probe we
corresponding
stringent
and
IL-la
of
we compared
IL-1f3
cDNA
not detect
transcript4
expressed
in these
cells
and
in tetradecanoyl
phorbol
acetate
(TPA)-stimulated
HL 60 cells (Fig 4, right
panel,
lanes 1, 3, and 4, respectively).
In conclusion,
we have assigned
the human
IL-la
gene to
(Figs 2 and 3). There
were no other highly
of silver grains
in the other
chromosomal
to chromosome
In contrast,
respectively).
observed
three
the
in 5637 cells,’7 as well as in
4, left panel,
lanes 1 and 4,
(DNAX
Research
Institute,
Palo
Alto, CA) and C. Vaquero
(INSERM
U.152, Paris, France)
for
providing
us with the human IL-Ia and fi cDNA probes; to Drs A.
Hagemeijer
and C. Mawas
for helpful
discussions;
to Drs D.
Birnbaum,
F. Birg, and C. Lipcey for reviewing
the manuscript;
to I.
Lantin and M. Girond for editorial
assistance,
and to J. Ingrand,
M.
Gum, and J. Caramelli
for the illustrations.
We are grateful
to Drs N. Arai
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1989 73: 104-107
The human interleukin-1 alpha gene is located on the long arm of
chromosome 2 at band q13
M Lafage, N Maroc, P Dubreuil, R de Waal Malefijt, MJ Pebusque, Y Carcassonne and P Mannoni
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