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Author(s)
CHROMOSOMAL ASSIGNMENT OF HUMAN GENES
FOR GASTRIN, THYROTROPIN (TSH)-β SUBUNIT AND
C-erbB-2 BY CHROMOSOME SORTING COMBINED
WITH VELOCITY SEDIMENTATION AND SOUTHERN
HYBRIDIZATION
Fukushige, Shinichi
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http://hdl.handle.net/11094/36004
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Osaka University
Osaka University
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 477-483
Vol. 134, No. 2,1986
January 29,1986
CHROMOSOMAL ASSIGNMENT OF HUMAN GENES FOR GASTRIN, THYROTROPIN (TSH)-~
SUBUNIT AND C-erbB-2 BY CHROMOSOME SORTING COMBINED WITH VELOCITY
SEDIMENTATION AND SOUTHERNHYBRIDIZATION
Shinichi Fukushige, Tomoaki Murotsu and Kenichi Matsubara
I n s t i t u t e for Molecular and C e l l u l a r Biology,
Osaka U n i v e r s i t y , Yamada-oka, Suita 565, Japan
Received November 15, 1985
Human genes for g a s t r i n , thyrotropin (THS)-B subunit and c-erbB-2 were
assigned to s p e c i f i c chromosomes using a s i n g l e - l a s e r cell s o r t e r .
For
this purpose, condensed human chromosomes prepared from a k a r y o t y p i c a l l y
normal lymphoblastoid cell l i n e were p r e l i m i n a r i l y fractionated by v e l o c i t y
sedimentation, and then sorted using a fluorescence-activated cell s o r t e r .
DNA was then extracted from the chromosomes, cleaved by r e s t r i c t i o n
enzymes, and subjected to Southern hybridization
using gene-specific
radioactive probes. When the assignment of specific chromosomes was not
possible due to chromosomal size overlapping, sorted chromosomes from cell
lines carrying chromosomal translocation or from hybrid cells carrying
known human chromosomes were used in addition. The results indicate that
human genes for gastrin, TSH-~, and c-erbB-2 are located on chromosomes 1 7 ,
1 and 17, r e s p e c t i v e l y .
® 1986A e a d e m i e P . . . . . Inc.
Chromosomal gene mapping is becoming increasingly important in basic,
as well
either
as applied research in biology and medicine.
by
in
situ
hybridization
(I)
or
It
is usually done
by discordance
analysis
using
somatic hybrid cell panels (2).
Both techniques
present some drawbacks,
requires time to obtain a decisive
since
conclusion,
in
situ
hybridization
and the hybrid cell
panel
method is subject to inconsistent results mostly due to the u n s t a b i l i t y of
human chromosomes. In contrast
cell
sorter
to
fractionate
to these two techniques, analysis
chromosomes (3
7) seems
using a
to
be the most
chromosomes
can not be
powerful method for rapid and correct gene assignments.
However, with
sorted out
sorting
into
a single-laser
individual
sorter,
components. We have overcome this
chromosomes from cell
Abbreviations:
cell
problem by
lines that carry translocated chromosomes or
TSH, t h y r o t r o p i n ;
base pair.
NaDodSO4 , sodium dodecylsulfate;
bp,
0006-291X/86 $1.50
477
Copyright © 1986 by Academic Press, Inc.
All rights of reproduction in any form reserved.
VOI. 134, No. 2, 1986
from
human-rodent
Southern blot
BIOCHEMICAL
AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
hybrid
analysis
cells
using the
that
sorted
carry
known human chromosomes.
chromosomes (3,
4),
rather
than
b l o t t i n g d i r e c t l y the chromosomes (5 - 7) greatly improved the accuracy of
the assignments.
MATERIALS AND METHODS
I.
Cell lines and chromosome preparation
Two human lymphoblastoid cell lines GMOI31: 46, XX and GM3197: 46, XX,
t(17;22)
(17qter-17p13::22qll-22qter;22pter-22qll::17p13-17pter)
(8) were
obtained from "The Human Genetic Mutant Cell Repository (Camden, NJ)".
These cell lines were grown in RPMI I~40 medium supplemented with 10% fetal
c a l f serum under 5% C09/95% a i r at 37 C.
. .
Human-mouse hybrid c e l l s TAIA and TA3A (9) were kindly provided by Dr.
N. Schimizu at Keio U n i v e r s i t y .
TAIA carries human chromosomes I , 4, 7,
13, 15, 18, 19 and TA3A carries human chromosomes 3, 13, 14. These hybrids
were grown in Dulbecco's modified Eagle's medium supplemented with 10%
fetal c a l f serum.
The c e l l s were then incubated for 16 hrs
in the
presence of colcemid (O.2~g/ml).
Chromosome suspensions were prepared
according to the polyamine method of S i l l a r and Young (I0) with some
modifications:
The cells containing condensed chromosomes were harvested
by c e n t r i f u g a t i o n , resuspended in 75mM KCI, and kept on ice for 30 min.
They were then collected by c e n t r i f u g a t i o n
for I0 min, washed with
polyamine buffer ( I 0 ) , resuspended in the same buffer containing 0.1%
d i g i t o n i n , followed by vigorous a g i t a t i o n for 1 min using a Vortex mixer to
release chromosomes.
2.
Velocity sedimentation and sorting
The chromosome suspensions (50ul) from 5 x 105 mitotic c e l l s were
layered over a 16 ml l i n e a r sucrose gradient (5 - 30%, W/V) in po~yamine
b u f f e r , centrifuged using a swing bucket at 900 rpm for 30 min at 4 C, and
then fractionated into sixteen 1 ml a l i q u o t s .
To each f r a c t i o n , ethidium bromide (50ug/ml) was added and the stained
chromosomes were sorted using a fluorescence-activated cell sorter CHROSS 1
(Japan Spectroscopic Co., LTD.) monitored by the MULTI 16 computer
(Mitsubishi E l e c t r i c , Tokyo).
For e x c i t a t i o n , a Spectra-Physics model
2025-05 laser at 488 nm/3OOmW was used. The flow rate of sorting was I000 2000 chromosomes/sec.
The preliminary f r a c t i o n a t i o n by sucrose gradient
sedimentation f a c i l i t a t e d rapid sorting.
To obtain m~ningful signals in
Southern blot analysis of chromosomal DNA, about 4 x I0 sorted chromosomes
were used.
3.
DNA extraction
he sorted chromosomes were collected by c e n t r i f u g a t i o n at
5 mi .
DNA was prepared by incubating the samples for 5 hrs
extraction buffer (75mM NaCI, 24mM EDTA, lOmM Tris.HCl, pH 7.5)
proteinase K (lOOug/ml), yeast c a r r i e r tRNA (lO~g/ml) and I%
followed by phenol extraction and ethanol p r e c i p i t a t i o n ( I I ) .
~
14 krp~ for
at 37UC in
containing
NaDodSO4 ,
4.
DNA probes
The following probes were used: human g a s t r i n , a 260 bp H i n d l l l H i n d l l l fragment from cDNA (12); human TSH-B, a 210 bp EcoRV-Rsal fragment
from genomic DNA (13); human c-er___bbB-2, a 440 bp Kpnl-Xbal fragment from
genomic DNA (14).
5.
Southern hybridization
The DNA from sorted chromosome was digested with EcoRl ( g a s t r i n , TSHB) or Pvull (c-er___bbB-2), placed in 2mm slots in a 0.7% agarose gel and
478
Vol. 1 3 4 , No. 2, 1 9 8 6
B I O C H E M I C A L A N D B I O P H Y S I C A L RESEARCH C O M M U N I C A T I O N S
elect~phoresed. B l o t t i n g onto n i t r o c e l l u l o s e f i l t e r s 8and h y b r i d i z a t i o n
with
P-labeled DNA probe ( s p e c i f i c a c t i v i t y 2 - 5 x lO cpm/ug) were done
as described by Southern (15).
RESULTS AND DISCUSSION
TO sort chromosomes, metaphase chromosomes in a cell
which
has
a
normal karyotype
(46,
XX)
lysate of GMOl31
w e r e fractionated
by
velocity
sedimentation through sucrose (7, 16), and each f r a c t i o n was then subjected
to
flow s o r t i n g .
histograms.
Fig.
l
shows r e l a t i o n s
between sucrose
fractions
and
By combining sedimentation with flow s o r t i n g , the sorting rate
increased by a factor of 5 - lO.
1.0
Eight chromosome fractions (A to H:
ii ~,i
i i
i~4
i i
i6i
18-16
i
i
i
i
i
ilo)
see
>z
0.5
S
o
o
m
(J o . c
10
5
Fraction
:~
::
i12-9
i
15
Number
i
!
i
i
:
. i ~ 8 - ! 6 1 $ ) ........... ) ........i
I'~
<aX:
7,~. . . .....i
i
+
12-9
i
i
::
i
i
! 71
J122~
i
.......i........
. . . . . . . . .
..............i
:: 15-13!
8:;J ........i -~
::
::
L
) 8i
::
..... i........ i..... i ....... i..._~,_.L
i i i i
!
i
i
i
[
!
i
!
:3
s
i
.................
i ........ ¸
Figure I .
Sedimentation profile (Panel A) and flow histograms of human
chromosomes prepared from the cell line GMOl31. Chromosomes were
preliminarily separated into 7 fractions
(5 to l l )
by velocity
sedimentation through sucrose.
Each fraction was mixed with ethidium
bromide and analysed by a fluorescence-activated cell sorter,
ordinate:
relative number of chromosomes; abscissa: relative fluorescence intensity.
Panel B is the histogram from an unfractionated sample. Panels 5 - II show
histograms of the corresponding sucrose fractions.
Numbers in the
histograms represent the positions of human chromosomes.
For example,
sucrose fraction 5 is enriched with chromosomes l , 2 and 3 to 5.
479
Vol. 134, No. 2, 1986
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
GM0131
A
B
I
!
H G
i 12-9
F E
D
C
B
A
T
ORIGIN
23Kb
2,
,~-i~
9.4
~i ..... ::.....
6.5
4.3
H'-I't'I °° I"
"
GM3197
D
C
h
g
f
•
d
¢
b
•
-.4
t (17;22
t(17;22)
"1' '1
Figure 2.
Assignment of human gastrin gene to chromosome 17.
(A) Flow
histogram of normal human chromosomes from the cell line GMOI31. Numbers
in the figure represent the positions of human chromosomes. Fractions A to
H were separately obtained as described in the text.
(B) DNA was prepared
from the sorted chrom~.omes, digested by EcoRI, and subjected to Southern
blot analysis using
P-labeled human gastrin gene (12) as a probe. Lane
T: total human lymphocyte DNA. Lanes A to
H; DNA from each sorted
chromosome fraction. The arrow indicates the position of the DNA fragment
hybridizing to the probe. Positions of marker DNAs using Hindlll-digested
phage DNA are shown on the l e f t . The gastrin gene is in fraction F that
contains chromosomes 16, 17, 18. (C) Flow histogram of chromosomes from a
human cell l i n e GM3197 that carries a reciprocal translocation t(17;22).
Chromosomes were sorted into fractions a to h as shown at the bottom of the
histogram. The arrow head indicates the position of the t(17;22)(17qter17p13::22qll-22qter) chromosome. (D) DNA was prepared, cleaved with EcoRl,
and subjected to Southern blot analysis as in (B). The gastrin gene is in
fraction e that contains chromosomes 13, 14, 15, t(17;22) as well as in
fraction f that contains chromosomes 16, 17, 18. Therefore, chromosome 17
should carry the gastrin gene.
Fig.
2A) were obtained in a two-day o p e r a t i o n and were used in one f i l t e r
assay.
DNA was e x t r a c t e d
appropriate
restriction
h y b r i d i z a t i o n which gives
from
each
enzymes,
chromosome f r a c t i o n ,
and
then
unambiguous s i g n a l s ,
480
analysed
digested
by
as compared to
with
Southern
spot
blot
Vol. 134, No. 2, 1986
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
analysis (5 - 7).
The f i l t e r
was hybridized to nick-translated human genes
including g a s t r i n , TSH-B or c-erbB-2.
The
gastrin
probe
showed a
positive
signal
in
fraction
corresponds to the pool of chromosomes 16, 17 and 18 (Fig.
next
step,
similarly.
chromosomes from
This cell
translocated
another
cell
carries a reciprocal
line,
translocation
fractions
e and f
demonstrate that the human gastrin
the
same way, a c e l l u l a r
(Fig.
In the
sorted
t(17;22)
is
f r a c t i o n e that contains chromosomes 13, 14 and 15 (Fig.
in
2B).
GM3197 were
chromosome, (17qter-17p13::22qll-22qter)
DNA showed signals
F that
and the
collected
2C).
in
Analysis of
2D). These results
clearly
gene is located on chromosome 17.
In
oncogene, c-erbB-2 was mapped on chromosome 17
(data not shown).
The human TSH-~ gene probe hybridized to fraction A containing sorted
chromosomes from GMOI31 c e l l s .
In
the
next
step,
This f r a c t i o n contains chromosomes 1 and 2.
we used a
contains human chromosome I ,
human-rodent
but not 2.
human chromosomes 1 and 2 (see
Southern blot
detected
hybrid
in
cell
analysis
the
(Fig.
3B).
corresponding
TA3A that
Fig.
does
hybrid
The fraction
3A)
In this
translocated
case, positive
contain
that corresponds to
obtained
were detected
line
by
signal
was
another
1
or
2.
GM3876 that carries
supported
in
signal
from
human chromosomes
chromosomes t ( l ; 2 0 )
signals
TAIA that
no positive
chromosome f r a c t i o n
Independent studies on chromosomes from the cell
reciprocally
line
showed a positive
In contrast,
not
cell
this
conclusion.
two f r a c t i o n s ,
one that
contains normal human chromosomes
1 and 2, and another that contains the
translocated
shown).
chromosomes (data not
These results
indicate
that
human TSH-B gene is located on chromosome I .
At
present,
hybrid c e l l s .
with
a panel
sorting
most of
In p r i n c i p l e ,
of
hybrid
the
gene mapping
studies
are
performed
using
chromosome sorting is unnecessary when working
cells.
However,
technique is more powerful
we found that
the
since in many hybrid cell
chromosome
lines,
human
chromosomes are unstable, r e s u l t i n g in frequent loss of the o n c e - i d e n t i f i e d
481
Vol. 134, No. 2, 1 9 8 6
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
T
T
A
A
3
o~
.Q
A
E
0 R I G I N --
1
A M
T
H
G
F
E
D
C
B A
~,~~mF:~
>
6 : 5 --
4.3~
~
E
o
2.3-2.0--
Relative
Fluorescence
Figure 3.
Assignment of human TSH-B gene to chromosome I .
(A) Flow
histogram of chromosomes from a human-mouse hybrid cell TAIA that carries
human chromosome l ,
but not 2.
TAIA carries, in addition, human
chromosomes 4, 7, 13, 15, 18, 19. The chromosome fractions corresponding
to the position for human chromosomes l and 2 (shown by a horizontal arrow)
were po3e2!ed. (B) EcoRl analysis of the DNA from sorted chromosome samples
using
P-labeled human TSH-B gene (13) as a probe. Lanes A to H contain
DNA samples prepared in the same way as those shown in Fig. 2.
Human
chromosomes l and 2 ( i f they are present) in TAIA and TA3A, as recovered
from the fraction shown by an arrow in (A), were analysed s i m i l a r l y . Lanes
T and M: total human DNA and total mouse DNA, respectively.
An arrow
indicates the position of the fragment which hybridizes to the human TSH-B
gene probe. Thus, the TSH-B gene must be located on chromosomeI .
chromosomes or in
fact,
frequent translocations to rodent chromosomes (6).
we tested 7 hybrid cell
Therefore,
when hybrid
corresponding
lines,
cells
and obtained inconsistent r e s u l t s .
are
chromosomal f r a c t i o n
In
used,
the
using a cell
purification
sorter
greatly
of
the
helps
to
reduce the complexity of these problems.
Recently, further development of sorting techniques using a dual-laser
cell
sorter
has made i t
chromosomal types
(6).
possible
to
Our technique,
sort
viz.
chromosomes into
combination
of
21
unique
preliminary
assignment with chromosome sorting from translocated or hybrid cell
lines,
is another practical way to assign genes to chromosomes.
ACKNOWLEDGEMENTS
We thank Dr. N. Shimizu (Keio U n i v e r s i t y ) for providing us with human
-mouse hybrid cell l i n e s , Dr. T. Yamamoto (Tokyo U n i v e r s i t y ) , Dr. K. Kato,
and Dr.
Y. Hayashizaki for providing us with human c-erbB-2, g a s t r i n , and
TSH-~ gene probes,
respectively.
This work was supported in part by
s c i e n t i f i c grants from the M i n i s t r y of Education, Science and Culture of
Japan.
482
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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