Isolation and characterization of a stage-specific transforming gene, Tlym-I, from T-cell lymphomas

Abstract

A cellular transforming gene detected by transfection of mouse T-cell lymphoma DNA has been isolated by molecular cloning. This gene (designated Tlym-I) is homologous to a small conserved family of sequences present in normal mouse and human DNAs but is not related to any of the previously described viral or cellular transforming genes.

Full text

Proc.
Natl.
Acad.
Sci.
USA
Vol.
81,
pp.
2227-2231,
April
1984
Medical
Sciences
Isolation
and
characterization
of
a
stage-specific
transforming
gene,
Tlym-I,
from
T-cell
lymphomas
(gene
family)
MARY-ANN
LANE*t,
ADRIENNE
SAINTEN*,
KEVIN
M.
DOHERTY*,
AND
GEOFFREY
M.
COOPERtt
Laboratories
of
*Molecular
Immunobiology
and
of
WMolecular
Carcinogenesis,
Dana-Farber
Cancer
Institute,
and
tDepartment
of
Pathology,
Harvard
Medical
School,
Boston,
MA
02115
Communicated
by
Matthew
S.
Meselson,
December
29,
1983
ABSTRACT
A
cellular
transforming
gene
detected
by
transfection
of
mouse
T-cell
lymphoma
DNA
has
been
isolated
by
molecular
cloning.
This
gene
(designated
Tlym-I)
is
ho-
mologous
to
a
small
conserved
family
of
sequences
present
in
normal
mouse
and
human
DNAs
but
is
not
related
to
any
of
the
previously
described
viral
or
cellular
transforming
genes.
Activated
cellular
transforming
genes
have
been
detected
by
transfection
of
NIH
3T3
cells
with
DNAs
from
avian,
ro-
dent,
and
human
neoplasms
of
a
variety
of
cell
types
(re-
viewed
in
ref.
1).
In
some
cases,
activated
transforming
genes
have
been
found
to
be
members
of
the
ras
gene
family.
Three
different
members
of
this
gene
family
(rasH,
rasK,
and
rasN)
have
been
detected
as
activated
transforming
genes
in
a
small
fraction
(10-20%)
of
multiple
different
types
of
tu-
mors,
including
carcinomas,
sarcomas,
neuroblastomas,
lymphomas,
and
leukemias
(2-13).
Activation
of
ras
genes
therefore
does
not
appear
to
correlate
with
either
tissue
type
or
stage
of
differentiation
of
neoplasms.
In
contrast,
we
previously
reported
detection
of
three
dif-
ferent
stage-specific
transforming
genes
within
the
B-lym-
phoid
lineage
and
two
additional
stage-specific
transforming
genes
within
the
T-lymphoid
lineage
(14).
Each
of
these
genes
was
activated
in
80-100%
of
B-
or
T-cell
neoplasms
representative
of
the
same
stage
of
differentiation
(14-17).
Activation
of
these
transforming
genes
thus
appeared
to
rep-
resent
highly
regular
and
specific
events
in
the
development
of
lymphoid
tumors.
One
such
gene,
Blym-J,
has
been
isolated
from
the
chick-
en
bursal
lymphomas
(18)
and
human
Burkitt
lymphomas
(17),
which
are
representative
of
the
intermediate
stage
of
B-
cell
differentiation.
Sequence
analysis
of
chicken
Blym-1
has
indicated
it
is
unusually
small
[0.6
kilobase
(kb)]
and
encodes
a
protein
that
shares
partial
homology
with
the
amino-termi-
nal
region
of
members
of
the
transferrin
family
(18).
Hybrid-
ization
studies
have
indicated
that
Blym-J
is
a
member
of
a
family
of
six
to
eight
genes
and
that
this
gene
is
not
homolo-
gous
to
previously
identified
cellular
transforming
genes,
in-
cluding
members
of
the
ras
family
(17,
18).
We
report
here
the
isolation
and
initial
characterization
of
a
second
stage-specific
transforming
gene,
Tlym-I,
from
a
T-
cell
neoplasm
representative
of
an
intermediate
stage
of T-
lymphocyte
differentiation.
Tlym-I
is
not
homologous
to
pre-
viously
described
transforming
genes,
including
ras
and
Blym-J.
Hybridization
of
Tlym-I
to
mouse
and
human
DNAs
indicates
that
it
is
a
member
of
a
small
family
of
evolutionari-
ly
conserved
genes.
MATERIALS
AND
METHODS
Cell
Lines.
T-lymphoma
cell
lines,
NIH
3T3
cells,
and
transformed
NIH
cells
were
described
previously
(14).
Library
Construction.
DNA
of
NIH
cells
transformed
by
S49
T-lymphoma
DNA
was
digested
with
BamHI
and
trans-
forming
sequences
were
enriched
by
sucrose
gradient
cen-
trifugation
as
described
by
Lane
et
al.
(19).
The
two
DNA
fractions
containing
enriched
transforming
activity
were
pooled
and
ligated
to
BamHI
arms
of
X
Charon
30.
DNA
was
packaged
in
vitro
(20)
to
generate
a
recombinant
library
of
12,000
phage.
Library
Screening.
The
library
was
first
amplified
as
a
plate
lysate
on
Escherichia
coli
strain
JC
7623
and
then
in
liquid
culture
on
E.
coli
strain
LE392.
JC
7623
is
a
rec
B-
rec
C-
strain
(21)
and
was
used
to
prevent
selection
of
X+
phage
during
growth.
In
the
initial
screen,
phage
were
pelleted
through
a
25%
sucrose
cushion
at
35,000
rpm
for
120
min
at
50C
(18);
1
x
109
phage
were
added
per
100-mm
plate
of
NIH
3T3
cells
as
a
calcium
phosphate
precipitate
in
the
presence
of
60
pug
of
NIH
3T3
DNA
as
carrier.
Transfection
assays
were
carried
out
as
described
(18).
Subsequent
screening
was
carried
out
by
using
5
x
108
phage
plus
20
pg
of
carrier
DNA
per
60-mm
dish.
Foci
were
enumerated
12-14
days
fol-
lowing
transfection.
Restriction
Endonuclease
Digestions.
X
phage,
plasmids,
and
cell
DNAs
were
digested
with
restriction
endonucleases
(New
England
BioLabs)
under
the
conditions
recommended
by
the
supplier.
Plasmid
Construction.
The
4.7-kb
BamHI-HindIII
frag-
ment
from
A
IT
was
subcloned
into
compatible
sites
in
pBR322.
This
plasmid,
designated
p3IT,
contained
the
entire
transforming
sequence.
Two
additional
plasmids
were
then
constructed
from
p3IT-one
which
contained
transforming
sequence
(psIT)
and
one
which
contained
flanking
sequence
(pobIT).
psIT
was
prepared
by
digestion
of
p3IT
with
EcoRI
and
ligation
of
the
EcoRI
site
in
pBR322
to
the
EcoRI
site
in
cell
DNA,
reducing
the
cell
DNA
insert
size
to
1.1
kb.
pobIT
was
constructed
by
digesting
p3IT
with
BamHI
and
Bgl
II
and
then
cross-ligating
these
two
sites,
leaving
a
1.2-kb
in-
sert
of
cell
DNA.
Blot-Hybridization
Analysis.
Cellular
DNAs
were
digested
with
restriction
endonucleases,
electrophoresed
in
agarose
gels
and
transferred
to
GeneScreen
Plus
(New
England
Nu-
clear)
under
conditions
described
by
the
supplier.
Fragments
of
plasmid
DNAs
to
be used
as
probes
were
isolated
by
agar-
ose
gel
electrophoresis.
Fragments
were
electroeluted
from
gel
slices,
extracted
with
phenol/chloroform,
concentrated
by
three
butanol
extractions,
and
precipitated
with
ethanol
in
the
presence
of
0.3
M
NaCI.
DNA
fragments
were
32P-la-
beled
by
nick-translation
(22)
to
specific
activities
of
1-5
x
108
cpm/,ug
of
DNA.
Prehybridization,
hybridization,
and
washes
were
carried
out
as
described
by
the
suppliers
of
GeneScreen
Plus,
except
that
washes
were
carried
out
in
the
presence
of
4x
concentrated
SET
buffer
to
detect
partially
homologous
sequences
in
human
DNA
under
relaxed
condi-
tions
of
stringency.
Abbreviation:
kb,
kilobase(s).
2227
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.

2228
Medical
Sciences:
Lane
et
al.
RESULTS
Molecular
Cloning
of
Tlym-I.
A
biologically
active
trans-
forming
gene
was
isolated
from
DNA
of
NIH
cells
trans-
formed
by
DNA
of
the
BALB/c
T-lymphoma
cell
line
S49
by
using
the
strategy
of
sib
selection
described
by Goubin
et
al.
(18).
However,
this
isolation
was
considerably
facilitated
by
enrichment
of
the
transforming
sequence
prior
to
construc-
tion
of
a
phage
library.
DNA
of
NIH
cells
transformed
by
S49
DNA
was
digested
with
the
restriction
endonuclease
BamHI,
an
enzyme
previ-
ously
shown
not
to
inactivate
the
transforming
activity
of
this
tumor
DNA
(14).
DNA
was
then
fractionated
by
sucrose
gradient
centrifugation
and
assayed
by
transfection
of
NIH
3T3
cells.
A
peak
of
transforming
activity
was
identified
in
the
8-
to
9-kb
region
of
the
gradient
(Fig.
1).
Transforming
activity
of
the
DNA
contained
in
the
two
peak
fractions
(nos.
13
and
14)
constituted
an
enrichment
of
15-fold
over
unfrac-
tionated
DNA
(Table
1).
These
findings
indicated
that
the
transforming
region
migrated
as
a
discrete
unit
and
suggest-
ed
that
the
transforming
unit
itself
was
not
larger
than
8-9
kb.
The
biologically
active
sucrose
gradient
fractions
enriched
for
transforming
activity
were
then
used
to
construct
a
re-
stricted
phage
library.
DNA
from
the
8-
to
9-kb
region
of
the
sucrose
gradient
was
ligated
to
BamHI
arms
of
X
Charon
30.
The
ligated
DNA
was
packaged
in
vitro
to
generate
a
library
of
-12,000
recombinant
phage.
This
phage
library
was
then
screened
by
sib
selection
using
transfection
of
NIH
3T3
cells
as
an
assay
to
identify
recombinant
phage
populations
that
contained
a
biologically
active
transforming
sequence.
The
library
was
initially
divided
into
30
pools
of
400
phage
each.
Each
pool
was
amplified
on
E.
coli
strain
JC
7623,
first
as
a
plate
lysate
and
then
on
E.
coli
strain
LE392
in
liquid
culture.
Freshly
pelleted
phage
from
each
pool
were
assayed
for
transforming
activity
by
transfection
of
NIH
3T3
cells.
32
-
28
23.7
9.4
6.6
4.2
K
b
24
20
f
o
I
;.
0
2
8
10
Fraction
12
14
16
FIG.
1.
Sucrose
gradient
fractionation
of
BamHI-digested
DNA.
BamHI-digested
DNA
(1
mg)
of
NIH
cells
transformed
by
S49
DNA
was
centrifuged
through
10-40%
sucrose
gradients.
Sixteen
frac-
tions
were
collected
from
each
of
three
gradients
and
homologous
fractions
were
pooled.
DNA
concentrations
were
ascertained
by
ab-
sorbance
and
DNA
size
(kb;
indicated
by
arrows)
was
determined
by
agarose
gel
electrophoresis
relative
to
HindIII-digested
X
DNA
markers.
One-third
of
each
fraction
was
assayed
for
transforming
activity
in
the
presence
of
carrier
DNA,
by
transfection
of
four
re-
cipient
cultures
of
NIH
3T3
cells.
Total
foci
obtained are
shown
for
each
fraction.
Table
1.
Isolation
of
X
IT
Number
Phage
positive/
Foci
per
Ag
Screen
screened,
no.
total
pools
of
cell
DNA
NIH
(S49)
-
0.15
BamHI
8-kb
fraction
2.2
Library
First
12,000*
5/30
80
Second
800t
3/20
360
Third
80f
4/40
4,800
Fourth
5
3/5
10,000
Transforming
efficiencies
of
cell
DNA,
gradient-enriched
cell
DNA,
and
phage
pools
or
single
phage
DNA
were
measured
by
transfection
of
NIH
3T3
cells.
Values
expressed
for
phage
pools
and
phage
DNA
are
normalized
to
the
amount
of
cellular
DNA
based
upon
an
assumed
cellular
insert
size
in
X
Charon
30
of
8.0
kb.
*30
pools
of
400
phage.
t2O
pools
of
40
phage.
*40
pools
of
2
phage.
Five
positive
pools
were
identified
that
contained
transform-
ing
activity.
The
pool
demonstrating
the
highest
transform-
ing
activity
was
selected
for
further
analysis.
The
original
plate
lysate
of
this
pool
was
divided
into
20
pools
of
40
phage
each,
amplified,
and
assayed
as
described.
Three
positive
pools
were
identified
and
1
of
these
was
divided
into
40
pools
of
2
phage
each.
These
were
then
amplified
and
assayed
by
transfection.
Four
positive
pools
were
identified.
One
posi-
tive
pool
was
selected
and
the
individual
phage
plaques
that
constituted
this
pool
were
separately
amplified
and
assayed.
Three
of
5
positive
phage
plaques
were
identified
and
further
plaque
purified.
The
isolated
clone
has
been
designated
X
IT.
The
isolation
of
X
IT
is
summarized
in
Table
1.
At
each
step
of
sib
selection,
the
transforming
activity
of
positive
phage
pools
increased
as
expected
relative
to
the
decreasing
complexity
of
the
phage
population.
The
final
transforming
activity
of
X
IT
was
104
transformants
per
pg
of
cell
insert
DNA,
representing
an
increase
of
7
x
104
relative
to
the
ini-
tial
transforming
activity
of
cellular
DNA.
Structure
of
X
IT.
X
IT
was
characterized
by
cleavage
with
a
series
of
restriction
endonucleases.
The
clone
contains
a
cellular
BamHI
fragment
of
8.7
kb,
which
includes
recogni-
tion
sites
for
Bgl
I,
EcoRI,
and
HindIII
(Fig.
2).
Transform-
ing
activity
of
the
S49
T-lymphoma
DNA
was
previously
shown
to
be
inactivated
by
cleavage
with
EcoRI
but
not
with
HindII1
(14).
This
suggested
that
the
biologically
active
Tlym-I
gene
was
localized
to
the
4.7-kb
fragment
of
X
IT
ex-
tending
from
the
left-terminal
BamHI
site
to
the
left-most
HindIII
site,
since
this
fragment
contains
the
only
EcoRI
site
in
the
X
IT
cell
insert
(Fig.
2).
This
4.7-kb
fragment
of
X
IT
was
therefore
subcloned
in
pBR322.
This
plasmid,
designat-
ed
p3IT,
retains
the
transforming
activity
of
the
original
X
IT
clone
(Table
2).
The
cloned
transforming
gene
isolated
in
p3IT
was
com-
pared
to
the
transforming
gene
initially
detected
by
transfec-
tion
of
S49
T-lymphoma
DNA
by
analysis
of'the
sensitivity
of
transforming
activity
to
cleavage
with
restriction
endonu-
cleases.
Transforming
activity
of
p3IT
was
inactivated
by
cleavage
with
EcoRI
or
Xho
I
but
not
with
BamHI
or
HindIII
(Table
2).
This
is
the
same
as
the
pattern
observed
for
S49
lymphoma
DNA
(14),
indicating
that
the
cloned
transforming
gene
is
the
same
as
that
previously
detected
by
transfection
of
whole
cell
DNA.
The
cellular
insert
of
p3IT
contained
a
single
Xho
I
site
within
-100
nucleotides
of
the
terminal
BamHI
site
(Fig.
2).
Since
cleavage
with
Xho
I
and
EcoRI
inactivate
transforming
activity,
one
end
of
the
Tlym-I
gene
is
quite
close
to
the
ter-
minal
BamHI
site
of
p3IT
and
the
gene
extends
for
at
least
1
kb
to
the
EcoRI
site.
The
size
of
the
gene
was
further
esti-
Proc.
Natl.
Acad
Sci.
USA
81
(1984)

Proc.
NatL
Acad.
Sci.
USA
81
(1984)
2229
Charon
30
XIT
*-
8.7
Kb
-'
L-end
R-end
FIG.
2.
Restriction
map
of
Tlym-I.
Cleavage
sites
for
the
indicat-
ed
restriction
endonucleases
were
mapped
in
X
IT
and
pBR322
sub-
clones
containing
all
of
the
Tlym-I
transforming
sequence
(p3IT),
a
fragment
from
the
transforming
region
(psIT),
and
a
flanking
se-
quence
fragment
(pobIT).
mated
by
digestion
with
EcoRV,
which
cleaves
p3IT
at
a
single
site
-2
kb
from
the
BamHI
terminus
(Fig.
2).
Since
cleavage
with
EcoRV
did
not
affect
transforming
activity
(Table
2),
the
size
of
the
biologically
active
Tlym-I
gene
is
between
1
and
2
kb.
Analysis
of
Tlym-I
Sequences
in
Cellular
DNAs.
Two
sub-
clones
of
p3IT
were
constructed
to
facilitate
analysis
of
ho-
mologous
sequences
in
human
and
mouse
DNAs.
One
sub-
clone,
designated
psIT,
contained
1.1
kb
of
cell
DNA
within
A
B
C
D
E
F
G
H
I
18.0.
%No
8.6-
Table
2.
Restriction
endonuclease
sensitivity
of
Tlym-I
in
pBR322
Restriction
endonuclease
Foci
per
gg
of
p3IT
DNA
4.6
x
103
EcoRI
<50
HindIll
4.2
x
103
BamHI
4.8
x
103
Xho
I
50
EcoRV
4.6
x
103
DNA
from
the
plasmid
p3IT
was
digested
with
the
indicated
en-
zymes
and
transfected
onto
NIH
3T3
cells
to
assay
transforming
activity;
0.1
jig
of
DNA
plus
20
tig
of
carrier
DNA
were
transfected
onto
each
of
four
assay
plates.
Results
are
expressed
as
foci
per
Mg
of
total
plasmid
DNA.
the
transforming
region
from
the
terminal
BamHI
site
to
the
EcoRI
site
(Fig.
2).
This
subclone
hybridized
to
highly
repet-
itive
mouse
sequences
and
was
therefore
inappropriate
for
analysis
of
mouse
DNA
but
useful
for
analysis
of
human
DNA.
A
second
subclone
contained
1.2
kb
of
cell
DNA
from
the
HindIII
site
at
the
other
terminus of
the
cell
insert
of
p3IT
to
the
BgI
II
site
in
cell
DNA,
which
was
cross-ligated
to
the
terminal
BamHI
site
(Fig.
2).
This
flanking
sequence
clone,
designated
pobIT
(Fig.
2),
was
useful
as
a
probe
for
hybridization
to
mouse
DNA.
NIH
cells
transformed
by
mouse
T-cell
lymphoma
DNAs
were
analyzed
by
blot-hybridization
to
determine
whether
they
contained
additional
exogenous
Tlym-I
sequences.
Since
these
cells
are
transformed
by
transfer
of
mouse
donor
DNA
to
a
mouse
recipient,
detection
of
novel
sequences
in
transformed
cells
is
dependent
on
DNA
rearrangements
that
occur
only
rarely
during
the
transfection
process
(23).
There-
fore,
DNAs
of
eight
independent
lines
of
NIH
cells
trans-
formed
by
DNAs
of
five
different
lymphomas
(14)
were
ana-
lyzed
by
cleavage
with
both
BamHI
bands
and
HindIII
(Fig.
3).
32P-labeled
pobIT
probe
hybridized
to
two
BamHI
bands
of
8.7
and
18
kb
in
DNAs
of
normal
NIH
3T3
cells
and
trans-
formed
NIH
cell
lines
(Fig.
3
Left).
The
8.7-kb
band
repre-
sents
the
genomic
size
of
the
cellular
insert
cloned
in
Charon
30,
whereas
the
larger
band
most
likely
contains
a
second
hybridizing
member
of
the
Tlym
gene
family.
Digestion
of
the
same
DNAs
with
HindIII
and
hybridization
to
pobIT
A
B
C
D
E
F
G
H
I
30.
._
.
V_
o
12_..
_
w
_____
_~~~
FIG.
3.
Tlym-I
sequences
in
DNAs
of
NIH
3T3
cells
and
NIH
cells
transformed
by
mouse
T-lymphoma
DNAs.
DNAs
(15
Yg)
were
digested
with
BamHI
(Left)
or
HindIII
(Right),
subjected
to
electrophoresis
in
agarose
gels,
transferred
to
GeneScreen
Plus
filters,
and
hybridized
with
32P-labeled
obIT
flanking
sequence
probe.
Band
sizes
are
indicated
in
kb.
Lanes:
A,
DNA
of
NIH
cells
transformed
by
S49
BALB/c
T-
lymphoma
DNA;
B,
DNA
of
NIH
cells
transformed
by
KKT2
AKR
T-lymphoma
DNA;
C
and
D,
DNAs
of
two
independent
foci
of
NIH
cells
transformed
by
SL3
AKR
T-lymphoma
DNA;
E
and
F,
DNAs
of
two
independent
foci
of
NIH
cells
transformed
by
L691
C57
leaden
T-
lymphoma
DNA;
G
and
H,
DNAs
of
two
independent
foci
of
NIH
cells
transformed
by
SL7
AKR
T
lymphoma
DNA;
and
I,
NIH
3T3
DNA.
Medical
Sciences:
Lane
et
aL
46

2230
Medical
Sciences:
Lane
et
al.
identified
in
NIH
3T3
cell
DNA
and
in
the
transformant
DNAs
a
band
of
6.2
kb
and
a
band
of
:"30
kb
(Fig.
3
Right).
These
findings
indicated
that
large
pieces
of
DNA
had
been
integrated
that
shared
conserved
restriction
sites
between
donor
and
recipient
DNA.
However,
DNA
from
one
trans-
formant
analyzed
by
digestion
with
HindIll
gave
evidence
of
a
recombination
event
between
host
and
donor
DNA,
which
gave
rise
to
a
novel
fragment
of
-12
kb
(Fig.
3
Right,
lane
C).
This
new
fragment
most
likely
arose
as
a
result
of
a
re-
combination
event
that
occurred
in
DNA
flanking
the
cellu-
lar
insert
of
X
IT,
as
new
bands
were
not
detected
in this
transformant
DNA
following
digestion
with
BamHI
(Fig. 3
Left,
lane
C).
Detection
of
an
event
of
this
frequency
(1/8)
is
comparable
to
that
observed
following
random
integration
of
Rous
sarcoma
virus
DNA
during
transfection
of
NIH
3T3
cells
(23).
Therefore,
these
findings
further
indicate
that
the
cloned
Tlym-I
transforming
gene
is
the
same
as
that
detected
by
transfection
of
whole
cell
DNAs
from
T
lymphomas.
Hybridization
of
the
32P-labeled
obIT
probe
to
DNA
from
the
BALB/c
T-lymphoma
S49
digested
with
either
BamHI
or
Xho
I
indicated
no
amplification
of
sequences
in
tumor
DNA
(Fig.
4,
lanes
A
and
C)
as
compared
with
BALB/c
liver
DNA
(Fig.
4,
lanes
B
and
D).
A
comparison
of
bands
from
S49
and
BALB/c
liver
further
indicated
that
activation
of
the
Tlym-I
gene
did
not
occur
as
a
result
of
gross
DNA
rearrangement
in
this
tumor.
Hybridization
of
32P-labeled
psIT
or
the
1.1-kb
fragment
from
this
plasmid
under
conditions
of
moderate
stringency
to
human
DNA
following
digestion
with
EcoRI,
BamHI,
or
HindIII
again
identified
a
small
number
of
bands
(Fig.
4,
lanes
E-G).
These
observations
are
consistent
with
Tlym-I
being
a
member
of
a
small
gene
family.
These
findings
fur-
ther
suggest
evolutionary
conservation
of
this
gene
family
between
mouse
and
human
species.
Tlym-I
Is
Not
Homologous
to
Previously
Identified
Trans-
forming
Genes.
p3IT
probe
did
not
hybridize
to
molecular
clones
of
the
retroviral
transforming
genes
abl,
erb,
fes,
fms,
mos,
myb,
myc,
ras
H,
rasK,
rel,
sis,
and
src,
indicating
that
Tlym-I
is
not
homologous
to
the
transforming
genes
of
these
sarcoma
and
acute
leukemia
viruses.
In
addition,
Tlym-I
did
not
hybridize
to
molecular
clones
of
the
Blym-J
transforming
gene
isolated
from
either
chicken
or
human
B-cell
lympho-
mas.
Tlym-I
is
thus
distinct
from
the
previously
isolated
neo-
plasm
transforming
genes
ras
and
Blym-1
(data
not
shown).
A
B
C
D
E
F
G
or
N
w-.
_
A*:'
..
A.*
>
w
_
f
at
...
....
b;
sit
1
tseS
...
..
H.k
I,+
..f
_
_
*
..
a.
FIG.
4.
Tlym-I
sequences
in
mouse
and
human
DNAs.
DNAs
(15
,ug)
were
digested
with
BamHI
(Left),
Xho
I
(Center),
or
EcoRI,
BamHI,
and
HindI11
(Right,
lanes
E,
F,
and
G,
respectively),
elec-
trophoresed
in
agarose
gels,
transferred
to
GeneScreen
Plus
filters,
and
hybridized
with
32P-labeled
obIT
flanking
sequence
probe
(Left
and
Center)
or
32P-labeled
sIT
transforming
sequence
probe
(Right).
Marker
bars
represent
DNA
sizes
of
23.7-,
9.5-,
6.7-,
4.3-,
2.3-,
and
2.0-kb
BALB/c
mouse
S49
T-lymphoma
DNA
(lanes
A
and
C),
BALB/c
mouse
liver
DNA
(lanes
B
and
D),
and
human
spleen
DNA
(lanes
E-G).
To
determine
whether
activation
of
Tlym-I
in
the
S49
T
lymphoma
involved
region-specific
integration
of
murine
leukemia
virus
DNA
(24),
a
molecular
clone
(pmlsp)
of
the
long
terminal
repeat
sequence
of
Moloney
sarcoma
virus
(25)
was
32P-labeled
and
hybridized
to
p3IT.
No
hybridization
was
detected,
indicating
that
the
biologically
active
Tlym-I
gene
was
not
associated
with
retroviral
DNA
(data
not
shown).
DISCUSSION
We
previously
reported
the
identification
of
three
different
stage-specific
transforming
genes
activated
in
neoplasms
of
the
B-lymphocyte
lineage
and
of
two
such
genes
in
neo-
plasms
of
the
T-lymphocyte
lineage
(14).
In
the
present
re-
port,
we
describe
the
molecular
cloning
and
initial
character-
ization
of
a
transforming
gene,
Tlym-I,
activated
in
neo-
plasms
representing
an
intermediate
stage
of
T-lymphocyte
differentiation.
The
Tlym-I
gene
is
small
(1-2
kb)
and
repre-
sents
a
transforming
gene
that
is
not
homologous
to
the
transforming
genes
of
retroviruses
or
to
Blym-1.
The
size,
restriction
map,
and
lack
of
homology
of
Tlym-I
to
molecular
clones
of
rasH
and
rasK
also
distinguish
Tlym-I
from
rasN.
The
lack
of
homology
between
Tlym-I
and
Blym-1
is
of
particular
interest
since
T
and
B
lymphocytes
are
thought
to
develop
from
a
common
stem
cell.
However,
lymphocytes
of
the
T
and
B
lineages
have
many
distinctive
features.
They
arise
in
different
primary
lymphoid
organs,
colonize
sepa-
rate
areas
of
the
secondary
lymphoid
tissues,
mediate
dis-
tinct
types
of
immunological
function,
and
express
a
differ-
ing
array
of
characteristic
surface
markers
(26).
In
addition,
stem
cells
that
give
rise
to
the
T-lymphocyte
lineage
diverge
early
in
hematopoiesis
from
other
stem
cell
populations
that
give
rise
to
the
B-lymphocyte
lineage
and
to
cells
of
the
my-
eloid,
erythroid,
and
granulocytic
series
(27).
The
lack
of
re-
lationship
between
Tlym-I
and
Blym-1
further
indicates
that
distinct
transforming
genes
are
involved
in
neoplasms
of
T
and
B
lymphocytes.
Blot-hybridization
analysis
of
mouse
and
human
DNAs
in-
dicates
that
Tlym-I,
like
Blym-J
(17),
is
a
member
of
a
small
family
of
evolutionarily
conserved
genes.
Although
the
transforming
gene
activated
in
mature
T-cell
neoplasms
is
distinct
from
Tlym-I
(14),
further
studies
to
identify
a
third
gene
within
the
T
lineage
in
tumors
classified
as
less
pheno-
typically
mature
than
those
within
the
intermediate
T
catego-
ry
have
proved
unsuccessful.
Thus
far,
two
mouse
and
one
human
pre-T-cell
neoplasms
have
been
found
to
contain
acti-
vated
Tlym-I
genes,
based
upon
restriction
endonuclease
sensitivity
(unpublished
observations).
It
is
therefore
rea-
sonable
to
speculate
that
the
transforming
gene
identified
in
mature
T-lymphocyte
neoplasms
may
be
a
different
member
of
the
Tlym-I
gene
family.
The
leukemogenic
process
is
thought
to
involve
several
steps.
Involvement
of
murine
leukemia
virus
in
T-cell
lym-
phomas
in
susceptible
strains
of
mice
has
been
indicated
by
numerous
investigators,
although
these
viruses
are
present
throughout
life,
whereas
leukemias
develop
only
following
long
latent
periods.
One
mechanism
proposed
for
viral
initia-
tion
of
disease
is
that
a
virus
that
replicates
in
T
cells
can
bind
specifically
to
the
surface
receptors
present
on
a
subset
of
thymic
T
cells
and
cause
this
population
to
expand
by
a
mechanism
analogous
to
immunologic
stimulation
(28).
An-
other
possibility
is
that
region-specific
integration
of
viral
DNA
activates
adjacent
cellular
genes
(24).
In
either
case,
it
is
clear
from
the
present
studies
that
Tlym-I
shares
no
ho-
mology
with
viral
genes
and
is
not
linked
to
viral
DNA,
sug-
gesting
that
viral
events
and
activation
of
Tlym-I
represent
different
stages
within
the
leukemic
process.
It
is
attractive
to
speculate
that
virus-related
events
may
be
involved
early
in
the
disease
process
and
that
activation
of
Tlym-I
may
oc-
Proc.
NatL
Acad
Sci.
USA
81
(1984)
t,
'@
S.
*,

Proc.
Natl.
Acad.
Sci.
USA
81
(1984)
2231
cur
during
progression
of
proliferative
T-cell
populations
to
neoplasia.
It
is
of
interest
to
note
that
the
stage-specific
genes
Tlym-I
and
Blym-1
are
not
promiscuous
in
their
activation
and
have
not
been
encountered
outside
of
their
respective
lineages,
whereas
the
less
restricted
ras
family
members
have
been
observed
without
specificity
in
occasional
carcinomas,
sar-
comas,
neuroblastomas,
and
leukemias.
Recent
reports
have
described
activation
of
the
rasN
transforming
gene
in
a
single
Burkitt
lymphoma
cell
line (12)
and
in
4
of
22
hematopoietic
tumors,
including
two
T-cell
leukemias
(13).
Activation
of
rasK
in
one
T-cell
leukemia
was
also
detected
(13).
In
con-
trast,
activation
of
Blym-J
has
now
been
detected
in
24
of
24
neoplasms
of
intermediate
stage
B
lymphocytes
of
chicken,
mouse,
and
human
origin
(14-17).
Similarly,
we
have
detect-
ed
activation
of
Tlym-I
in
10
of
12
mouse
and
human
neo-
plasms
of
early
and
intermediate
stage
T
lymphocytes
(ref.
14;
unpublished
observations).
Since
ras
genes
are ex-
pressed
and
may
function
in
multiple
stages
of
differentiation
within
many
different
cell
lineages
(29,
30),
they
may
play
an
occasional
role
in
a
variety
of
different
neoplastic
diseases.
In
contrast,
stage-specific
transforming
genes
such
as
Tlym-I
and
Blym-1
may
function
at
specific
stages
of
differentiation
within
discrete
cell
lineages
and
contribute
more
reproduci-
bly
to
the
development
of
specific
types
of
neoplasms.
Note
Added
in
Proof.
Recent
results
indicate
that
Tlym-I
shares
ho-
mology
with
class
I
major
histocompatibility
complex
genes
in
the
Qa/Tla
region.
We
are
grateful
to
G.
Vande
Woude
for
the
plasmid
pmlSP
and
to
R.
Kolodner
for
suggesting
and
supplying
the
bacterial
strain
JC
7623.
The
secretarial
skills
of
C.
Suther
are
gratefully
acknowl-
edged.
This
work
was
supported
by
Grant
CA33108
from
the
Na-
tional
Cancer
Institute.
G.M.C.
holds
a
faculty
research
award
from
the
American
Cancer
Society.
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