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(Received for publication, November 17, 1995) From the
As shown previously, mutants of the Y1 mouse adrenocortical
tumor cell line that resist agonist-induced desensitization of adenylyl
cyclase have elevated levels of a 68-kDa protein (designated p68),
suggesting a possible relationship between p68 and the regulation of
adenylyl cyclase activity. In the present study, cDNA cloning and
sequencing were used to identify p68 as mouse transketolase. Cells
overexpressing p68 exhibited a 17.4-fold increase in transketolase
enzymatic activity relative to parental Y1 cells and a 28-fold
amplification of the transketolase gene as determined by Southern blot
hybridization analysis. Using fluorescent in situ hybridization analysis, the transketolase gene was mapped to mouse
chromosome 16B1 and to human chromosome 3p21.2. Transketolase gene
amplification was associated with telomeric fusion of the chromosome 16
pair together with the appearance of multiple copies of the
transketolase gene throughout a different chromosome. The relationship
between overexpression of transketolase and desensitization resistance
was evaluated in somatic cell hybrids formed between a
desensitization-resistant adrenal cell line and a
desensitization-sensitive rat glial cell line. In these hybrids,
transketolase overexpression behaved dominantly, whereas
desensitization resistance behaved recessively. These results
dissociate the desensitization resistance phenotype from overexpression
of transketolase and suggest that desensitization resistance may have
resulted from disruption of an essential regulatory gene in conjunction
with the amplification event.
In a variety of cell types, the chronic stimulation of adenylyl
cyclase by hormones and neurotransmitters often desensitizes the
enzyme, rendering it refractory to further stimulation. In our
laboratory, this phenomenon has been investigated extensively using Y1
mouse adrenocortical tumor cells and in a family of
desensitization-resistant (DR) ( A potential insight into regulation of the
desensitization pathway came from our observations that the DR
phenotype is associated with the overexpression of a 68-kDa protein
designated p68(1, 6, 7, 8) . Among
18 independent subclones of the Y1 adrenal cell line, the level of p68
correlated with the level of ACTH-responsive adenylyl cyclase activity
and those with high levels of p68 desensitized more slowly and
recovered from the desensitized state more quickly than clones with low
levels of p68(1, 6) . Inasmuch as p68 has not been
identified, we have undertaken the cloning and sequencing of the cDNA
encoding this protein. We report that p68 is the mouse transketolase
(EC 2.2.1.1; TKT). We show that TKT activity in the DR mutant is
20-fold higher than in parental Y1 cells and that the overexpression of
TKT results from amplification of a chromosome segment derived from
mouse chromosome 16. Using somatic cell hybridization analyses, we are
able to dissociate TKT overexpression from the DR phenotype, suggesting
that the DR phenotype likely resulted from a reciprocal gene deletion
that accompanied amplification of the TKT gene.
Figure 1:
Isolation of cDNA clones encoding p68. A, the thick upper line represents the 1872 bp of p68
coding sequence; 5`- and 3`-untranslated sequences are represented by
the thin flanking segments. Also indicated are sites for the
restriction endonucleases ApaI (A), BamHI (B), EcoRI (RI), NcoI (N), PstI (P), SacI (SI), and SacII (SII). The sequence was determined from
overlapping cDNA fragments isolated by expression screening of a
Figure 3:
Fluorescent in situ hybridization
analysis of the mouse TKT gene. Chromosome spreads were prepared from
mouse splenocytes and probed with biotinylated TKT genomic DNA. Signals
were amplified and detected with FITC-avidin (panel A). Panel B shows the same mitotic spread stained with DAPI. The
fluorescent banding patterns obtained were used to identify the
chromosome labeled with the TKT probe as chromosome
16.
Figure 2:
Epitope selection and Northern analysis
using a p68 cDNA clone. In the epitope selection experiment (left), purified p68 and extracts from parental Y1 cells
(Y1
Mouse
chromosomes were probed with a partially characterized 20-kb TKT
genomic clone isolated from an EMBL3 mouse genomic library. Human
chromosomes were probed with mouse TKT cDNA from nucleotide 153 to
nucleotide 2062. Probes were labeled with biotinylated dATP, hybridized
to the chromosome spreads, and detected with FITC-avidin. Signals were
amplified by incubation with biotinylated goat anti-avidin followed by
a second round of incubation with FITC-avidin. Chromosome banding
patterns were obtained with the chromatin-binding fluorescent dye
4`-6-diamidino-2-phenylindole (DAPI). Chromosomal localization of TKT
was made by superimposing photographs of the hybridization signals with
photographs of the DAPI banding patterns.
The identity of p68 as TKT was further confirmed by
demonstrating that extracts from the DR mutant exhibited a 17.4-fold
higher level of TKT activity (0.40 ± 0.04 units) compared to
extracts from parental Y1 cells (0.023 ± 0.002 units),
consistent with the observed amplification of p68 in DR clones.
Figure 4:
Chromosomal locations of mouse and human
TKT. Mouse and human chromosome spreads were probed for the TKT gene
and banded with DAPI. The resultant fluorescent signals were
photographed separately and the images were superimposed. The TKT
signals (filled circles) from 10 mitotic figures are
superimposed on schematic representations of the banding patterns of
mouse chromosome 16 (left) and human chromosome 3 (right) respectively. The schematic representations of
chromosome banding patterns were adapted from (35) and (42) .
Figure 5:
Southern blot analysis of the TKT gene. In panel A, genomic DNA from DS and DR cells was digested to
completion with the restriction endonucleases indicated,
electrophoresed on 0.8% agarose, blotted onto a Hybond
In chromosome
spreads prepared from parental Y1 mouse adrenocortical tumor cells, TKT
signals also were observed on chromosome 16, and there was no evidence
of TKT gene amplification (data not shown). In the DR mutant, however,
the TKT gene seemed to be amplified over a large region on a single
chromosome (Fig. 6). Since the level of amplification was very
high, the morphology of the affected chromosome was completely changed
and identification of the affected chromosome was not possible. TKT
signals also were evident on the chromosome 16 pair, which showed an
abnormal telomeric fusion in the DR mutant (Fig. 6). Additional
faint signals seen scattered throughout the chromosome spread are not
reproducible and represent background.
Figure 6:
Fluorescent in situ hybridization
analysis of the TKT gene in DR cells. Metaphase spreads were prepared
from mitotically arrested DR cells, probed with biotinylated TKT
genomic DNA and stained with DAPI. The arrows show an
amplified TKT fluorescent signal (panel A) and the
corresponding affected chromosome (panel
B).
As determined by Southern
blot hybridization analysis (Fig. 5), other genes associated
with the proximal region of mouse chromosome 16, i.e. immunoglobulin
To further
address the relationship between TKT overexpression and desensitization
resistance, we evaluated the linkage of these two phenotypes in somatic
cell hybrids formed between a DR derivative,
Kin-8HGPRT As
determined from Southern blot hybridization analysis using the mouse
TKT probe, the TKT genes in two independently isolated
Kin-8HGPRT
Figure 7:
Amplification of the TKT gene in adrenal
As shown in Table 1, the hybrid clones responded to ACTH,
isoproterenol, and NaF with increases in adenylyl cyclase activity. The
response to ACTH reflected the contribution of Kin-8HGPRT
Figure 8:
Desensitization of adenylyl cyclase in
adrenal
In order to further understand the biochemical and molecular
causes of the DR phenotype in mutant Y1 adrenocortical tumor cells, we
sought to identify p68 and determine the basis for its overexpression
in DR cells. Based on cDNA sequencing results and direct assays of
enzymatic activity, we have established that p68 is the mouse TKT. TKT
is a thiamine-requiring enzyme that is part of the pentose phosphate
metabolic pathway responsible for the synthesis of pentoses and for the
generation of NADPH(32) . Defects in TKT have been described in
a population of alcoholic patients and may contribute to the
neuropathological disturbances associated with WernickeKorsakoff
syndrome(33, 34) . Chromosomal mapping experiments
localized human TKT to chromosome 3p21.2 (Fig. 4) and mouse TKT
to the B1 region of chromosome 16 ( Fig. 3and Fig. 4), a
region that appears to be poorly defined and not yet established as
syntenic with human chromosome 3p21(35) . Previous gene mapping
studies also had localized the human TKT to chromosome
3p(34, 36) ; however, the earlier results had placed
the TKT gene at 3p14 (36) rather than in the adjacent 3p21.2
region as reported here. As evidenced from Southern blot and
fluorescent in situ hybridization analyses ( Fig. 5and Fig. 6), the overexpression of TKT in DR mutant clones resulted
from an approximate 28-fold amplification of the TKT gene. In most
examples of gene amplification, the regions involved (referred to as
amplicons) are very large and can involve as much as 10,000 kb of
DNA(37) . Other markers of chromosome 16 proximal to the
centromere, such as the immunoglobulin To further explore
the relationship of TKT gene amplification to desensitization
resistance, we examined the linkage of these two phenotypes in somatic
cell hybrids between a DR isolate and desensitization-sensitive C6
glioma cells. The hybrids acquired the TKT amplicon and overexpressed
TKT (Fig. 7) but failed to resist ACTH-induced desensitization
of adenylyl cyclase (Fig. 8). These results clearly dissociate
TKT gene amplification from the DR phenotype and indicate that
desensitization resistance behaves recessively in the hybrid. On the
basis of these results, we suggest that desensitization resistance may
have resulted from a recombination event that disrupted or mutated a
gene required for the desensitization process rather than from
amplification of TKT itself or from coamplification of a closely linked
gene.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U05809[GenBank].
Volume 271,
Number 9,
Issue of March 1, 1996 pp. 4993-4998
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)Y1 mutants (1, 2, 3, 4, 5) . We have
shown that the DR mutation in Y1 cells not only affects desensitization
from the endogenous ACTH receptor but also affects desensitization from
wild-type mouse 
-adrenergic and human dopamine D-1
receptors when genes encoding these receptors are transfected into the
mutant cell line(2, 3, 4) . Using ligand
binding analyses, we demonstrated that the DR mutation did not affect
receptor internalization, a late step in the desensitization pathway,
but prevented receptor uncoupling from its guanyl nucleotide-binding
regulatory protein(2, 3, 5) . On the basis of
these findings, we have suggested that the DR mutation does not reside
within the ACTH receptor; rather, it affects an early component of the
desensitization pathway that is shared among different receptor
signaling systems.
cDNA Library Screening
Custom cDNA libraries in
the bacteriophages gt11 and
gt10, respectively (Clontech
Laboratories, Inc., Palo Alto, CA) were prepared from
poly(A)
RNA (9) isolated from Y1 mouse
adrenocortical tumor cells (10) and from the Y1 derivative,
Kin-8(11) . The
gt11 library was probed for expression of
p68 using a rabbit polyclonal p68 antiserum (8) and
I-labeled protein A as described(12) . Subsequent
screenings of the
gt11 and
gt10 libraries were performed by
DNA hybridization as described by Maniatis et al.(13) using the 600-bp EcoRI fragment from
gt11 clone 16 (Fig. 1). The probe was labeled by nick
translation in the presence of [
-P]dCTP
using a kit from Life Technologies, Inc. (Canadian Life Technologies,
Inc., Burlington, Ontario, Canada).
gt11 library (gt11-16), hybridization screening of
gt11 and
gt10 libraries and 5`-RACE techniques. B, cDNA fragments
were sequenced from both strands in the presence of dGTP and
C
dITP. 5`- and 3`-untranslated regions are indicated by lowercase letters, the coding region is indicated by uppercase letters and the translation start (ATG) and stop
(TAG) sites are indicated in bold.
Rapid Amplification of 5` cDNA ends (5`-RACE)
The
5`-RACE procedure (14) was used to clone the 5` end of the p68
transcript. Total RNA was prepared from DR cells overexpressing p68 (1) using guanidine thiocyanate for extraction, followed by
centrifugation through CsCl(15) . cDNA was synthesized with
SuperScript(TM) reverse transcriptase and C-tailed with terminal
deoxynucleotidyltransferase and dCTP using a 5`-RACE System from Life
Technologies, Inc. The oligodeoxynucleotide primer used for first
strand cDNA synthesis (5`-GGTATGGAAAAACAGGACAGCCAT-3`) was
complementary to the mouse p68 (TKT) sequence from positions
233-256 (Fig. 3). The cDNA was then amplified by
polymerase chain reaction (PCR) using a kit containing AmpliTaq®
DNA polymerase (Perkin Elmer (Canada) Ltd., Rexdale, Ontario, Canada)
together with the Anchor Primer provided with the 5`-RACE System and an
internal primer (5`-CATGATCTCGGCAGCGCTGCAGCATGATGT-3`; Kronem Systems
Inc., Missisauga, Ontario, Canada) complementary to the p68 sequence at
positions 206-235. PCR was carried out for 36 cycles with a hot
start(16) ; the timing for each cycle consisted of 1-min
incubations at 94, 54, and 72 °C. At the end of the reaction,
samples were incubated at 72 °C for 10 min to ensure that cDNA
synthesis went to completion. An aliquot of the reaction was amplified
for an additional 35 cycles under similar conditions, except that the
reaction contained the universal amplification primer from the 5`-RACE
system instead of the Anchor Primer and the annealing temperature was
raised to 65 °C. The PCR product was end-filled with Klenow DNA
polymerase (Pharmacia, Baie d'Urfe, Quebec, Canada) and cloned
into the SmaI sites of pBluescript SK and
KS
(Stratagene, La Jolla, CA).
DNA Sequence Analysis
Sequences from p68 cDNA
downstream from the internal EcoRI site (Fig. 1) were
determined using fragments generated following digestion with EcoRI and BamHI; sequences 5` of the internal EcoRI site were determined using nested deletion fragments
generated by digestion with exonuclease III and mung bean nuclease
(Stratagene). All DNA fragments were subcloned into the phagemids
pBluescript SK and KS
, and
single-stranded templates were prepared following propagation in Escherichia coli JM101 cells with VCR-M13 helper phage
(Stratagene). DNA was sequenced by the dideoxynucleotide chain
termination method (17) using Sequenase 2.0 (U. S. Biochemical
Corp.) with T7 primers (U. S. Biochemical) or T3 primers (Pharmacia).
Reactions carried out in the presence of dGTP or c
dITP were
compared to resolve sequence ambiguities. Data base searches were
carried out using the FASTA algorithm(18) , and alignments were
performed using Geneworks® version 2.21 (IntelliGenetics, Inc.,
Mountain View, CA).Western, Northern, and Southern Blot Analyses
For
Western blot analysis, purified p68 and adrenal cell extracts were
separated by electrophoresis on polyacrylamide gels, blotted onto
nitrocellulose using a Bio-Rad Transblot apparatus and probed with an
affinity-purified p68 antiserum and I-protein A
essentially as described(8, 19) . The
affinity-purified p68 antiserum was obtained by passing a polyclonal
p68 antibody (8) over nitrocellulose filters containing the
epitope expressed in purified bacteriophage
gt11 clone 16 (Fig. 1) as detailed previously(12) . For Northern blot
hybridization analysis, total RNA from adrenal cell cultures was
prepared as described above, fractionated on a 1.2% agarose, 2.2 M formaldehyde gel, transferred to a Nytran Plus nylon filter
(Xymotech Biosystems, Toronto, Ontario) and probed with a
P-labeled cDNA probe prepared by nick translation of the
600-bp EcoRI fragment from the bacteriophage
gt11 clone
16 using a kit from Canadian Life Technologies Inc. For Southern blot
hybridization analysis, high molecular weight DNA was prepared as
described by Wigler et al.(20) and collected by
spooling onto glass rods. DNA samples digested with restriction
endonucleases were fractionated by electrophoresis on 0.7% agarose
gels, blotted onto nylon membranes, and hybridized with a
P-labeled mouse TKT cDNA from nucleotide 153 to nucleotide
2062 (Fig. 2) or with genomic probes for the mouse
immunoglobulin
light chain gene and the mouse immunoglobulin
5 gene.
) and a cell line overexpressing p68 (Y1
)
were resolved and Western blotted with affinity-purified p68 antiserum
as described under ``Materials and Methods.'' The positions
of the 75-kDa and 50-kDa markers are indicated. In the Northern blot
hybridization experiments (right), total RNA from parental Y1
cells and a cell line overexpressing p68 was electrophoresed on an
agarose-formaldehyde gel and probed with the cDNA insert from
gt11-16. The electrophoretic position of the endogenous 2.2-kb
ribosomal RNA subunit is indicated. In each experiment, reactive bands
were visualized by fluorography using intensifying
screens.
Transketolase Activity
Transketolase activity was
assayed in a coupled enzymatic assay as described
previously(21) . Briefly, cell monolayers were rinsed with
phosphate-buffered saline, scraped in buffer containing 50 mM potassium phosphate, pH 7.8, 1 mM EDTA, 1 mM 2-mercaptoethanol, and 0.1% (w/v) Triton X-100. Cells were
homogenized using a Dounce homogenizer with a tight-fitting pestle, and
homogenates were centrifuged 100,000 
g for 90 min at 4
°C to obtain cell supernatant fractions. Assays were carried out in
quartz cuvettes in a 1-ml reaction volume containing 1 mM MgCl, 0.1 mM NADH (Boehringer Mannheim
Canada, Laval, Quebec), 2.5 mMD-xylulose-5-phosphate
(Sigma), glycerol-phosphate dehydrogenase (1 unit) and triose-phosphate
isomerase (11 units) (Sigma), 100 mM Tris-HCl, pH 7.6, and
5-30 µg of cell supernatant. The assay mixture was
equilibrated at room temperature for 5 min, and the reaction was
initiated by adding ribose 5-phosphate (Sigma) to a final concentration
of 2.5 mM to the sample cuvette. Reactions were monitored by
measuring the change in absorbance at 340 nm over a period of 20 min;
results were converted to units of activity, where 1 unit of activity
represents 1 µmol of NADH oxidized min
mg protein. Activity was linear with
respect to both enzyme protein and incubation times.
Gene Mapping
Gene mapping was performed by SeeDNA
Biotech Inc. (North York, Ontario) as described
previously(22, 23) . Normal mouse chromosomes were
prepared from synchronized cultures of splenic
lymphocytes(24) . Human chromosomes were prepared from
synchronized cultures of lymphocytes isolated from cord
blood(22) . Chromosomes from parent and mutant mouse
adrenocortical tumor cell lines were prepared from cells arrested in
metaphase with 2 µg/ml vinblastin for 18 h (25) .Adenylyl Cyclase Activity
Adenylyl cyclase
activity was measured in cell homogenates by measuring the conversion
of [2,8-
H]ATP to cAMP in a 5-min reaction at 37
°C as described previously(26) . The reaction mixture
contained 1 mM disodium ATP (approximately 1.6
10
cpm; DuPont Canada), 2 mM MgCl, 6
mM theophyllin, 50 µg of albumin, 12.5 mM Tris-HCl, pH 7.7, and approximately 140 µg of enzyme protein
in a final volume of 85 µl.
Isolation of p68 cDNA
A gt11 cDNA library
prepared from Y1 cells was screened for p68 cDNA by expression using a
polyclonal p68 antiserum(8) . An immunoreactive isolate,
gt11-16, was plaque-purified, and found to contain a 900-bp insert
with an internal EcoRI restriction site that encoded an open
reading frame (Fig. 1). The identity of the insert as a p68 cDNA
clone was confirmed using epitope selection and Northern blot
hybridization techniques (Fig. 2). As shown in Fig. 2,
p68 antiserum affinity-purified by passage over the epitope expressed
in
gt11-16 reacted with purified p68, and specifically recognized
single bands of 68 kDa in Y1 cell extracts on Western blots. The
signals obtained when cell extracts were probed with the
affinity-purified antiserum correlated with the differential levels of
expression of p68 expected in parental Y1 cells and mutant DR cells
overexpressing p68. Similarly, Northern blots probed with a cDNA
fragment from the
gt11-16 isolate gave a signal at 2.2 kb that was
more intense in mutant DR cells overexpressing p68 than in the parental
Y1 cell line (Fig. 2). The cDNA from the
gt11-16 isolate
was then used as a probe to clone three additional p68 cDNA fragments
from mouse adrenal cell libraries prepared in
gt11 and
gt10
as described under ``Materials and Methods.'' These cDNA
fragments spanned 1910 bp of p68 cDNA plus the 3` poly(A) tail.
Isolation of an additional 152 bp of cDNA including the initiator ATG
from the 5` end of the p68 transcript was achieved using the 5`-RACE
procedure (Fig. 1).
Identification of p68 as Mouse TKT
The p68 cDNA
fragments were sequenced completely from both DNA strands and shown to
encode a large open reading frame corresponding to 623 amino acids with
a calculated molecular mass of 67,553 Da (Fig. 1). A search of
the GenBank(TM) (27) data bank showed that the p68 sequence
was 83% identical with human TKT at the DNA level, 94% identical at the
protein level and included conserved amino acids implicated in binding
thiamine pyrophosphate(28) . The sequencing data thus indicate
that p68 is the mouse TKT. Of the 38 amino acids that differed between
the mouse and human TKT, the substitutions S30T, S31T, V46E, and A426P
in the mouse protein also are seen as polymorphisms in the human gene (28) .Chromosomal Localization of TKT
Strong TKT signals
were detected on more than 80% of mitotic figures examined and were
localized to mouse chromosome 16 and human chromosome 3; background
signals were minimal, and positive signals were not detectable on any
other chromosomes. A representative example for mouse TKT is shown in Fig. 3. Detailed characterization of 10 mitotic figures further
localized the TKT gene to the B1 region of mouse chromosome 16 and to
the p21.2 region of human chromosome 3 (Fig. 4).
Basis for Overexpression of TKT in DR Mutant
Clones
In order to gain further insight into the relationship
between the DR phenotype and overexpression of TKT, we examined TKT
gene copy number by Southern blot hybridization using mouse TKT cDNA as
a probe. As shown in Fig. 5, Southern blotting genomic DNA from
DS and DR cells using TKT cDNA gave similar bands of hybridization that
ranged in size from 1 to 12 kb, depending on the restriction
endonuclease used for digestion. In each case, hybridization signals
for the TKT gene were, on average, 28-fold more intense in the DR
mutant than in parental Y1 cells, suggesting that overexpression of TKT
resulted from gene amplification. In control experiments, hybridization
of enzyme-digested genomic DNA from DS and DR cells with cDNA probes
for the ACTH receptor (26) or the regulatory subunit of the
type 1 cAMP-dependent protein kinase (29) gave signals of
approximately equal intensity (data not shown).
nylon membrane, and hybridized to a TKT cDNA probe. In panel
B, EcoRI-digested genomic DNA from DS and DR cells was
electrophoresed on a 0.4% agarose gel, blotted and probed for mouse
immunoglobulin
light chain (
1) and mouse immunoglobulin
5. Fragment sizes were estimated using HindIII-digested
bacteriophage
and HaeIII-digested
X-174 as
standards.
1 and mouse immunoglobulin
5 (30) , were not amplified in the DR mutant clone.
Effects on Adenylyl Cyclase Activity
The
identification of p68 as TKT raises interesting questions about its
contribution to the DR phenotype and the mechanisms responsible for its
overexpression in DR cells(6) . We considered the possibility
that this enzyme, when overexpressed, protects adenylyl cyclase from
agonist-induced desensitization. TKT has not been implicated in the
regulation of the adenylyl cyclase system previously, and its function
in this regard was not readily apparent. We were unable to modify
adenylyl cyclase activity in broken DR cells by treatment with TKT
antiserum or by adding back purified enzyme to homogenates of parental
Y1 cells (data not shown), suggesting that increased expression of TKT per se is not responsible for the DR phenotype., and the rat glioma cell line,
C6TK
(25) . As we reported previously, Kin-8
cells, like the DR parent, resist ACTH-induced desensitization and
produce elevated levels of p68 (approximately 10% of total protein; (1) ), whereas in C6 cells, the adenylyl cyclase system is
readily desensitized upon continuous exposure to
-adrenergic
agonists such as isoproterenol (31) and the levels of p68 are
low (approximately 0.1% of total protein; (8) ).
C6TK hybrid
clones, H7 and H8, were amplified to the same extent and gave the same
restriction patterns as the Kin-8HGPRT
fusion partner (Fig. 7A) and parental DR cells (Fig. 5). Under these
same conditions of hybridization stringency, the mouse TKT probe did
not give a detectable signal for the TKT gene from the rat glial cell
line. As determined by Northern blot hybridization (Fig. 7B), TKT transcripts were markedly abundant in the H7
and H8 hybrids, reaching levels comparable to those seen in
Kin-8HGPRT
and parental DR cells; these levels of TKT
transcript were much higher than those seen in the C6TK
fusion partner or in DS cells. These results indicate that the
hybrid clones acquired and expressed the amplified TKT gene from the DR
parent and that TKT overexpression behaves dominantly in the hybrids.
glial hybrids. In panel A, Southern blots were
prepared from EcoRI-digested genomic DNA from DS cells, the DR
subclone Kin-8HGPRT, C6TK
, and the
hybrids H7 and H8. In panel B, Northern blots were prepared
from total RNA isolated from the same clones and from parental DR
cells. The blots were hybridized with a TKT cDNA probe as described
under ``Materials and Methods.'' Fragment sizes were
estimated using end-labeled HindIII-digested bacteriophage
and HaeIII-digested
X-174 as
standards.
cells, whereas the response to isoproterenol reflected the
contribution of C6TK
(Table 1). Despite the
presence of the amplified TKT gene and overexpression of TKT
transcripts, adenylyl cyclase in the hybrid clones was rapidly
desensitized upon exposure to ACTH (Fig. 8). Within 1 h of
exposure to ACTH, the hybrid clones lost 85% of their
hormone-responsive adenylyl cyclase activity. In contrast, the
Kin-8HGPRT
parent resisted ACTH-induced
desensitization and retained 70% of its ACTH-responsive activity after
6 h of continuous exposure to the hormone (Fig. 8). The
desensitization induced by ACTH in the hybrid clones was homologous,
since the hybrids retained 85-90% of their
isoproterenol-stimulated adenylyl cyclase activity after treatment with
ACTH (not shown).
glial hybrids. Kin-8HGPRT cells
(
) and the adrenal glial hybrids H-7 (
) and H-8
() were incubated with 7.5 nM ACTH
for the times indicated. After incubation, cells were washed free
of hormone, homogenized and assayed for ACTH-responsive adenylyl
cyclase activity in the presence of maximally effective concentrations
of ACTH
(20 µM). Results are
expressed as a percentage of the ACTH-responsive adenylyl cyclase
activity observed prior to desensitization (Table 1) and are the
average of two experiments carried out in
duplicate.
genes, are not amplified
in the DR mutant (Fig. 5) and thus must be too far away from the
TKT gene to have been included in the amplicon. Although the basis for
amplification of the TKT gene is unknown, it is interesting that TKT
shares structural and functional homology with the RecP protein of Streptococcus pneumoniae, a protein required for genetic
transformation that functions to promote insertion-duplication
mutations in the prokaryotic chromosome(38, 39) . As
reviewed elsewhere (40, 41) , gene amplification may
occur through a number of different mechanisms, may involve
recombination events (including gene insertions, deletions, and
inversions), and is sometimes associated with telomeric fusions (e.g.Fig. 6). Amplified genes can exist as
self-replicating minute chromosomes or as arrays of amplified segments
on one or more chromosomes(40) , as seen in the case of TKT
amplification in the DR mutants (Fig. 6).
)
Dr. Vera Mihajlovic-Madzarevic participated in the
isolation and partial sequencing of the gt10 clone 18. MaryKay
Greer and Luba Foux provided technical assistance. Iodinated protein A
was generously provided by Dr. C. C. Yip, the TKT 256-233
oligodeoxynucleotide was synthesized by S. DeLeon, and genomic probes
for mouse immunoglobulin
light chain and mouse immunoglobulin
5 were gifts from Dr. Gillian Wu (University of Toronto, Toronto,
Ontario, Canada). ACTH
(Cosyntropin) from Organon
Inc., West Orange, NJ and ACTH
(Acthar) from
Rhone-Poulenc Rorer Canada Inc., Montreal, Quebec were provided as
generous gifts from the manufacturers.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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