(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) ()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.
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
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.
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) 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.
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 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.
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 1 and mouse immunoglobulin
5 (30) , were not amplified in the DR mutant clone.
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, 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) ).
As
determined from Southern blot hybridization analysis using the mouse
TKT probe, the TKT genes in two independently isolated
Kin-8HGPRT
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.
Figure 7:
Amplification of the TKT gene in adrenal
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.
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 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).
Figure 8:
Desensitization of adenylyl cyclase in
adrenal 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.
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 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).
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].