(Received for publication, February 7, 1994; and in revised form, October 7, 1994)
From the
In primary rodent cells transformed by the E1A region of the
highly oncogenic adenovirus type 12, repression of transcription
mediated by the far upstream TATA-like element was observed only in
conjunction with either possible juxtaposition of a CAA repeated
element in the presence of E1A and was dependent upon the relative
arrangement of both the TATA-like and CAA repeated motifs in both
homologous and heterologous promoter constructs. A gel shift
competition study demonstrated that the TATA-binding protein (TBP) or a
TBP-like protein can bind to both the upstream TATA-like sequence and
the regular TATA box on the H-2K basal
promoter. Moreover, employing immunoselection and cyclic amplification
and selection of targets (CASTing) methods with nuclear extracts
derived from Ad12-E1A transformants, we have identified a high affinity
binding site in the H-2K
class I promoter
for E1A-associated DNA-binding proteins. The sequences of the binding
sites were identified and were found to contain both the upstream
TATA-like motif and the CAA repeated motifs. Our results suggest that
the TATA-like sequence in the far upstream region of the H-2K
gene is one of the elements that is
required for Ad12-E1A-mediated negative repression.
Primary rodent cells transformed by the E1A region of the highly
oncogenic adenovirus type 12 (Ad12) but not by the same region of the
non-oncogenic virus Ad5 (or Ad2) (1) express the reduced level
of the products of major histocompatibility complex (MHC) ()class I genes(2, 3) . The lower levels of
MHC class I mRNA in Ad12-transformed cells reflect decreased rates of
transcription of the endogenous class I genes. This down-regulation of
transcription is mediated by the 1,266-amino acid 13S product of the
Ad12-E1A
region(4, 5, 6, 7, 8, 9, 10) .
The resultant decrease in cell-surface levels of MHC class I antigens
is reflected by the lower susceptibility of Ad12-transformed cells to
allogenic cytotoxic T cells and NK cells, suggesting a model by which
Ad12-transformed cells escape immune surveillance and develop into
proliferating and evasive
tumors(4, 5, 11, 12) .
Control of
the initiation of transcription of the mouse H-2K MHC class
I gene has been studied extensively, and the regulatory region has been
shown to contain the common CCAAT and TATA regions as well as multiple cis-acting regulatory elements. The best characterized of
these elements are RI, RI`, and R2 in the CRE/IRS
region(3, 13, 14, 15) , which have
also been defined by a footprinting study in
vivo(16) . Several transcriptional factors have been shown
to bind to these
elements(17, 18, 19, 20, 21, 22, 23, 24, 25, 26) .
The level of binding to the R1 element has been shown to be higher in
the case of extracts from Ad5-transformed cells than in the case of
extracts from Ad12-transformed
cells(27, 28, 29) . The R2 binding activity
is significantly higher in extracts from Ad12-transformed cells than in
extracts from Ad5-transformed cells or parental
lines(7, 28, 30) . The poor enhancer activity
of the R1 site in Ad12-transformed cells is correlated with increases
in the extent of binding of nuclear factors to the R2 element,
suggesting the presence of a repressor whose effects are mediated via
the R2 site. Kralli et al.(31) demonstrated recently
that the putative R2-binding repressor protein, designated R2BF, is
similar to members of the family of thyroid hormone/retinoic acid
receptors.
We showed previously that a distal 5`-element
(-1,521 to -1,837 relative to the start site of
transcription) that contributes to E1A-mediated negative regulation is
included in the promoter region of the H-2K gene(9) . The CAA repeated sequences in regions
-1,736 to -1,689 and -1,616 to -1,535 are both
necessary for full negative regulation of the H-2K
gene by E1A. We report here that an additional element, a
TATA-like sequence, in the far upstream region of the 5`-flanking
sequence (-1,773 to -1,767) is also a key element in the
negative regulation of expression of the MHC class I H-2K
gene by E1A in conjunction with
either upstream or downstream CAA repeats. A gel shift competition
assay demonstrated that the factor that binds to the far upstream
TATA-like sequence can also bind to the TATA-box sequence in the basal
promoter. Using an immunoselection and cyclic amplification and
selection of targets (CASTing) method, we also obtained direct evidence
that Ad12-E1A can associate with the proteins that bind to the
TATA-like sequence and to the CAA repeats in Ad12-E1A-transformed
cells.
Plasmids-The deletion and substitution CAT
mutants, namely, del-4ml, del-4m2, del-5ml, and del-5m2 were
constructed as follows. The BspHI/RsaI DNA fragment
of a distal 5`-flanking sequence of the H-2K gene, including the element CTGTAAGCCAGACCC or the TATA-like
sequence TATTAAA, was mutated to either ATGTAAGAAAGAAAC or TAGCGAA,
respectively, and ligated to the RsaI/DdeI DNA
fragment (-1,735/-1,188) or the BstNI/Sau3A fragment of a distal 5`-flanking sequence
of the H-2K
gene
(-1,615/-1,534) with 4-6-bp BamHI or HindIII linkers of both ends of the DNA. The resultant
fragments were inserted into the BamHI site or the HindIII site of pH-2K
(367)CAT (32) to
generate appropriate CAT derivatives in the correct or reversed
orientation. The TATA-like sequence in the BspHI/RsaI
DNA fragment was converted to various TATA-like sequences found in the
promoters of the H-2K
, adenovirus major
late promoter (AdML), hsp70, c-fos, histone,
-globin,
adenoviruses E3 and E4, and SV40 early genes (see (33) ) by
site-directed mutagenesis (using a kit from Amersham Japan, Tokyo,
Japan) and the appropriate primers. In each case, overhang was
generated by EcoRI, and the DNA fragment was either inserted
into the EcoRI site of pH-2K
(367)CAT(CAAu) or
coinserted with the DNA fragment RsaI/DdeI
(-1,735/-1,688) into either the EcoRI site of
pSV2CAT (34) or the HindIII site of pBLCAT2 (35) to generate various CAT plasmids ( Fig. 4and Fig. 5).
Figure 4:
Relative promoter activity of
pH-2KCAT constructs with various TATA-like sequences and a
CAA repeated motif. The histogram represents the CAT activity of the
pH-2K
CAT constructs in CYpAdC3 cells or Ad5-E1A-transformed
3Y1 cells, as indicated. All CAT values are the averages of results
from at least five transfections. Normalized CAT activity associated
with pH-2K
(367)CAT was taken arbitrarily as 1.0. The
standard deviation for each result is indicated by a bar.
Figure 5:
Effects of combinations of various
TATA-like sequences in the upstream region and various proximal
promoters in Ad12-E1A- or Ad5-E1A-transformed cells. Average results of
five independent experiments are shown in the histogram as CAT
activities relative to activity associated with
pH-2K(367)CAT in CYpAdC3 cells, which was arbitrarily taken
as 1.0. P+E is the H-2K
promoter/enhancer
region. The standard deviation for each result is shown by a bar.
Separate plasmids containing the H-2K minimum promoter (pH-2P) and the H-2
enhancer (pH-2E) joined to the basal promoters of the early SV40,
thymidine kinsase, and conalbumin genes were generated as described
elsewhere(10) . Sequences of constructs were verified by the
dideoxy chain termination method(36) . Plasmids pE1A,
pE1A12-12S, pE1A12-13S, pE1A5-13S,
pE1A5-12S(37, 38) ,
pH-2K
(2015)CAT(9) , and pRSV
-gal (39) have been described previously.
Figure 1:
Deletion
study of the E1A-responsive negative element in the upstream region of
the promoter of the H-2K gene. A
schematic representation of the CAT constructs derived from
pH-2K
(367)CAT (32) and their relative CAT
activities is shown. Dotted lines represent internal
deletions. Relative CAT activities of mutants were measured in
Ad12-E1A-transformed CYpAdC3 cells(9) , in Ad12-infected or
Ad5-infected baby mouse kidney cells, and in uninfected baby mouse
kidney cells. Normalized CAT activity associated with
pH-2K
(367)CAT was taken arbitrarily as 1.0. The boxes represent TATA-like sequences and the upstream and downstream CAA
repeated sequences (TATTAAA, CAAu, CAAd). Shaded boxes represent a mutated TATA-like sequence (TAGCGAA). Empty
oval, upstream element, 5`-CTGTAAGCCAGACCC-3` (-1,788 to
-1,774); striped oval, mutated upstream element,
5`-ATGTAAGAAAGAAAC-3` (-1,788 to -1,774). The construction
of CAT plasmids is described under ``Materials and
Methods.''
Figure 2:
Relative promoter activities of H-2K-CAT constructs with the upstream
TATA-like sequence and a version of the CAA repeated motif in 3Y1 cells (panel A) and E1A-transformants obtained with Ad12-13S
E1A (panel B), Ad12-12S E1A (panel C),
Ad5-13S E1A (panel D), and Ad5-12S E1A cDNA (panel E). Normalized CAT activity associated with
pH-2K
(367)CAT in 3Y1 was arbitrarily taken as 100. All CAT
activities are the averages of results from at least five
transfections, and the standard deviation for each is indicated. A, pH-2K
(2015)CAT ((9) ); B,
pH-2K
(367)CAT; C, del-4m1CAT; D,
del-5m1CAT.
Figure 3:
Effects of the nature of the upstream
TATA-like sequence in the promoter region of the H-2K gene. A, effects of the
relative positions and the orientations of the upstream TATA-like
sequence and the CAA repeated motif on the activity of the H-2K
promoter. The orientation of each
upstream TATA-like sequence and CAA repeated motif is indicated by an arrow. Average results of four independent experiments are
shown in the histogram as CAT activities relative to that expressed by
pH-2K
(367)CAT. Normalized CAT activities of
pH-2K
(367)CAT in CYpAdC3 cells and Ad5-E1A-transformed 3Y1
cells (lane 1) were taken arbitrarily as 1.0. B,
activity of the H-2 enhancer/silencer linked to heterologous promoters
or to the homologous basal promoter in Ad12-E1A- or Ad5-E1A-transformed
cells. All CAT values are the averages of results from at least four
transfections. Normalized CAT activity associated with
pH-2K
(367)CAT in CYpAdC3 cells was taken arbitrarily as
1.0. The orientations of the upstream TATA-like sequence and the CAA
repeats are indicated by arrows. TATA, upstream
TATA-like sequences (BspHI/RsaI); CAAu,
upstream CAA repeated motif (RsaI/DdeI); CAAd, downstream CAA repeated motif (BstNI/Sau3A). H-2E, H-2 enhancer; H-2P, H-2 promoter; H-2P/E, H-2 promoter/enhancer; TK, thymidine kinase promoter; SV, SV40 promoter; Cona, conalbumin promoter. The standard deviation for each
result is indicated by a bar.
Previous studies have
demonstrated that monomers and dimers of the CAA repeats do not
function as negative elements in Ad12-E1A-mediated repression of the H-2K gene(10) . Therefore, we next
examined whether the upstream TATA-like sequence might affect the H-2K
promoter activity of CAT plasmids with
oligomerized CAA repeated motifs. To our surprise, the effect of the
TATA-like sequence on the H-2K
promoter activity
was apparent in conjunction with monomeric or dimeric CAA motifs (data
not shown). This effect was also sensitive to the relative orientations
of the TATA-like sequence and the CAA motifs but not to the relative
positional effects of the two elements (data not shown).
Figure 6:
Gel shift studies. A, competition
in gel shift assays with oligonucleotide DNA probes that correspond to
the upstream TATA-like sequence (-1,755 to -1,776) and the
proximal TATA-box sequence (-8 to -28), using nuclear
extracts from CYpAdC3 cells. Lane 1, DNA probe labeled with T4
kinase; [P]TATAu,
5`-ggacgctggaTATAAAgtccacgcagcccgc-3`; lanes 2, 4,
and 5, [
P]TATAd DNA probe,
5`-agccagacccTATTAAAtgtctccctttaga-3`; lanes 3, 6,
and 7, [
P]TATAu DNA probe; lanes 4 and 6, 500 ng of TATAu competitor oligomer; lanes 5 and 7, 500 ng of TATAd competitor oligomer. An arrowhead indicates the protein-DNA complex. B,
effects of TBP-specific antiserum on the shifted complex in the gel
shift assay. Lane 1, free DNA probe (AccI/DdeI); lane 2, nuclear extract from
CYpAdC3 cells; lanes 3-5, 10
,
10
, and 10
dilutions of rabbit
antiserum against human TBP (referred as anti-24-28); lanes
6-8, 10
, 10
, and
10
dilutions of mouse antiserum against human TBP; lanes 9 and 10, anti-24-28 (10
and 10
dilutions, respectively) that had been
preincubated with TBP protein (24 µg), which had been purified as
described elsewhere(42) ; lanes 11 and 12,
preimmune rabbit control serum (10
and
10
dilutions, respectively). Arrowheads indicate protein-DNA complexes (B1, B2, and B3). C, in vitro translation of
[
S]methionine-labeled TBP in a rabbit
reticulocyte lysate. Lane 1, pGEM1 without the rTBP sequence; lane 2, 500 ng of rTBP/pGEM1; lane 3, 1 µg of
rTBP/pGEM1. The arrowhead on the right indicates the
recombinant TBP. D, binding properties of in vitro translated rTBP. Gel shift analysis used
P-radiolabeled oligonucleotides (0.1 pmol) that
corresponded to the upstream TATA-like sequence in the H-2K
promoter, as described
elsewhere(42) . Approximately 20 fmol of
[
S]methionine-labeled TBP (lanes
2-5) were used in gel shift assays in the presence or
absence of a 500-fold molar excess of synthetic oligonucleotide
duplexes that corresponded to TATAu, TATAd, and a mutated TATA sequence
(TATAm; 5`-agccagacccTAAATTAtgtctccctttaga-3`). An arrowhead indicates the rTBP-DNA complex.
Figure 7:
Immunoselection and CASTing studies.
Cloning of the binding sites in the H-2K
promoter region by immunoselection and CASTing is shown. A,
preparative gel shift assay using DNA fragments isolated by
immunoaffinity precipitation with Ad12-E1A-specific or Ad5-E1A-specific
antiserum and PCR cycling.
P-labeled DNA fragments (5
10
cpm) isolated from the sonicated DNA from the H-2K
promoter region by immunoselection
and PCR cycling were incubated with an extract from CYpAdC3 cells or
Ad5-E1A-transformed 3Y1 cells. B1 and B2, DNA-protein
complexes; F, free DNA probe. Lane 1, extract of
CYpAdC3 cells; lane 2, extract of 3Y1 cells; lane 3,
extract of Ad5-E1A-transformed 3Y1 cells after passage through a
heparin column (see (10) ). B, agarose gel 1.2%
electrophoresis of the DNA fragments after immunoselection with
E1A-specific antibody and the CASTing assay. Lanes 1-6,
isolation of binding site DNAs; lane 7, HaeIII digest
of
174; lane 8, HindIII digest of
. C, gel shift assays of representative DNA fragments
isolated by immunoselection and CASTing. Lanes 1-5, DNA
fragments with mutations in the TATA-like sequence (TATTAAA) to TAGCGAA
as the gel shift probe and the nuclear extract from CYpAdC3 cells; lanes 6-10, representative examples of DNA fragments
used as DNA probes for gel shift assays with a nuclear extract from
CYpAdC3 cells; lanes 11-15, DNA probes used in lanes
6-10 as probe and an extract from Ad5-E1A-transformed 3Y1
cells. An arrowhead indicates the protein-DNA complex. D, effects of Ad12-E1A-specific antiserum on the shifted
complex. One of the representative DNA probes (5
10
cpm) isolated as described above was incubated with an extract of
CYpAdC3 cells in the presence of increasing levels of Ad12-E1A-specific
or Ad5-E1A-specific antiserum.
Lane 1, free DNA
probe; lane 2, nuclear extract from CYpAdC3 cells; lanes
3-6, 10
, 10
,
10
, and 10
dilutions of
Ad12-E1A-specific antiserum; lanes 7 and 8,
10
and 10
dilutions of
Ad5-E1A-specific antiserum (M73); lane 9, 10
dilution of Ad12-E1A-specific antiserum that had been
preincubated with Ad12 E1A protein (10
µg).
Figure 8:
Nucleotide sequence analysis of DNA clones
isolated by immunoselection and CASTing. A, the major or minor
binding sites indicate the nucleotide sequences of the putative cis-element of the DNA fragments cloned in pBluescript. The
frequency of the clones containing insert DNAs of the major binding
sites is more than 25% in total recombinant clones obtained. A number indicates the distance from the cap site of the H-2K gene. B, size distribution
of the DNA fragments isolated by immunoselection and CASTing. The sizes
of DNA fragments cloned in pBluescript were analyzed statistically. Upper panel, for the extract from CYpAdC3 cells; lower
panel, for the extract from Ad5-E1A-transformed 3Y1 cells.
Putative cis-elements are represented in the
figure.
We previously identified the CAA repeats in the far upstream
region of the mouse MHC class I H-2K gene as
negative regulatory elements by using the product of E1A gene of
adenovirus type 12(9, 10) . We have now provided
evidence that the TATA-like sequence located between -1,773 and
-1,767 is also required for the E1A-dependent negative regulation
of the MHC class I H-2K
gene in conjunction with
one of two CAA repeated elements. A TATA-like element with either an
upstream CAA repeat or a downstream CAA repeat is functional (see Fig. 1). However, whereas such a TATA-like element can function
independently of its relative position and distance from the promoter,
its function is dependent on its relative orientation with respect to
the CAA repeats and the TATA-like sequence. A TATA-like sequence in an
inverted orientation plus one of two CAA repeats is associated with
decreased E1A-dependent negative regulation (Fig. 3A).
We failed to observe such a negative function of a TATA-like element in
an orientation-dependent but position- and distance-independent manner
in normal cells and Ad5-E1A-transformed cells (Fig. 3A). Moreover, we found that the combined
negative activity of the upstream TATA-like sequence with CAA repeats
appeared to be specific to the 13S E1A from Ad12 and not to the 12S E1A
or the 13S E1A from Ad5 (Fig. 2).
To determine the way in which the repression of the MHC class I gene functions in the context of heterologous basal promoters, a DNA fragment containing one CAA repeated sequence was linked, in separate constructs, to the SV40 early, herpes simplex virus type 1, and chicken conalbumin basal promoters. We did not detect any major difference in the suppressive activity on these heterologous promoters as compared with the H-2 basal promoter-enhancer combination in Ad12-E1A transformants (Fig. 3B). Our results are consistent with the previous report by Ge et al.(30) . Thus, the H-2 TATA-box sequence per se may not be important since its conversion to other TATA-box sequences in the promoter failed to affect transcription of the H-2 gene in Ad12-E1A-transformed cells. However, the suppressive activity of these elements on heterologous promoters was less significant in Ad5-E1A-transformed cells (Fig. 3B).
We next examined the nature of the optimum TATA-like sequence in the
upstream region for E1A-mediated negative regulation. Examples of
endogenous cellular and viral genes that are activated as a result of
the E1A trans-activation process include the heat-shock gene
for hsp70(46, 47) , the gene for hsp89(48) ,
the gene for -tubulin (49) , the c-fos gene(50) , the viral E1B gene(51) , and the long
terminal repeat of human HIV(52) . A binding study in vitro demonstrated that Ad-E1A binds to TFIID(53, 54) .
The correlation between the much greater trans-activation by
the 13S E1A protein than by the 12S E1A protein (56, 57) and the greater affinity of TFIID for 13S-E1A (53, 54, 55, 56) suggests that the
E1A-TFIID interaction plays a fundamental role in E1A trans-activation in nononcogenic E1A-transformed cells.
Therefore, we attempted to change the far upstream TATA-like sequence
of the H-2 promoter to the TATA-box sequences from heterologous genes
to examine the effects of these elements on E1A-mediated negative
repression. As shown in Fig. 4, TATA-box sequences from H-2K
and Ad-ML (as well as from hsp70 and c-fos genes) were all able to function as upstream TATA-like
elements in mediating the E1A-dependent repression of the H-2K
promoter (lanes A, B, and C). Moreover, it is clear that the upstream TATA-like sequence
is preferentially functional in conjunction with either an upstream or
a downstream CAA element ( Fig. 1and Fig. 5). In
addition, the results of our immunoselection and CASTing studies
suggest that nuclear proteins present in Ad12-E1A-transformed cells can
bind to DNA fragments that contain the TATA-like sequence and the basal
TATA-box sequence in cooperation with E1A molecules ( Fig. 7and Fig. 8). To our surprise, we found that the majority of DNA
fragments isolated by the immunoaffinity and CASTing method by use of
an extract from Ad12-E1A transformants contained the upstream TATA-like
sequence and the CAA repeated motif. By contrast, most of the DNA
fragments isolated by use of an extract of Ad5-E1A transformants
contained the basal TATA-box and H-2 enhancer (for example, CRE/IRS
(R1/R2) sequences) (Fig. 8B). Addition of antiserum
against Ad12-E1A to the reaction mixture used for the gel shift assays
with a DNA fragment that contained the upstream TATA-like sequence as
probe resulted in the disappearance of shifted bands (Fig. 7D). Addition of antiserum against Ad5-E1A did
not have such an effect (Fig. 7D).
What is the
nature of the binding proteins that associate with the DNA sequence of
the far upstream CAA repeats and the TATA-like element, and how do
these proteins interact with TBP (or a TBP-like protein) to achieve
negative regulation of the H-2K gene in Ad12-E1A
transformants but not in Ad5-E1A transformants? Gel shift experiments
using competitive oligonucleotides designed on the basis of binding
sites, recombinant TBP, and TBP-specific antiserum revealed that
similar or identical molecules, possibly including TBP, bound to both
the upstream TATA-like sequence and the basal TATA-box sequence in
vitro and in vivo (Fig. 6). We speculate that the
similar or identical TBP (or TBP-like protein) can bind to both TATA
sequences in parent 3Y-1 cells and Ad5-E1A-transformed cells. Several
lines of experimental evidence have suggested that E1A-CR3 of
nononcogenic adenovirus binds in vitro specifically and stably
to the isolated TBP of
holo-TFIID(53, 54, 55, 56) . Recent
studies support this model and provide direct evidence that trans-activation is mediated through a direct physical
association between the E1A-CR3 domain and TBP in the holo-THIID
complex(55, 56) . We also observed that the affinity
of Ad12-13S E1A for TBP is much stronger than that of
Ad5-13S E1A protein for TBP in vitro (data not shown).
The affinity of TBP (or TBP-like protein) for the region of the
upstream TATA-like element and the CAA repeated motifs is weak in
Ad5-transformed cells, about 10-fold lower than that in Ad12-E1A
transformants (see (10) ). Recently we succeeded in
characterizing the recombinant proteins that bound to the CAA repeats
and were associated with Ad12-E1A protein in vitro (data not
shown). In the presence of Ad12-E1A, E1A associates with TBP on the
upstream TATA-like element and the TATA-box to form a protein complex
that includes TBP, proteins that bind to CAA repeats, and E1A.
Alternatively, E1A may modify the binding affinities within complexes
that include TBP to allow more efficient binding to the upstream
TATA-like sequence. In this way, the basal transcriptional activity of
the H-2K
gene can be decreased. The formation of a
large protein complex including TBP and proteins that bind to CAA
repeats might be critical for association with E1A in Ad12-E1A
transformants but not in Ad5-E1A transformants. We also cannot exclude
the possibility that the distinct sequences of respective E1A proteins
may be involved in the binding to the TBP or to a TBP-like protein to
cause the conformational changes that alter the transcriptional
machinery of the H-2K
gene. Preliminary studies by
glycerol gradient centrifugation indicate that a large complex of these
components derived from Ad12-E1A transformants but not from Ad5-E1A
transformants or parent cells is formed in vitro. (
)We speculate that the ability to form such a large
complex, which includes TBP, the proteins that bind to CAA repeats, and
E1A, might be critical for regulation of the H-2K
gene. Kralli et al.(31) and Ge et al.(30) reported that the R2 class I enhancer element is
involved in Ad12-E1A-mediated and not in Ad5-E1A-mediated repression in
transformed cells. We cannot rule out such a possibility since we
detected moderate repression of the promoter activity of H-2 constructs
with the R1/R2 region in the presence of Ad12-E1A (see (10) ).
At this time we cannot explain the possible functional relationship
between R2BP(31) , COUP-TF, which has been identified more
recently by Liu et al.(58) , and the factors with
which we have been dealing here. It is possible that dual regulation,
involving proximal and far upstream elements, might be important for
the full negative control of the activity of the H-2 promoter by
Ad12-E1A.
The Ad12-E1A-dependent negative repression was independent
of the nature of the heterologous promoters tested but was
preferentially observed with the specific common sequence TAT(A/T)AA.
Recent studies by Brou et al.(59) demonstrated that
distinct TFIID complexes mediate the effects of different types of
transcriptional activation. Similarly, different complexes that
included TBP may be formed and may bind to the upstream TATA-like
sequence or to the proximal TATA sequence in different ways. Further
characterization of the genes that encode the proteins that bind to the
TATA-like sequence and to the basal TATA-box should help us to
elucidate the mechanism of regulation of transcription of the MHC class
I H-2K gene.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X15104[GenBank].