ABSTRACT
Alterations in the amino acid sequence of the estrogen receptor (ER) have been
shown to have dramatic effects on its function. Recently, mutant ERs have been
isolated from both clinical samples and established breast cancer cell lines,
primarily through the use of the polymerase chain reaction (PCR). All
previously reported mutations have given rise to either alterations or
truncations of the ER protein. We determined the structure of a novel 80 kDa ER
which is expressed in an estrogen independent subclone of the MCF-7 human breast cancer cell line (MCF-7:2A). This 80 kDa ER was initially detected by Western blot
analysis using a variety of ER specific antibodies. PCR mapping and partial PCR
mediated subcloning of the ER cDNA were used to demonstrate that this protein
was an ER containing an in-frame duplication of exons 6 and 7. This type of duplication has not been
previously described for any members of the steroid receptor superfamily.
Karyotype analysis coupled with fluorescence
in situ
hybridization (FISH) demonstrated that MCF-7:2A cells contained 4-5 copies of the ER gene in contrast to 2 copies in MCF-7:WS8 cells. The ER gene was localized by FISH analyses in
both the MCF-7:WS8 and MCF-7:2A cells on chromosome 6, which is the source of the ER in normal
human cells. The relative expression level of 2:1 is consistent with DNA gene
dosage analysis. Genomic PCR was then used to demonstrate that the 80 kDa ER
mRNA was not derived from the trans-splicing of two ER mRNAs but was the result of a genomic rearrangement in
which exons 6 and 7 were duplicated in an in-frame fashion. This variant ER may prove to be useful in elucidating the
mechanism of estrogen action in breast cancer cells.
The estrogen receptor (ER) is a ligand activated, transactivating protein that
is involved in the growth control of ER positive breast cancer cells and other
estrogen target tissues (
1
). In order to study the mechanisms responsible for the development of estrogen
independent growth in the human breast cancer cell line MCF-7, we have derived a subclone (i.e., MCF-7:2A) which grows maximally in the absence of estradiol (
2
). The long term growth of MCF-7 cells in the absence of estrogens leads to the development of a uniform
phenotype characterized by estrogen independent, but antiestrogen sensitive,
growth and elevated expresssion of the ER (
3
,
4
). The MCF-7:2A clone shares these characteristics in addition to the expression of a
unique 80 kDa ER. In this paper we describe the molecular structure and genomic rearrangement
responsible for the genesis of this mutant ER.
The cloning of the human ER made possible the analysis of its molecular
structure and examination of the expression and function of variant ERs (
5
-
7
). The wild type ER is a 66 kDa protein coded by a 6.6 kb mRNA, comprised of
five functional domains derived from eight exons. The three domains of the ER
which show the greatest degree of sequence homology are the A domain which, in
concert with the B domain, contains the transactivation function #1 (AF-1), the C domain which contains two zinc-fingers and is responsible for DNA binding and the E domain which is
responsible for ligand binding as well as the ligand inducible transactivation
function #2 (AF-2) (
8
).
A vast literature exists describing mutant ERs with one or more changes
(reviewed in ref.
9
). Generally, these mutants were derived from either of two sources, mutants
found in clinical or laboratory samples that have been initially identified
using RT-PCR (
10
-
15
) or variant ERs constructed through site directed mutagenesis (
6
,
16
-
22
). Both sources have given insight into the functions of the ER protein.
A number of splicing mutants have been isolated from clinical samples. These
mutant mRNAs demonstrated the loss of one or more exons. The activity of some
of the proteins derived from these mutant mRNAs were subsequently studied
through their expression in heterologous systems. Examples include an exon 3
deletion which abrogates DNA binding and causes complete inactivation of the ER
as a sequence specific transcriptional activator. Deletion of exon 5 leads to
an ER of ~41 kDa that acts as a constitutive activator of estrogen responsive genes
when expressed in a yeast system. The exon 7 deletion mutant gives rise to a ~52 kDa ER that behaves as a dominant negative regulator of the wild type ER
function in a similar yeast system (
23
).
In this paper we describe a novel mutant 80 kDa ER that was isolated from a
subclone of the widely studied MCF-7 human breast cancer cell line. Our finding is unique in that this mutant
protein is expressed at readily measurable levels in the MCF-7:2A cells and has not been seen in any other human breast cancer cell
line.
MCF-7 cells were obtained from Dean Edwards (at the San Antonio Breast Cancer
Group, TX) (originally obtained from the Michigan Cancer Foundation Detroit,
MI). T47D (
24
) and MDA-MB-231(
25
) cells were obtained from American Type Culture Collection, Rockville, MD. All
tissue culture components were obtained from GIBCO Laboratories, Grand Island,
NY unless otherwise stated. MCF-7:WS8 and T47D:A18 cells were grown in RPMI 1640 containing 10% heat
inactivated fetal bovine serum (Bioproducts for Science Inc., Indianapolis, IN)
6 ng/ml bovine insulin, 2 mM l-glutamine, 100 U/ml penicillin, 100 [mu]g/ml streptomycin and 250 ng/ml amphotericin B (fully estrogenized
medium). MCF-7:2A and MDA-MB-231:10A cells were routinely grown in estrogen-free medium which substitutes phenol-red free RPMI and 3* dextran-coated charcoal treated fetal bovine
serum. Cells were passed at 1:10-1:20 dilutions once per week using 0.1% trypsin.
Whole cell extracts were prepared by direct lysis of PBS washed cells in sample
buffer (10% glycerol, 150 mM Tris-HCl pH 6.8, 0.5 mM EDTA, 0.125% SDS, 1% [beta]-mercaptoethanol and 5 [mu]g/ml bromphenol blue) followed by immersion in a boiling
water bath for 5-10 min. Equal amounts of protein were run in a standard Western blot as
described previously (
2
) with the following changes. The secondary antibody used was a horseradish
peroxidase (HRP) conjugated goat anti-rat antibody, (HyClone Laboratories, Logan, UT) and visualization was
accomplished using the ECL visualization kit (Amersham Corp., Arlington
Heights, IL) as per the manufacturers directions. The membrane was then wrapped
in plastic film and exposed to Kodak X-Omat film for 15 s and developed.
MCF-7:WS8 and MCF-7:2A cells were prepared for FISH by incubating actively growing
cultures with 0.1 mg/ml of colcemid for 3 h. After exposure to a hypotonic
solution of 0.075 M KCl at 37oC for 20 min, the cells were then fixed in 3:1 methanol-glacial acetic acid three times. Slides were made by dropping the
fixed cells onto cold wet slides and air dried. FISH was performed on both
interphase and metaphase cells from MCF-7:WS8, MCF-7:2A and normal human peripheral blood, using a digoxingenin-labeled ER DNA probe (Oncor, Gaithersberg, MD). The protocol
was based on the manufacturer's recommendations with some modifications.
Briefly, the fixed slides were denatured in a solution containing 70% formamide
and 2* SSC pH 7.0 for 2 min at 72oC, dehydrated in a pre-chilled ethanol series and allowed to air dry. A hybridization
mixture consisting of 65% formamide in 2* SSC, 0.03 mg/ml herring sperm DNA and 10 mg/ml of digoxingenin-labeled ER DNA probe was applied to each slide. The slides were
then overlaid with a coverslip and incubated overnight at 37oC. Post-hybridization washes consisted of rinsing the slides for 20 min in
50% formamide in 2* SSC at 43oC, followed by two changes in 2* SSC for 10 min each at 37oC. The hybridized probe was detected with an
immunodetection system using a commercial fluorescein-conjugated anti-digoxingenin antibody. Chromosomes and nuclei were counterstained
with propidium iodide and the slides were mounted in antifade solution.
Hybridization signals were visualized using an MAX-BX40 Olympus fluorescence microscope equipped with a dual color filter. A
minimum of 50 metaphases and 100 interphases were examined for each cell line.
Total RNA was prepared from MCF-7:WS8 and MCF-7:2A cells grown in estrogen-free medium by lysis in 4 M guanidine isothiocyanate followed
by discontinuous cesium chloride gradient centrifugation through a 5.7 M
cushion. Total RNA, 5 [mu]g per reaction, was reverse transcribed using MMLV reverse transcriptase
primed with oligo (dT)
12-18
(GIBCO BRL, Gaithersburg, MD). PCR was then performed using ~5% of this reaction and 100 ng of each appropriate primer (Oligos Etc.,
Wilsonville, OR) in a 50 [mu]l reaction with 2.5 U of
Taq
polymerase (Boehringer Mannheim, Indianapolis, IN). PCR was run for 40 cycles, 1
min at 60oC, 2 min at 72oC and 94oC for 1 min in a DNA Thermal Cycler (Perkin-Elmer-Cetus, Foster City, CA). A portion of this reaction
was then run on a 1.4% agarose gel containing 0.5 [mu]g/ml ethidium bromide and photographed. Specific PCR was performed using the
same conditions with a downstream primer (D80) that was specific for the
junction of the 3' end of exon 7 and the 5' end of exon 6.
Following PCR using the primer set U4-D4, the ~800 bp band was gel purified and subcloned into pUC18 using the Sure
Clone Ligation kit (Pharmacia Biotech, Piscataway, NJ). Clones containing
appropriate inserts were then sequenced using a standard dideoxy chain
termination method. The universal and reverse primers were specific for regions
flanking the multiple cloning site in pUC18 and were purchased from Pharmacia.
The sequencing reaction was performed using Sequenase
®
T7 DNA Polymerase (version 2.0, United States Biochemical, Cleveland, OH) as
per the manufacturers instructions.
High molecular DNA was prepared using standard methods (
26
). Total genomic DNA, 100 ng per reaction, was amplified using the XL PCR kit as
per the manufacturer's directions (Perkin-Elmer-Cetus, Foster City, CA). Fifteen cycles were run using 93oC for 1 min followed by 68oC for 12 min. This was followed by 16 additional cycles as
described above with a 15 s per cycle extension. A final 20 min extension
segment at 72oC was then performed and the samples were run on a 1% TBE agarose gel
containing ethidium bromide. The gel was illuminated on a Foto/Prep I (Fotodyne
Inc, Hartland, WI) and photographed.
The expression of the ER can be readily measured through the use of standard
Western blotting techniques of unfractionated whole cell extracts from human
breast cancer cell lines. As shown in Figure
1
, the MCF-7:WS8 and T47D:A18 cell lines express a single ER protein that migrates at
66 kDa in a denaturing SDS polyacrylamide gel. The ER negative cell line MDA-MB-231:10A displays no ER signal, as expected. The MCF-7:2A subclone, however, clearly expresses both the wild type
66 kDa ER as well as a species that migrates at ~80 kDa. This species has been shown to react with three other antibodies to
the ER (ref.
2
and data not shown).
In order to investigate the chromosomal location of the ER genes, karyotype
analysis was performed on both the MCF-7:2A and MCF-7:WS8 cells. Giemsa banding studies confirmed that the MCF-7:WS8 and MCF-7:2A cells are both very evolved hypotetraploid lines
with model chromosomes numbers of 68 and 69, respectively. While 2/3 of these
chromosomes are marker chromosomes, ~70% of these markers are found in both cell lines, strongly indicating that
both cell lines are derived from a common parent line (data not shown). The
human ER has been previously mapped to the long arm of chromosome 6 at band 6q25.1 (
27
; Fig.
2
A). (The accepted designation for the estrogen receptor gene in cytogenetic
studies is ESR. However, in this section we will continue to use the abbreviation ER in order to maintain consistency with
the rest of the paper.) Karyotpe analysis determined that each cell line
expressed four copies of chromosome 6. Of these four copies, one is
cytogenetically normal and the other three have dramatic alterations. These
data verify the common lineage of the MCF-7:WS8 and MCF-7:2A cells.
A
Table 1
Northern analysis of the MCF-7:2A cells was unable to resolve a larger mRNA which could code for the 80
kDa ER. An RT-PCR based strategy was subsequently utilized to analyze the structure of
the 80 kDa ER mRNA. Using primer sets that encompass the entire coding sequence
of the human ER (Fig.
3
A and Table
1
) PCR was performed on oligo(dT) primed MCF-7:WS8 and MCF-7:2A cDNAs (Fig.
3
B). Primer set #1 typically gave poor results due to the very high (>65%) G + C
content in this region of the ER cDNA. Subsequent studies using a modified PCR
protocol (
28
) demonstrated no differences in the sizes of the PCR product in this region of
the ER cDNA from MCF-7:WS8 or MCF-7:2A cell lines (see Discussion). The PCR products using primer sets
#2 and #3 were indistinguishable in MCF-7:WS8 and MCF-7:2A groups. The products from primer sets #4 and #5, however, show
numerous extra bands in the MCF-7:2A lanes that are not present in the MCF-7:WS8 lanes. Together these two primer sets amplify the entire
ligand binding domain of the ER. The smaller of the additional bands from
primer sets #4 and #5 in the MCF-7:2A groups are ~300-400 bp larger than the wild type product. This is consistent
with an insertion necessary for a 14 kDa increase in the size of the ER
protein. Primer sets #4 and #5 amplify products that overlap by ~70 bases in the wild type 66 kDa ER. The fact that both sets gave rise to
larger products in addition to the expected products suggested that the
insertion was in the region of their overlap. These data demonstrated the
existence of a unique mRNA in the MCF-7:2A cells which contains an insertion in the ligand binding domain.
To address the possibility that the mRNA coding for the 80 kDa ER is the product
of a trans-splicing event (
31
,
32
), XL PCR (
28
) was performed on genomic DNA from the MCF-7:WS8 and MCF-7:2A cells. Primers were designed for this study that would give
rise to products containing a single intron separating two neighboring exons.
An upstream primer (U7) that bound in exon 7 (see Table
1
and Fig.
6
B) was paired with a downstream primer that bound in exon 6 (D6). This set would
amplify sequences separating exon 7 and exon 6' in the 80 kDa ER gene and should not amplify any sequences in the wild
type ER gene. The primer U5 was paired with D4 to amplify the sequences
separating exons 6 and 7 (or 6' and 7'). In the wild type ER gene this intron has been shown to be >24
kb (
7
). Finally, PCR using the primers U7 and D5 would amplify the intron separating
exons 7 and 8 in the MCF-7:WS8 ER gene and exons 7' and 8 in the MCF-7:2A ER gene. This set of primers should give rise to
identical products in both the MCF-7:WS8 and MCF-7:2A groups. As shown in Figure
6
, primers U7 and D5 give rise to identical ~4.2 kb products in both the MCF-7:WS8 and MCF-7:2A groups. The PCR utilizing primers U5 and D4 did not give
rise to any visible product. This is presumably due to poor amplification of
sequences of this length (>24 kb). PCR using primers U7 and D6 gives rise to a
specific band in the MCF-7:2A group that is ~6 kb. This fragment contains 274 bp of exon sequence, therefore the
intron separating exons 7 and 6' can be estimated to be ~5.7 kb. This is clearly longer than the wild type intron downstream
of exon 7 (3.5 kb) and also much shorter than the 17 kb wild type intron
upstream of exon 6. Consequently, the sequence separating exon 7 from exon 6' appears to be the site of the gene rearrangement responsible for the
genesis of the 80 kDa ER. This also confirms that the 80 kDa ER mRNA is not the
result of a trans-splicing event.
We identified a novel 80 kDa ER that is expressed in the MCF-7:2A human breast cancer cell line in addition to the wild type 66 kDa ER.
This 80 kDa protein is the result of translation of an ER mRNA that contains a
duplication of exons 6 and 7. While many mutant ER mRNAs have been discovered
using the techniques of RT-PCR and RNAse protection, to our knowledge no mutant ER protein has been
detected in the cells from which the mutant mRNA was originally isolated. The
80 kDa ER is easily detected using Western blot analysis of unfractionated
whole cell extracts. Uniquely, it was Western blot analysis which led to the
initial discovery of the 80 kDa ER. While the 80 kDa ER is a small proportion
of the total ER complement in the MCF-7:2A cells it may account for enough transcriptional activity to regulate
proliferation in the absence of their primary mitogen (estradiol). We have
previously described the growth characteristics of these cells as well as their
ability to induce the transcription of a luciferase reporter gene and both
these activities, while considerably greater than that of the wild type MCF-7:WS8 cell in the absence of estrogen, were significantly less than the
maximal activity seen in MCF-7:WS8 cells in the presence of estrogen (
2
). We surmise that the selection process has allowed the outgrowth of a clone
which can use this marginal activity in order to allow sustained growth in
estrogen free media.
The Western blot presented in Figure
1
demonstrates the presence of the mutant ER in the MCF-7:2A cells and not the quantitative expression of the wild type and mutant
ERs in these cell lines. We have performed detailed studies on the steady state
expression of the ER mRNA and protein in these two cells lines as well as the
T47D:A18 cell line (
33
). We have shown that changes in the levels of the ER are quite complex,
particularly in the MCF-7:2A cell line in which the two forms of the ER appear to be
differentially regulated. For the study presented in Figure
1
we used whole cell extracts from cells which had been grown in estrogen-free media for 5 days. This treatment leads to an increase in the steady
state ER levels in the MCF-7:WS8 cells, however this is the standard culture media for the MCF-7:2A cells and the ER is already maximal. The ratio of the 80 kDa ER
to the 66 kDa ER does not reflect the apparent gene copy number of the mutant
versus wild type ER. We have observed that this ratio can fluctuate between
nearly equal expression (
2
) to the ~ 1/10 ratio seen here. It is unclear as to what is the primary determinant
of these alterations in expression, however the expression of the ER has been
shown to be regulated by sequences in the coding sequence of the ER (
34
) and the mutation which has caused the appearance of the 80 kDa ER may have
given rise to aberrant regulation of this protein which causes its lower steady state expression level.
Further studies will be necessary to determine the mechanism of regulation of
the two ERs in the MCF-7:2A cells.
In studies described here we have used the technique of PCR mapping to establish
the gross structure of the 80 kDa ER cDNA. The principal change in the
structure of the 80 kDa ER cDNA was observed using primers that amplified the
steroid binding domain of the cDNA. In primer set 4 (U4-D4) (see Fig.
3
) we observed a number of additional bands in the MCF-7:2A group. We believe that only one of these additional bands is
responsible for the 80 kDa ER. Subsequent studies demonstrated that the ~800 bp band contains an ER cDNA with a duplication of exons 6 and 7. This
observation can explain the presence of the additional larger bands in primer
sets 4 and 5. Templates that contain duplicated sequences can give rise to
additional spurious products due to the phenomenon of long range jumping in
PCR, whereby incompletely extended products anneal to the duplicated sequences
that lie outside the original area of interest. This hybridization leads to
undesired amplification and the appearance of these larger bands (
35
).
Functional studies on an artificially created 80 kDa ER was not possible by
simply inserting the duplicated exon sequences into the wild type ER expression
vector due to the lack of suitable cloning sites in the regions flanking exons
6 and 7 in the wild type ER. Additionally, this duplication could not be
definitively shown to be the only change in the coding sequence of the 80 kDa
ER until the entire cDNA had been sequenced. Subsequent to the studies
presented here we have succeeded in cloning the entire coding sequence of this
mutant ER, as well as a wild type ER from the MCF-7:2A cells through the use of XL-PCR. The functional properties of this mutant ER are described in a
submitted report (
36
) in which we show that the 80 kDa ER does not appear to function as a
stimulatory transcription factor but does appear to inhibit the activity of the
wild type ER in transient transfection studies.
The alteration in conditions used with this procedure also allowed us to confirm
that the size and DNA sequence of the fragments in primer sets 1 and 2 were the
same in the wild type and 80 kDa ER. More importantly, the entire coding
sequence of both ERs have been sequenced and have been found to be wild type
other than the exact duplication of exons 6 and 7 in the 80 kDa ER (
36
).
The presence of the smaller (~300 bp) band in primer set 5 (U5-D5) can be attributed to the deletion of exon 7 from the ER mRNA.
This amplified product has been consistently observed in the PCR of MCF-7 cDNAs by ourselves and others as well as in clinical samples including
breast cancers and meningiomas (
37
,
38
). Fuqua
et al
. have investigated the function of this mutant and have shown that it acts as a
dominant negative repressor of normal ER function in yeast (
11
). While we can demonstrate the presence of this cDNA in both the MCF-7:WS8 and MCF-7:2A cells by PCR we do not observe any measurable amounts of this
protein in whole cell extracts by Western blot analysis using the monoclonal
antibody H226, which recognizes an epitope in the A/B domain of the ER (
6
). We believe that this type of splicing error occurs rarely and therefore the
amount of protein which is present is below the level of detection of the
Western blot, however, the exquisite sensitivity of PCR allows the detection of
these rare transcripts. The biologic activity of mutant ERs expressed at these
low levels is unclear.
Karyotype and FISH analysis confirm that the MCF-7:2A and MCF-7:WS8 cells share a common lineage. These data, along with genomic
PCR data, suggest that the MCF-7:2A cells have undergone a chromosomal rearrangement which has caused a
duplication of the entire ER gene as well as a duplication of the portion of
the gene which contains exons 6 and 7. The most likely scenerio would be a
recombination event between ER genes in which intron 5 exchanged with intron 7
causing a duplication of exons 6 and 7 and the exchange of exon 8. This would
also suggest that at the time of the recombination a gene missing exons 6 and 7
arose. However, this deletion has not been observed in any RT-PCR that we have performed with the MCF-7:2A RNA. It is possible that the gene missing exons 6 and 7 was
lost during the growth and selection of the MCF-7:2A cells. It also appears from our data that the 80 kDa ER is coded by
only one of the four copies of the ER gene present in the MCF-7:2A cells. The remaining three copies do not appear to be significantly
different from the wild type 66 kDa ER. This is confirmed by Southern blot data
which suggests that the wild type gene is in such excess that the altered
fragments cannot be resolved using this technique.
We also performed genomic PCR on the MCF-7:WS8 and MCF-7:2A cells and found that the segment of the wild type ER gene from
exon 7 to exon 8 could be amplified. This segment has been shown to contain the
smallest (~3.5 kb) ER intron (
7
). The majority of introns in the ER gene are >10 kb and are not readily
amplified using XL PCR. Genomic PCR of the MCF-7:2A ER gene, however, did demonstrate that the segment of the 80 kDa ER
gene from exon 7 to exon 6' does include an intron of ~5.9 kb. This suggests that this mutant gene includes either part of
the wild type intron which separates exons 5 and 6 (~17 kb in the wild type ER) or else an addition to the intron which
separates exons 7' and 8 (~3.5 kb in the wild type ER). This confirms that the 80 kDa ER
protein is not the result of a trans-splicing event, but is a consequence of the genetic rearrangement of an ER
gene, most likely located on the derivative chromosome
The fact that the 80 kDa ER protein is present in a cell line that has developed
estrogen independent growth suggests that it may be involved with the evolution
of this phenotype. The finding that the 80 kDa ER is expressed in all 10
subclones of the MCF-7:2A cell line thus studied and has been maintained in the MCF-7:2A cells for over 100 passages further supports the importance of
this protein (
2
). However, the presence of an excess of 66 kDa ER in this cell line makes
direct elucidation of the activity of the 80 kDa ER quite difficult. While
expression of mutant ERs in heterologous systems has proven useful in
investigating the properties of these proteins it does not directly address the
function of these proteins in the cells from which they originate. Further
analysis of the MCF-7:2A cell line should provide greater understanding of the properties of
this protein in its `natural' environment.
The appearance of a mutant ER containing a duplication of two exons is an
extremely unusual event that has never been observed in any members of the
steroid hormone receptor superfamily. Study of the changes associated with this
extraordinary genetic rearrangement should provide unique insights into the
phenomon of ER regulated growth and the biochemical properties of the ER.
We thank Professor Pierre Chambon for the ER cDNA probe and Abbot Laboratories
for the monoclonal ER antibodies H222 and H226. We thank Dr David Boothman for
helpful discussions and critical reading of the manuscript. We thank Jay Pink,
Pharmacia Biotech for helpful discussions regarding PCR, cloning and
sequencing. We also thank Matt Bong, Jim Holsen, Kyle Hansen and Michelle Mucks
for excellent technical assistance. This work was supported by NIH Grant CA
32713 to V.C.J. and in part to a fellowship to J.J.P. from the Department of
Human Oncology, NIH training grant 5T32-CA09471. We are grateful for the support of the Lynn Sage Foundation for
M.M.B.
Primer
a
Position
b
Sequence
c
U1
351-370
GCC ACG GAC CAT GAC CAT GA
D1
726-707
CTG CAG GAA AGG CGA CAG CT
U2
565-584
AAC GCG CAG GTC TAC GGT CA
D2
1047-1028
AAT GGT GCA CTG GTT GGT GG
U3
831-850
ACG CCA GGG TGG CAG AGA AA
D3
1317-1298
CAA GGC ACT GAC CAT CTG GT
U4
1211-1230
GAG ACA TGA GAG CTG CCA AC
D4
1756-1737
GGG TGC TGG ACA GAA ATG TG
U5
1684-1703
GAA GAG GAG TTT GTG TGC CT
D5
2167-2148
TGT GGG AGC CAG GGA GCT CT
D80
d
1910-1891
TAC ACA TTT TCC CTG GTT CCT CA
U7
e
1748-1768
CCA GCA CCC TGA AGT CTC TGG
D6
e
1705-1685
GAG GCA CAC AAA CTC CTC TCC
(2022-2002)
f
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