European Molecular Biology Laboratory (G.F., V.S-B., F.G.)
D-69117, Heidelberg, Germany
National Diagnostic
Centre (C.G., M.K.) University College Galway,
Ireland
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous studies on the structure and organization of the human ER
(hER
) gene indicated that the transcriptional activity of this gene
was probably controlled by more than one promoter (23, 26, 27). We
reported that a portion of the 5'-flanking region of the ER
gene, at
approximately -1.9 kb upstream from the formerly assigned
transcription start site (5), presented a high homology with the 5'-end
of the rat ER
cDNA and exon 1 of the mouse ER
gene (26). This
predicted the existence of a second hER
mRNA, which would arise from
splicing of an upstream exon to the 5'-untranslated region (UTR) of the
previously designated exon 1. Such a transcript was demonstrated by
RT-PCR (26). Further investigation into the expression of these two
hER
mRNAs suggested the likely existence of additional hER
transcripts. Using the rapid amplification of cDNA ends (RACE)
methodology, we have now identified four new hER
mRNAs,1 which together
with those previously characterized, give rise to a total of six hER
mRNA isoforms (AF hER
mRNAs). All of these transcripts are
generated by differential promoter usage and differ in their 5'-UTR
because of an alternative splicing event. Furthermore, these
transcripts are differentially expressed in human tissues or cell
types.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In the corresponding experiment with a probe specific for transcript C,
two protected fragments, in addition to the expected protected fragment
detected at the splice site position (+163), were observed (Fig. 1B).
Taking into account the intronic region -1860 to +162 (previously
described in Ref. 26), the sites identified by the 5'-ends of the two
longest fragments are located at 1974 and 2000 bp upstream from the
transcription start site characterized by Green et al. (5).
The same two sites were mapped by a primer extension analysis using
either primer
(Fig. 1C
) or a specific primer for transcript C (data
not shown), thus confirming that they correspond to the transcription
start sites of C hER
mRNA isoform. It should be noted that the
expression level in MCF7 of the hER
mRNAs containing exon 1C
sequences (<10% of the total hER
mRNA level), was nevertheless
much lower than suggested by a two transcript explanation of the S1
nuclease analysis of the hER
mRNAs/probe A hybrid results where
approximately 50% of hER
transcripts diverged from A hER
mRNA at
the splice site position. Moreover, the proportion of hER transcripts
detected by probe C at the splice site position (at least 90% of the
total hER mRNA level in MCF7) was also more than the 50% predicted
from transcripts A expression level. These results suggested the
existence of additional hER
mRNA isoforms in MCF7 obtained by the
splicing of unidentified upstream exon(s) to the acceptor splice site
of exon 1A at position +163. This hypothesis was strengthened by a
primer extension experiment using the primer
, which was designed to
hybridize hER
mRNAs in a region downstream of the splice site
position and was thus able to be extended to all the 5'-extremities of
hER
transcripts. Results showed indeed several other extension
products in addition to those related to A and C hER
mRNAs (Fig. 1C
). These products might arise from new hER
transcripts and partial
extension on hER
mRNAs. Interestingly, these results suggested also
that the hER
gene exhibits alternative splicing in a tissue-specific
manner since the primer extension pattern obtained from MCF7 was
different from that detected in endometrium, especially for the C
hER
mRNA isoform whose expression was relatively higher in
endometrium than in MCF7.
In conclusion, these data clearly indicated the existence of more than
two hER mRNA isoforms whose expression level may be controled in a
tissue-specific manner.
Four Novel hER mRNA Isoforms: B, D, E, and F hER
mRNAs
To amplify new 5' mRNA extremities of the hER gene, a variation
of the inverse PCR technique was performed (30). The 5'-ends of three
additional hER
mRNA isoforms (B, D, and F hER mRNA isoforms) were
thus cloned from MCF7 total RNA, together with the two previously
described (A and C hER
mRNA isoforms) (Fig. 2
). In agreement with the S1 nuclease
analysis of the hER
mRNAs/probe A hybrids, the most frequent cloned
isoform was A hER
mRNA. Clones containing B, C, D, and F mRNA
sequences were much less frequent (
810% for each type). All of
the 5' hER
cDNA ends contained common exon 1A sequences up to the
splice site position that is located 5' to the translational initiation
codon of the hER
gene. All the cDNAs diverged from each other,
however, immediately upstream from this position. Due to the RACE
technique used to generate the 5'-terminal hER
cDNA regions (nick
translational replacement of the mRNA was used to synthesize the second
strand cDNA), the sequences of the new B, D, and F hER
mRNAs are
probably incomplete at their 5'-ends. This possibility is supported by
the fact that clones containing A and C mRNA sequences were deleted
of some nucleotides at the 5'-end of these transcripts.
|
|
Proximal Promoters of the B, D, E, and F hER mRNA Isoforms and
Preliminary Organization of the 5'-Region of the hER
Gene
The evidence of previouly unidentified upstream exons that were
alternatively spliced to the acceptor site of exon 1A (at position
+163) prompted us to further investigate the 5'-genomic organization of
the hER gene.
The specific 5'-cDNA end sequences of B hER mRNA isoform were
totally homologous to a genomic region located between exon 1A and 1C
that had previously been sequenced (32). This genomic region was
therefore part of a new exon whose 3'-end was positioned at 168
nucleotides upstream from the transcription start site characterized by
Green et al. (5). This new exon is referred to as exon 1B
(Figs. 2
and 3
). Several transcription start sites of B hER
mRNA
were determined by S1 nuclease protection analysis using probe B
spanning the region -150 to +283 (numbering from one of the main start
sites of B hER
mRNA isoform) (Fig. 2
) and further verified by a
primer extension experiment with primer B (data not shown).
|
Using a rapid genomic walking technique (see Materials and
Methods), the 5'-flanking regions of the 5'-RACE products obtained
for D and F hER isoforms, as well as the 5'-flanking region of the
exon shared by isoforms E and F (exon 1E), were amplified. The sizes of
these genomic PCR products were approximately 430, 400, and 170 bp,
respectively. Figure 2
shows the hER
5'-untranslated and flanking
sequences obtained from these PCR products. The 5'-flanking sequences
of exon 1E were identical to the sequences of the 5'-hER
cDNA end
recently identified (31). Therefore, this result demonstrated that the
5'-end of isoform E was encoded by only one exon (exon 1E), whereas
that of isoform F contained two exons of which the upstream one (exon
1F) is spliced to an acceptor site located in exon 1E (Fig. 2
). The
sequences adjacent to this site in exon 1E correspond to the splicing
consensus acceptor sequences (AG).
Using MCF7 and liver RNA, the localization of the transcription start
sites of E and F hER mRNA isoforms was determined both by S1
nuclease protection analysis using the probes E and F spanning the
corresponding 5'-untranslated and cloned flanking sequences (see
Materials and Methods for the probe preparation) and by
primer extension with the long primer E/F (Fig. 2
and data not shown).
The 5'-extremity of D hER
mRNA isoforms was only detected by primer
extension using a long primer (primer D). S1 nuclease mapping
experiment specifically designed to map the 5'-end of D isoform failed
to detect any signal.
As previous studies suggested the existence of a further transcription
start site upstream of exons 1C at position -3090 (27), S1 nuclease
experiments on MCF7 RNA were performed using probes spanning the region
-2500 to -3250. No transcription start sites were detected and the
probes were fully protected. As this result could be due to residual
DNA fragments, an RT-PCR experiment using a primer 5' to position
-3090 (primer C6) and a primer in exon 1A (primer VI) was performed
(see Fig. 3 for primer location). An RT-PCR product, the size of which
corroborated with a splicing event between exons 1C and 1A, was
obtained indicating that the genomic region located upstream of -3090
(i.e. between exons 1C and 1D) could also be transcribed and
spliced to the body of the hER gene. This could be due to some of the
pre-D hER
mRNAs that were not spliced as expected using the normal
splicing donor sequence of exons 1D, but by utilizing those of exons
1C. Similar observations were obtained for the genomic region located
between exons 1C and 1B (data not shown), but the S1 nuclease and
primer extension experiments demonstrated clearly that both C and B
isoforms existed independently of such read through.
Taken together, all of these results demonstrated that the hER gene
is a complex genomic unit controlled by at least six promoters (AF)
and exhibiting alternative splicing of five upstream exons
(1B1F).
Differential Expression of the hER mRNA Isoforms
To get an overview of the pattern of expression of the hER mRNA
isoforms in various human tissues and cell lines, an RT-PCR analysis
was performed. Single-stranded cDNAs were synthesized from total RNA of
various sources using a hER
gene-specific primer (I) chosen from the
3'-UTR region of the hER
gene (exon 8) (Fig. 4A
). The different hER
cDNAs were
amplified by two rounds of PCR (30 cycles each) utilizing a common
3'-primer (II) and nested primer (III) located upstream from primer I
in exon 8, in combination with 5'-primers and nested primers specific
for the different hER
mRNA 5'-extremities (Fig. 4A
). Thus,
approximately all the coding region of the hER
transcripts was
amplified. Results from this study showed a differential expression of
hER
mRNA isoforms among the tested samples (Fig. 4B
). The D hER
mRNA isoform displayed a more restricted expression pattern than the A,
B, and C hER
mRNA isoforms, whereas the E and F hER
mRNA were the
only forms detected in most tissues and cell lines tested. HeLa, which
is usually considered to be an ER-negative cell line, expressed
PCR-detectable amounts of the isoforms B, E, and F. It should also be
noted that isoforms E and F were the only hER
transcripts detected
by RT-PCR in the two osteoblast cell lines, HOS TE 85 and SAOS.
|
To estimate quantitatively the relative abundance of the different
hER mRNA isoforms in the various RNA samples, S1 nuclease mapping
experiments were performed using single-stranded DNA probes specific
for each hER
transcript (probes A', B', C', D', E', and F') (Fig. 5A
). Due to the common sequence 3' to the
splice site position, these probes were also able to measure the
residual expression resulting from the sum of the expression of the
other isoforms [for example (
- A) hER
mRNA in Fig. 5A
]. To
distinguish between undigested probes and specific protected fragments,
all of the probes contained additional sequences in their 3'-ends that
originated in the vectors used for the single-stranded probe
preparation. Finally, the integrity of the RNA from the different
tissues and cell lines tested was checked by performing a S1 nuclease
protection assay on ubiquitin mRNA, a housekeeping messenger. It
should be noted that, as for other housekeeping messengers, ubiquitin
mRNA may present some tissue variation in its expression level, and it
is generally more highly expressed in cultured cells than in
tissues (33, 34). As shown by the results in Fig. 5B
and also their
quantification summarized in Table 1
(where the data are expressed as percentage of the
total hER
mRNA expression detected in MCF7 cells), there was tissue
specificity in the level of expression of the different hER
mRNA
isoforms. Briefly, transcript A was the major form (
50%) expressed
in the mammary gland or the cell lines derived from this tissue (MCF7,
T47D, and ZR 751). In endometrium, the predominantly expressed forms
were the A and C hER
mRNA isoforms, whereas the C and F were the
main two isoforms detected in ovary. Finally, high levels of the E
hER
mRNA isoform were restricted to the liver (see upper strong band
with E' probe and middle and strong band with F' probe), which
confirmed the RT-PCR results previously published (31). It should be
noted that no protected fragments corresponding to isoform D were
obtained after the hybridization of probe D' with the different RNA
samples followed by a S1 digestion even though it represented 8% of
the clones selected by RACE performed on MCF7 total RNA and was readily
detected by primer extension using a long primer (primer D). The S1
nuclease result does not exclude a major role for the isoform D since
it may be more highly expressed in other tissues or at specific
physiological stages.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Overall Organization of the hER Gene
Using a RACE methodology, we have isolated and characterized
several new hER cDNA isoforms and demonstrated that these hER
transcripts are produced from a single hER
gene by the use of
multiple promoters. The hER
mRNA isoforms are generated by splicing
of five alternative upstream exons or leader sequences (1B1F) to a
common acceptor site that is situated 70 nucleotides upstream of the
translation start site in the previously assigned exon 1(A) (5).
Preliminary analysis of the 5'-organization of the hER
gene by
genomic walking techniques, S1 nuclease mapping, and primer extension
studies showed that each of the hER
mRNA isoforms is transcribed
using an isoform-specific promoter. Four of these promoters (AD) were
located within the 4-kb region upstream of the ER translational
initiation codon. Southern blot hybridization and genomic PCR
experiments suggested that the two others (E + F) were much further
upstream.
The conservation of the acceptor sequences of the exon 1A splice site
in other species, such as rodent, chicken, or Xenopus (data
not shown), suggests that the expression of the ER gene in these
species also involves complex alternative splicing of upstream exons.
The existence of similar phenomena for the rat (r) ER
gene has
recently been demonstrated by the identification in RACE experiments of
new rER
5'-cDNA ends diverging from each other immediately upstream
from the predicted alternative splice site (35, 36, 37). Likewise, in
chicken, isoforms of ER mRNA that vary in their 5'-UTR sequences have
also been recently isolated in our laboratory (38). Although highly
probable, the multiplicity of leader sequences remains to be
demonstrated for other species.
Features of the hER mRNAs and the Corresponding Promoters
As demonstrated by the RACE and RT-PCR experiments, the hER
mRNA isoforms contain common sequences from exon 8 up to the acceptor
splice site position located upstream from the AUG of the hER
coding
region in exon 1A but diverged from each other immediately upstream
from this position. The presence of a stop codon in exon 1A, in the
part common to all hER
mRNAs, and upstream and in-frame with the
translational initiation codon of hER
protein precludes the
possibility that any of the hER
RNA isoforms described in this paper
alter the N-terminal sequence of the ER
protein. Therefore, all
hER
mRNA isoforms encode a common ER
protein. Sequence analysis
showed that several of the 5'-UTRs of the hER
mRNA isoforms contain
short ORFs (sORFs) (Fig. 2
and Refs. 5, 26). The significance of
these sORFs remains to be elucidated, but similarly placed sORFs in
other messengers, such as GCN4 or the BCR/ABL oncogene mRNA, have been
shown to be involved in the translational control of their expression
(39, 40). It should also be noted that all of the hER
5'-UTR
sequences could be folded, using the algorithm of Zuker and Stiegler
(41), into more or less stable secondary structures [
G
values were
-40 (E hER
mRNA) to -100 (A hER
mRNA)
kcal/mol], which could also have an impact on the stability of the
isoforms. Therefore, one possible function of these alternatively
spliced 5'-UTR exons might be the regulation of the hER synthesis by
controlling the turnover and/or the translation efficiency of the
hER
mRNA isoforms. In keeping with this hypothesis is the fact that
the 5'-UTR sequences of A and C hER
mRNA isoforms are highly
homologous with the identified 5'-UTR sequences of the chicken and
rodent ER
mRNA, respectively (26, 42). Although transcript B was not
known to exist when the initial comparison were performed between
chicken and hER genomic sequences (42), the results presented in the
study showed the existence of a conserved region that corresponds to
the leader sequences of isoform B. This region was shown recently to be
also transcribed in chicken (38). These data suggest that a functional
role for some of the 5'-UTRs has been preserved during evolution.
Sequence alignment of the 5'-UTR of the D, E, and F hER
mRNA
isoforms with the known ER
5'-UTR sequences from other mammalian
species showed no significant homology.
An evolutionary conservation of transcriptional regulations of the
ER gene is also suggested by the high degree of homology that has
been shown between the human A and B promoter regions and the chicken
ER
promoters (38, 42) and between the human C promoter region and
the rodent ER
promoters (26). To date, no similar homology has been
found for the promoter sequences of D, E, and F isoforms.
Computer-assisted analysis of these new promoter sequences revealed an
half-estrogen-responsive element in F hER
mRNA promoter as well as
consensus and degenerate sequences for the binding of AP1 complex in
the promoters of D and F hER
mRNA, respectively. These data are of
particular interest given the fact that, in addition to its capability
to bind to the estrogen response element, ER
can also act in a
protein/protein related manner with the AP1 complex formed by c-Fos and
c-Jun (43) and thereby potentially autoregulate its expression. Further
studies are obviously necessary to characterize fully the different
promoters of the hER
gene to elucidate the transcriptional events
controlling the hormonal- and tissue-specific expression of the
different hER
mRNA isoforms.
Tissue Specificity
The results on the distribution of the hER mRNA isoforms showed
a differential pattern of expression of the hER
gene in human
tissues and cell types. As previously suggested, the predominant form
expressed in the mammary gland and in the breast cancer cell lines is
isoform A, accounting for approximately 50% of the total hER
mRNA.
Interestingly, this transcript (together with isoform B in MCF7) was
more abundant in the ER-positive breast tumor cell lines MCF7 and T47D
than in the healthy tissue, whereas the expression of other hER
mRNA
isoforms was not significantly altered (see Table 1
). This observation
suggests that the high ER
expression level in these breast tumor
cell lines might be due to an overactivity of the corresponding
promoters. Corroborating with this hypothesis is the fact that two
binding sites for ERF-1, a member of the AP2 family of developmentally
regulated transcription factors, have been located in the 5'-UTR of
exon 1A of the hER
gene and are required for its efficient
expression (44, 45). This transcription factor is highly expressed in
ER-positive breast carcinomas in contrast to normal human mammary
epithelial cells, where low levels of ERF-1 protein were detected (44).
The proximity of these two ERF-1 binding sites to the A and B promoter
regions might explain the comparative increase in the activity of these
two promoters in most of the ER-positive breast cancer cell lines.
Previous studies with the MCF7 breast cancer cell line have reported
either no change or up to a 60% decrease in hER
mRNA level after an
estradiol treatment, depending on the estrogen treatment history of the
cells (12, 24). The present study shows that estradiol down-regulates
the expression of each hER
mRNA isoform in a similar manner (see
Fig. 6
). This result is consistent with the fact that the main
mechanism responsible for the hER
mRNA down-regulation in MCF7 cells
was shown to be posttranscriptional (12) and may involve the 3'-UTR
that is common to all hER
mRNA isoforms (46).
In the endometrium, the expression pattern of the hER transcripts
differed from that in breast tissue due to an increase in the
proportion of the C hER
mRNA isoform to 40% of the total hER
mRNA, whereas the expression level of the other isoforms remains
relatively unchanged between endometrium and breast tissues. The
importance of ER
expression in the endometrium had previously been
shown by the phenotypic changes in the uteri of a mutant mouse line
with a insertional disruption of the ER
gene (47, 48). Likewise, our
results show a significant expression of ER
transcripts in human
ovaries with a predominance of C and F hER
mRNA isoforms. In rat,
examination of ER
mRNA expression at cellular level, by in
situ hybridization, showed that ER
mRNA was expressed at a low
level throughout the ovary with no particular cellular localization, in
contrast to ERß mRNA, which was expressed preferentially in granulosa
cells of small, growing, and preovulatory follicules (49).
Interestingly, high amounts of the E hER
mRNA isoform were
restricted to the liver, which suggests that this isoform is important
in some specific aspects of hepatic ER
regulation. In agreement with
this hypothesis, it has been reported that hER
gene expression was
differently regulated by estrogen in liver compared with endometrium
and breast tissue or cell lines (12, 13). Estrogen increased hER
mRNA expression level in the liver, whereas hER
mRNA expression was
down-regulated in the two other tissues. In addition, the hepatic ER
level was shown to be approximatively 5 times lower in immature than
mature mammalian females (50). Our results confirm these data by the
detection of higher levels of hER
mRNA in female compared with male
liver. In addition, the S1 nuclease protection experiment shows clearly
that the relative level of the predominant hER
mRNA isoform detected
in the liver (E hER
mRNA) is higher in adult females than males.
These data indicate that the tissue-specific expression of hER
transcript in liver is probably linked to a differential promoter
or/and 5'-UTR usage and are in keeping with different promoter
requirements for this important gene.
Several reports have suggested that estrogens also contribute to
skeletal muscle growth (19, 51). Using immunoblotting and
immunofluorescence microscopy approaches, recent work demonstrated that
rodent skeletal myoblasts contain the ER (19). Our S1 nuclease data
show that the human male skeletal muscle ER
was encoded mainly by
the C hER
mRNA isoform and more weakly by the A isoform.
Since estrogen deficiency has been recognized to be a cause of
postmenopausal bone loss, several studies have demonstrated the
presence of the ER protein and its mRNA in skeletal tissue,
especially in the osteoblasts and osteoclasts, the two bone cell types
involved in the formation and resorption of the bone mass (15, 16). To
determine which of the hER
mRNA isoforms were expressed in
osteoblasts, RT-PCR and S1 nuclease mapping analysis were performed
using total RNA from two osteoblast cell lines, HOS TE85 and SAOS. The
E and F ER
mRNA isoforms were detected in both cell lines by RT-PCR,
but none of the isoforms were present at sufficient levels to give a
signal using the S1 nuclease method in these preliminary experiments.
The low amounts of ER
transcripts detected in this study in the
osteoblasts might be explained by the fact that only a small population
of the cells that served as a source of RNA expressed ER
because of
the cell cycle-dependent regulation of the ER
gene (16). The fact
that only E and F hER
mRNA isoforms are expressed in the two
osteoblast cell lines might be related to this correlation between ER
expression and the cell cycle in osteoblastic cells, and these may have
control elements required for such a pattern of expression.
Finally, apart from the D hER mRNA isoform, all hER
transcripts
were detected in brain and pituitary. Nevertheless, S1 nuclease
protection analysis was unsuccessful in measuring hER
mRNA
expression probably due to a low proportion of ER-positive cell types
in these tissues and the fact that the RNA samples used in the
experiments were prepared from males. In situ hybridization
studies on female tissues using specific probes may be more appropriate
to investigate the differential expression patterns of the ER mRNA
isoforms in the pituitary and nervous system.
Importance and Possible Applications of Findings
As previously mentioned, the ER is a key component in the
signal transduction pathways controlled by the ovarian hormone,
estrogen. These pathways direct a variety of physiological processes,
such as establishment and maintenance of female sex differentiation
patterns, reproductive cycle and pregnancy, liver, fat, and bone cell
metabolism, cardiovascular and neuronal activity, and embryonic and
fetal development (1, 2, 3). It is also well established that some of
these pathways influence several pathological processes including
breast, endometrium, and ovarian cancers, osteoporosis,
arteriosclerosis, and Alzheimers diseases and that estrogen has both
desirable and harmful effects on these human pathological processes
(2, 3, 4). By providing evidence that the hER
gene is a complex genomic
unit exhibiting alternative splicing and promoter usage in a
tissue-specific manner, this study should have implications in the
basic, applied, and clinical research that involves ER biology. It
shows that it is not always appropriate to focus on the A promoter and
its messenger that were initially described (5) because in some tissues
other hER
transcripts are predominant and would be unaffected by
approaches that target A hER
mRNA isoform only. It also suggests
approaches in which the transcriptional or translational efficiency of
particular hER
transcripts might be altered: 1) by targeting
transcription factors that bind specifically to the promoters or 2) by
designing antisense oligonucleotides that are specific to each
transcript, while allowing another hER
transcript to be functional;
in this way, the benefical ER
-mediated effects are provided.
Finally, heterogeneity in the 5'-ends of mRNAs generated by alternative promoter usage and splicing seems to be a common feature among the members of the steroid/thyroid hormone/retinoic acid receptor family since several members of this family have also been reported to be transcribed from multiple promoters in a tissue-specific and developmental manner (52, 53, 54, 55). It is obvious that the increasing complexity of the organization and expression of the genes coding for the members of this family, which is emerging from this and others studies, is an appropriate means of achieving the differential and spatio-temporal expression of these important transcription factors, which may account to a large extent for the pleiotropic effects of their corresponding ligands in a wide range of physiological processes.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RNA Isolation
Total RNA from cell lines and tissues was extracted with TRIzol
(GIBCO-BRL) as described by the manufacturer. Human mammary gland total
RNA (a pool of RNA from six 16- to 35 yr-old Caucasian females), human
brain total RNA (derived from a 60-yr-old Caucasian male), human liver
total RNAs (derived from adult Caucasian male and females), and human
skeletal muscle total RNA (derived from a 32-yr-old Caucasian male)
were purchased from CLONTECH (Palo Alto, CA). Human pituitary RNA
(derived from a Caucasian male) was kindly provided by Professor J.
Duval (Université de Rennes, Rennes, France).
RACE
Alternative 5'-variants of hER cDNA were cloned by an inverse
PCR method (30). RT of MCF7 total RNA (10 µg) and second-strand
synthesis were performed using a commercial kit (GIBCO BRL) as
recommended by the manufacturer, except that the hER gene-specific
primer IV (5'-CTGGCCGTGGGGCTGCAGGAAA) was used instead of the usual
oligo (dT) primer (see Fig. 3
). Subsequently, the cDNA was circularized
in the presence of T4 DNA ligase and submitted to 35 rounds of PCR
amplification using the sense primer IX (5'-CGCAGGTCTACGGTCAGACC) and
the antisense primer VI (5'-TTGGATCTGATGCAGTAGGGC) (See Fig. 3
). PCR
included a denaturation step at 94 C for 1 min, an annealing step at 50
C for 1 min, and an elongation step at 72 C for 1 min, after an initial
denaturation at 94 C for 5 min and was followed by 7 min elongation.
One percent of the initial PCR product was reamplified under the same
conditions with the nested primers X (5'-ACTCAACAGCGTGTCTCCGAG) and VII
(5'-GGTCCGTGGCCGCGGGCAG) (See Fig. 3
). The PCR products were subcloned
in the TA cloning vector pCRTM2.1 (Invitrogen, San Diego, CA), and
colonies were screened with the oligonucleotides A2
(5'-GCTGCGTCGCCTCTAACCTC) and C2 (5'-CAAGCCCATGGAACATTTCTG), which are
specific for exons 1A and 1C, respectively (see Fig. 4A
for the
location of these two primers), and the oligonucleotide VIII
(5'-AAGGCTCAGAAACCGGCGGG), which is 3' to the splice site and
recognizes a common sequence of all hER cDNA 5'-ends (see Fig. 3
).
Colonies that did not hybridize to the A2 and C2 oligonucleotide probes
but hybridized to VIII probe were sequenced by the dideoxy chain
termination method.
Genomic Walking
The isolation and characterization of genomic regions further
upstream of exon 1D, 1E, and 1F sequences generated by the RACE
technique were performed using the Vectorette II starter pack from
Genosys Biotechnologies Inc. (U.K.) as recommended by the manufacturer.
Human genomic DNA was digested with different restriction enzymes
(BamHI, BglII, EcoRI,
HindIII, and TaqI) and then ligated to the
appropriate vectorette units to form vectorette libraries. Five
microliters of each vectorette library reaction were then used in two
rounds of 30-cycle PCR amplification. The 3'-primers and nested primers
used for the amplification of the genomic sequences further upstream of
exon 1D, 1E, and 1F were D3 (5'-TGGCTCTCTCAGGTGAAGA) and D4
(5'-GAAGAAGGGTAAAGATTGAT), E3 (5'-TGCTGATATTTGGTACCGCAGTCCC) and E4
(5'-GAGATCTTTGTGCTTACTCCTT), and F3 (5'-TTGAAGAGAAGATTATCACTA) and F4
(5'-CTGTCTTCTTATGCTATAGAA), respectively (See Fig. 3). The common
5'-primer and nested primer were specific for the vectorette unit and
provided by the manufacturer. Both rounds of amplification were
performed using the Expand long template PCR system (Boehringer
Mannheim, Indianapolis, IN). Ten microliters of each PCR reaction were
electrophoresed on a 1% agarose gel and tranferred to nylon membranes
(Hybond N+, Amersham, Arlington Heights, IL) with 20x saline sodium
citrate (SSC) as transfer solution. The membranes were incubated
in a prehybridization buffer containing 6x SSC, 5x Denharts
solution, 0.05% sodium pyrophosphate, 100 µg/ml sperm DNA, and 0.5%
SDS, at 37 C for 1 h. Then, the membranes were hybridized in 6x
SSC, 1x Denharts solution, 0.05% sodium pyrophosphate, 100 µg/ml
yeast tRNA with the oligonucleotide probes D5
(5'-TGGCTCCTCCGTTGAATGTG), E5 (5'-TCTTTGGTAGCTACAGAATATAATT), and F5
(5'-TAGAATGGGCAGGAGAAAGGAG), respectively (See Fig. 3
), which had been
end-labeled using T4 polynucleotide kinase and [
-32P]
ATP (3000 Ci/mmol). The most stringent wash was carried out for 20 min
at 55 C in 6x SSC, 0.05% sodium pyrophosphate. The specific PCR
products were then subcloned in the TA cloning vector pCRTM2.1
(Invitrogen) and sequenced. The genomic portion separating exon 1D from
the 3.2-kb identified genomic region upstream from exon 1A (27, 32) was
amplified by two rounds of PCR amplification using human genomic DNA
and the primers D1 (5'-CACATTCAACGGAGGAGCCA) and C4
(5'-CCTGGGACACTATGCAGTTACTGA) and the nested primer D2
(5'-ATCAATCTTTACCCTTCTTC) and C5 (5'-AATGTTAATGATGCCATCATGCAAAT) (see
Fig. 3
). To check the specificity, the PCR product was hybridized with
the oligonucleotide probe C6 (5'-AATACTGACTATGGAGAGAG) as previously
described, and then subcloned and sequenced.
Modified S1 Nuclease Mapping and Primer Extension
Biotinylated single-stranded DNA templates were used to prepare
highly labeled single-stranded DNA probes or long primers by extension
from a specific primer by the T7 DNA polymerase in the presence of
[-32P]dCTP (3000 Ci/mmol) (28, 29). These probes or
long primers were then hybridized with the appropriate RNA sample and
subjected either to an S1 nuclease digestion or to a reverse
transcriptase extension, respectively. The origin of probe A template
was a genomic PCR product obtained by amplification using the upstream
5'-biotinylated primer A0 (5'-AGCGACGACAAGTAAAGTAAAGT) with the
downstream primer IV (see Fig. 1A
). To prepare the templates used to
make probes C, A', B', C', D', E', and F' (see Figs. 1A
and 5A
), as
well as ubiquitin S1 probe, RT-PCR reactions were performed. The ER
upstream primers for amplification were C0 (5'-ACTCCCCACTGCCATTCAT)
(see Fig. 1A
), A2, B2 (5'-ATCCAGCAGCGACGACAAGT), C2, D2, E1
(5'-AGCCTCAAATATCTCCAAAATCT), and F1 (5'-TTCTATAGCATAAGAAGACAG),
respectively (See Figs. 4A
and 5A
). The ER common downstream primer was
primer IV. Ubiquitin primers were Ub1 (5'-GTAAAAACCCTTACGGGGAAG) and
Ub2 (5'-ACCACCACGAAGTCTCAACAC). The RT-PCR products were subcloned
downstream of T7 in the TA cloning vector pCRTM2.1 (Invitrogen), and
then PCR reactions were performed using a biotinylated T7 primer with
either primer IV for ER probes or M13 reverse primer for ubiquitin S1
probe. The templates for primers
, A, B, C, D, and E/F preparation
were obtained by RT-PCR using the common downstream primer IV with the
biotinylated upstream primers
1 (5'-ATGACCATGACCGTCCACAC), A1
(5'-CTCGCGTGTCGGCGGGACAT), B1 (5'-CTGGCCGTGAAACTCAGCCT), C1
(5'-TCTCTCGGCCCTTGACTTC), D1 and E/F1 (5'-AAGGAGTAAGCACAAAGATCTC)
(primer specific for E and F hER
mRNA isoforms and located in exon
1E), respectively. Finally, probe B, D, E, and F templates were
prepared by PCR using the biotinylated T7 primer with primer V
(5'-TCTGACCGTAGACCTGCG) and, for each reaction, two partially
overlapping templates (a + b) to link promoter sequences with the
corresponding 5'-cDNA end: 1) the TA cloning vector pCRTM2.1 containing
the genomic region further upstream of exon 1B, 1D, 1E, or 1F. For D, E
and F, these regions were generated by the genomic walking technique
(see previous section) and for B by genomic PCR using the primers B0
(5'-GCACACCCCATTCTATCT) and B3 (5'-ACTTGTCGTCGCTGCTGGAT) (see Fig. 3
);
2) the corresponding RT-PCR product obtained utilizing the upstream
primer B1, D1, E1, or F1 with the common downstream primer IV.
All biotinylated PCR products were bound to streptavidin-coated
magnetic beads (Dynal, Norway) as recommended by the
manufacturer, and the nonbiotinylated DNA strands were removed in 0.1
M NaOH. A, B, C, D, E, and F S1 probes and A, B, C, D, and
E/F primers were obtained by extending the VI primer annealed to the
corresponding biotinylated single-stranded template, whereas probes A',
B', C', D', and E' and primer were prepared using primer V.
Ubiquitin S1 probes was prepared by extending Ub2 primer. After elution
of the single-stranded DNA probes by alkaline treatment and magnetic
separation, 105 cpm of the probe or primer were
coprecipited with 30 µg of total RNA and then dissolved in 20 µl of
hybridization buffer (80% formamide, 40 mM PIPES, pH 6.4,
400 mM NaCl, 1 mM EDTA, pH 8), denatured
at 65 C for 10 min, and hybridized overnight at 55 C. The S1 digestions
and the reverse transcriptase extension were carried out as previously
described (56), and the samples were electrophoresed through a
denaturing polyacrylamide/urea gels.
RT-PCR Analysis
cDNAs were synthesized using 1 µg of total RNA from different
origins, an oligonucleotide primer (I) from the 3'-UTR (exon 8) of
hER mRNAs (5'-TTGGCTAAAGTGGTGCATGATGAGG) (see Fig. 4A
), and 50 U of
Expand reverse transcriptase (Boehringer Mannheim) under the conditions
recommended by the supplier. Of the 20-µl reverse transcriptase
reaction, 2.5 µl were then used in two rounds of 30-cycle PCR
amplification. The 5'-primers and nested primers used for A, B, C, D,
E, and F hER cDNA amplification were A1 and A2, B1 and B2, C1 and C2,
D1 and D2, E1 and E2 (5'-AATTATATTCTGTAGCTACCAAAGAAG), and F1 and F2
(5'-GAGTGATAATCTTCTCTTCAA), respectively (see Fig. 4A
). The 3'-primer
II (5'-ATTATCTGAACCGTGTGGGAG) and the nested primer III
(5'-CGTGAAGTACGACATGTCTAC) were from the common 3'-region of all hER
cDNAs, immediately upstream of the primer used for reverse
transcription. Both rounds of amplification were performed using the
Expand long template PCR system (Boehringer Mannheim) as recommended by
the manufacturer. Five microliters from each reaction were analyzed on
an 1% agarose gel and transferred to nylon membranes (Hybond N+,
Amersham). The membranes were hybridized as previously described (see
Genomic Walking) with the oligonucleotide probe Ex. 2
(5'-CCCTGGCGTCGATTATCTGAA) (see Fig. 4A
).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
This work was supported by a European Molecular Biology Organization long-term fellowship (to G.F.), the Irish-American Partnership (C.G.), the Irish Health Research Board (M.K.), and the Irish Cancer Research Advancement Board.
1 The nucleotide sequences reported in this paper
have been submitted to European Bioinformatics Institute Data
Bank with accession numbers AJ002559, AJ002560, AJ002561, and
AJ002562.
Received for publication January 27, 1998. Revision received August 14, 1998. Accepted for publication September 8, 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|