From the Sir William Dunn School of Pathology,
University of Oxford, South Parks Road,
Oxford OX1 3RE, United Kingdom, ¶ Nextran Inc.,
Princeton, New Jersey 08540, and the
Department of Pathology,
University of Cambridge,
Tennis Court Road, Cambridge CB2 1QP, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CD59 is a complement regulatory protein and may
also act as a signal-transducing molecule. CD59 transgenic mice have
been generated using a CD59 minigene (CD59
minigene-1). Although this minigene contained a 4.6-kilobase pair
5'-flanking region from the human CD59 gene as a promoter,
the expression levels of the CD59 mRNA were substantially lower
than those observed in humans, suggesting that CD59 gene
expression might also require other transcriptional regulatory elements
such as an enhancer. To investigate the transcriptional regulation of
the CD59 gene, we used three cell lines that express CD59
at different levels. We have identified DNase I-hypersensitive sites in
intron 1 in HeLa cells, which express CD59 at high levels, but not in
Jurkat (intermediate level) or Raji cells (low level). Furthermore,
cell line-specific enhancer activity was detected in a fragment
containing these DNase I-hypersensitive sites. The CD59 enhancer was
mapped to between The human complement regulatory protein CD59 is a 20-kDa
glycoprotein anchored to the membrane via glycosylphosphatidylinositol (1), which restricts human complement lysis by inhibiting assembly of
the complement membrane attack complex (2-4). Human CD59 transfectants generated with a rat T cell line (5) and a Chinese hamster ovary cell
line (6) were observed to develop resistance to the lytic activity of
human complement. These results suggested that the transfer of the
human CD59 gene together with genes encoding other
complement regulatory proteins (e.g. decay-accelerating factor and membrane cofactor protein) might be used to protect xenogeneic transplants at the endothelial interface. Indeed,
studies in CD59 transgenic animals have demonstrated that expression of human CD59 may offer some protection to xenografted tissues (7-9).
CD59 protein can be detected on cells of a wide range of living tissues
and on cell lines (2). It has also been reported that cells undergoing
apoptosis lose expression of CD59 and other complement regulatory
proteins (10), suggesting that expendable cells might also exploit
complement activation to assist their removal. CD59 is associated with
an intracellular protein kinase, p56lck (11), suggesting some
signaling role for CD59, perhaps in cell recovery from complement
attack (12). How the glycosylphosphatidylinositol-anchored molecules
associate with the intracellular protein kinase is unknown. Northern
blot hybridization of CD59 mRNAs from many cell lines suggests that
the expression of CD59 is controlled in a cell line-specific manner
(see "Results"). Transcriptional regulation of the CD59 gene might be important for these functions.
One goal in the creation of CD59 transgenic animals is to ensure
adequate expression in the tissue required for xenogeneic transplantation to prevent the deleterious effects of human complement. Although it may be possible to generate high expressor CD59 transgenic animals using a constitutive CD59 expression system (e.g.
controlled by a strong promoter such as cytomegalovirus), the
consequences of unrestricted expression of CD59 might be
disadvantageous if it also functioned as a ubiquitous signal transducer.
In order to achieve a greater understanding of the mechanisms
controlling CD59 gene regulation, we isolated the
CD59 gene and determined its structure (13). This gene
comprises a 5'-noncoding and three coding exons (13). In a previous
report, we estimated (by Southern blot hybridization) that the size of
intron 1 was larger than
35-kb.1 We have now cloned
this region and calculated it to be only 14 kb. Recently, an additional
alternative 5'-noncoding exon was identified in intron 1 (14). Five
different CD59 mRNAs were demonstrated as the products of
alternative polyadenylation (13).
In this paper, we have demonstrated that CD59 gene
expression is regulated by an enhancer located in intron 1. We have
identified a DNA sequence that functions as an enhancer element in HeLa
cells, which express CD59 at high level, but not in Jurkat
(intermediate level) or Raji cells (low level). Two lines of transgenic
mice were generated using CD59 minigenes that either
contained the enhancer or did not. High levels of CD59 mRNA
expression were observed only in transgenic mice generated with the
CD59 minigene containing the enhancer, suggesting that this
enhancer also functions in vivo.
Construction of CD59 Minigene and Generation of Transgenic
Mice--
CD59 minigene-1 and CD59 minigene-2
were constructed using CD59 genomic DNA fragments. The structure of
the minigenes is indicated in Fig. 1. The resulting minigenes were
micro-injected into fertilized mouse eggs to obtain transgenic mice.
Founder mice were C57BL/6 × SJL F2 crosses. Lines were
established by subsequent breeding back to C57BL/6. The copy number of
the CD59 minigenes was estimated by Southern blot hybridization.
Northern Blot Hybridization--
Total RNAs from transgenic mice
were isolated by the acid phenol extraction method (15), and total RNAs
from cell lines were isolated by guanidinium thiocyanate/CsCl method
(16). RNAs were electrophoresed on an agarose/formaldehyde gel and
transferred to a nylon membrane. The RNA filter was hybridized with
32P-labeled CD59 cDNA and DNase I-hypersensitive Assay--
Deoxyribonuclease I (DNase
I)-hypersensitive assays were performed as described previously (17).
Isolated nuclei were treated with DNase I (0, 1.5, 6.3, 9.4, 12.5, 25 units/ml) at 25 °C for 5 min in 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris-HCl, pH 7.4, 0.5 mM dithiothreitol, 5% glycerol, 10% sucrose, and the DNAs
were isolated. 20 µg of DNAs were digested with EcoRI and
analyzed by Southern blot hybridization using probe E1 and probe E2.
The position of the probes is indicated in Fig. 4A.
Luciferase and Chloramphenicol Acetyltransferase (CAT)
Assay--
To construct luciferase and CAT reporter plasmids, pGL3
Basic Vector (Promega) and pBLCAT2 (18) were used. The
3'-SacI site of the 1.4-kb SacI fragment was
introduced 5- bp downstream of the 5'-end of exon 2 using the
Exonuclease III/mung bean nuclease deletion method. The structures of
reporter plasmids are shown in Figs. 5A, 6A, and
7A.
1.5 × 107 cells were transfected with 3 µg (HeLa
and Jurkat) or 5 µg (Raji) of firefly luciferase reporter plasmids
with 0.3 µg (HeLa and Jurkat) or 0.5 µg (Raji) of internal control
plasmids (pRL-TK or pRL-SV40, Promega) by electroporation. After
48 h culture, cells were harvested, and luciferase activities were
analyzed by dual-luciferase reporter assay system (Promega). These
assays were repeated more than three times, and the firefly luciferase activities were normalized to Renilla luciferase activities.
10 µg of CAT reporter plasmids were transfected with 5 µg of a
Gel Mobility Band Shift Assay--
Nuclear extracts were
prepared using methods described previously (19). DNA-binding reactions
were carried out for 30 min at 25 °C in 20 µl of buffer containing
a 62-bp probe, 2 µg of poly(dI-dC)·poly(dI-dC), 5 µg of nuclear
extract, 12 mM Hepes, pH 7.8, 4 mM Tris-HCl, pH
7.8, 60 mM KCl, 1 mM EDTA, 1 mM
dithiothreitol, and 12% glycerol. For the competition assay, a
100-fold excess of unlabeled probe was additionally added. Probes were
amplified by polymerase chain reaction and purified from agarose gel.
After confirming the DNA sequence of the fragments, the probes were labeled with [ Expression of CD59 mRNA--
To investigate the
transcriptional regulation of the CD59 gene, transgenic mice
were generated using a CD59 minigene (CD59 minigene-1) (Fig. 1). The human
CD59 gene was detected by tail blot hybridization in seven
lines of transgenic mice. However, human CD59 mRNA was detected in
only four lines by Northern blot hybridization. The expression levels
of human CD59 mRNA in liver, heart, and kidney from these
transgenic mice were compared with that in the human cell line HeLa and
are shown in Fig. 2 with the copy number
of the transgene. Although this minigene contained a 4.6-kb 5'-flanking
fragment as promoter, expression levels of CD59 mRNAs were
extremely low. In HeLa cells, the CD59 probe hybridized to 0.7-, 1.3-, 1.9-, and 2.1-kb CD59 mRNA molecules, which differ in size from
their 3'-untranslated sequence produced by alternative polyadenylation
(13). In the transgenic mice, the probe only hybridized to an unusual
3.2-kb mRNA molecule (Fig. 2). This molecule may be produced by
alternative splicing of transcribed products from the transgene (see
"Discussion"). We have failed to generate high expressor CD59
transgenic mice using CD59 minigene-1, suggesting that this
minigene might lack important transcriptional regulatory elements.
We also observed by Northern blot hybridization that expression levels
of CD59 mRNA were regulated in a cell line-specific manner (Fig.
3). Extremely high level expression of
CD59 mRNA was observed in the fibroblast cell lines MRC5 and Hf19.
In addition, high level expression was detected in HeLa (epithelioid
carcinoma), HepG2 (hepatocyte carcinoma), and HWLCL (B cell) cells,
intermediate expression in JEG3 (colon carcinoma), Jurkat (T cell), and
NALM1 (B cell) cells, and low expression in Raji (B cell) cells. To investigate whether this cell line-specific expression of CD59 mRNA
was determined by promoter activity in these cells, a luciferase reporter assay was performed. The reporter plasmids were constructed using a 2-kb 5'-flanking fragment as the CD59 promoter and analyzed using cells expressing high (HeLa), intermediate (Jurkat), and low
level (Raji) CD59 mRNA. Since it is difficult to compare the promoter activities directly in different type cells, the CD59 promoter
activity in these three cell lines was compared with negative control
(pGL3-Basic Vector) and TK and SV40 promoter activities by luciferase
assay, as shown in Table I. CD59 promoter activity was detected in all cells. In HeLa cells (which express CD59
mRNA at high level), CD59 promoter activity was 5.6-fold and
4.2-fold weaker than TK and SV40 promoter activities, respectively. On
the other hand, in Jurkat cells (intermediate level), this promoter was
only 2.6- and 1.5-fold weaker than TK and SV40 promoter activities,
respectively. In Raji cells (low level), CD59 promoter activity was
3.6- and 2.4-fold weaker than TK and SV40 promoter activities,
respectively. Based upon these results, it appears that CD59 promoter
activity might not be reflected in the expression levels of CD59
mRNA. Taken together with results of CD59 expression levels in mice
transgenic for the CD59 minigene-1, these results suggest
that CD59 expression is regulated not only by a promoter but also by
other transcriptional regulatory elements such as an enhancer.
Cell Line-specific DNase I-hypersensitive Sites and an
Enhancer--
DNase I hypersensitivity assays were performed to
identify other transcriptional regulatory elements (Fig.
4A). Two DNase I-hypersensitive sites (HSs) were identified using probe E2 on EcoRI-digested DNA from HeLa nuclei but not from Jurkat and
Raji nuclei (Fig. 4B). These DNase I-HSs were mapped to
between 1.0- and 0.8-kb upstream of exon 2 (Fig. 4A) using
probe E2 with BamHI-, EcoRI/XbaI-,
BglII-, and PvuII-digested DNA (data not shown). To confirm these cell line-specific DNase I-HSs were not artifacts due
to the procedure, probe E2 was washed off the filter, and the same DNA
filter was hybridized with probe E1 (Fig. 4A). A cluster of
DNase I-HSs was detected in all three cell lines (Fig. 4C)
and mapped in the CD59 promoter region (Fig. 4A) using probe E1 with EcoRI/XhoI-digested DNA (data not
shown).
DNase I-HSs have been associated with a number of functionally specific
positions (e.g. promoters, enhancers, silencers, and origins
of replication) (20). To investigate whether CD59 expression is
controlled by the region containing the DNase I-HSs located upstream of
exon 2, we analyzed the enhancer activity of a 1.4-kb SacI
fragment (1.4-kb upstream region of exon 2 plus 5 bp of exon 2 and
containing the HSs) by luciferase reporter assays. The 1.4-kb fragment
was introduced into the downstream region of the luciferase gene in
plasmid CD59 P containing a 308-bp fragment ( Mapping and Nucleotide Sequence of CD59 Enhancer--
To establish
a more accurate position of the cell line-specific enhancer, deletion
mutants of the fragment were constructed and introduced downstream of
the luciferase gene in plasmid CD59 P (Fig.
7A). The nucleotide sequence
of the 1.4-kb fragment and positions of the 5'- and 3' (only P/5
d5)-ends of deletion mutants are shown in Fig.
8. In HeLa cells, a 2.8-fold enhancement
over CD59 promoter activity was observed when an enhancer fragment with
a 248-bp deletion was used (Fig. 7, P/5 d1). Disappearance of the
enhancer activity was caused by a further 266-bp deletion from P/5 d1
fragment (Fig. 7, P/5 d2), suggesting that the CD59 enhancer is present
in this 266-bp sequence (i.e. between Binding of Nuclear Factors to the CD59 Enhancer Region--
The
enhancer activity located between Generation of CD59 Transgenic Mice Using a CD59 Minigene Containing
the Enhancer Region--
We have identified and mapped a putative CD59
enhancer. To investigate activity of this element in vivo,
we created a second CD59 minigene construct (CD59
minigene-2) by adding an additional 0.85-kb fragment that includes the
enhancer element (Fig. 1, CD59 minigene-1 and
CD59 minigene-2). 10 lines of transgenic mice were generated, and CD59 mRNA was detected in all 10 lines (three of them are shown in Fig. 10 with the copy
number of the transgene). CD59 mRNA expression levels in all
transgenic mice generated using the minigene-2 were much higher than
those in mice transgenic for CD59 minigene-1. In
CD59 minigene-1 transgenic mice, mainly 3.2-kb CD59 mRNA
was observed. In CD59 minigene-2 transgenic mice, this
3.2-kb molecule was detected, and all alternative polyadenylation products (0.7-, 1.3-, 1.9-, and 2.1-kb mRNA molecules) were also observed. Taken together, these results suggest that CD59
gene expression is regulated by the enhancer.
CD59 with other complement regulatory proteins protects human
cells against bystander complement lysis. Therefore, expression of CD59
could be regulated in a wide range of cells. We have demonstrated that
CD59 gene expression is regulated by an enhancer. Extremely high level expression of CD59 was observed in two lung fibroblast cell
lines, MRC5 and Hf19. It is not clear why fibroblasts should express
this gene so well, but as they migrate into regions of tissue damage
and inflammation, they may be exposed to natural, potentially lytic,
complement activity. Their high level expression of CD59 probably
results from use of the enhancer element. Accordingly, we have
identified DNase I-HSs in the enhancer region in MRC5 cells (data not shown).
The CD59 enhancer seems not to be functional in the lymphocyte cell
lines Raji (B cell) and Jurkat (T cell). These cells might also
suppress gene expression of CD59 through activity of the negative
transcriptional elements in the upstream region of exon 2. Indeed, the
expression of CD59 in Raji cells was extremely low. It is conceivable
that some expendable cells (e.g. cancer cells,
virus-infected cells, damaged cells, and apoptotic cells) down-regulate
expression of the CD59 gene by turning off the enhancer and
using the negative transcriptional regulatory elements and thereby
allow complement-mediated cell destruction. Jones and Morgan (10)
reported that CD59 and decay-accelerating factor expression on the cell
surface was reduced by the apoptotic process. Perhaps signaling for
apoptosis, such as the activation of caspases (22), may down-regulate
CD59 gene expression by inactivation of the enhancer
activity and through harnessing this negative transcriptional
regulatory activity.
A 3.2-kb CD59 mRNA was detected in CD59 transgenic mice. In human
cells, 0.7-, 1.3-, 1.9-, 2.1-, and 5.8-kb mRNA molecules are
produced by alternative polyadenylation. This unusual 3.2-kb molecule
might appear to be due to lack of splicing between exon 1 and exon
2.2 An alternative
5'-untranslated exon (45-bp) was identified 5 kb downstream of exon 1 (in intron 1) (14). Our minigenes did not contain this alternative
exon. Therefore, it is possible that the 3.2-kb molecules were produced
by unusual alternative splicing due to the absence of the alternative exon.
We have mapped the CD59 enhancer to A 1.4-kb nucleotide sequence located upstream of exon 2 was screened
using the transcription factor data base (21). We have also
demonstrated that HeLa cell-specific or enriched nuclear factors bound
to the cell line-specific enhancer region ( We have demonstrated here that gene expression of the complement
regulatory protein CD59 is regulated by an enhancer. This information
will be of great value in the creation of CD59 transgenic pigs to
provide organs with enhanced resistance to lysis by human complement.
1155 and
888 upstream of the 5'-end of exon 2. To
investigate the enhancer activity in vivo, a new
CD59 minigene was constructed by the addition of the
enhancer fragment into CD59 minigene-1. High expressor CD59
transgenic mice were generated using the new minigene.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-actin cDNA as a probe.
-galactosidase plasmid by electroporation. For CAT assays, cell
extracts were prepared by freeze/thawing. CAT activity was measured by
quantifying the acetylation of [14C]chloramphenicol. CAT
activities were normalized to
-galactosidase activities.
-32P]ATP and T4 polynucleotide kinase
and purified using G-50 micro columns. Samples were analyzed by 4%
polyacrylamide gel containing 89 mM Tris borate, 2 mM EDTA, and 10% glycerol, pH 8.3.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (13K):
[in a new window]
Fig. 1.
Structure of human CD59
minigenes. Structures of CD59 minigenes are shown
with the gene structure of human CD59. Exons are indicated by
black boxes. Positions of the first ATG and the stop codon
(TAA) for CD59 gene expression are indicated.
Minigenes were constructed using the genomic fragments indicated by
solid lines. CD59 minigene-1 contains a 0.5-kb
5'-flanking region of exon 2. CD59 minigene-2 contains a
1.4-kb 5'-flanking region of exon 2. The 0.5-kb and 1.4-kb regions are
indicated.
View larger version (67K):
[in a new window]
Fig. 2.
Expression of CD59 mRNA in transgenic
mice generated using CD59 minigene-1. 10 µg of RNA
from liver, heart, and kidney of the CD59 minigene-1
transgenic mice were hybridized with the human CD59 cDNA. To
indicate amount of the loading RNA, ethidium bromide staining of the
RNA gel is shown below the Northern blot. 0.7-, 1.3-, 1.9-, and 2.1-kb CD59 mRNAs produced by alternative polyadenylation are
indicated. The copy number of the CD59 minigene in these
transgenic mice is indicated above the line
number.
View larger version (68K):
[in a new window]
Fig. 3.
Cell line-specific expression of CD59
mRNA. 10 µg of total RNAs from JEG3 (lane 1),
HeLa (lane 2), HepG2 (lane 3), MRC5 (lane
4), Hf19 (lane 5), Jurkat (lane 6), Raji
(lane 7), NALM1 (lane 8), and HWLCL (lane
9) were hybridized with a CD59 cDNA and a -actin cDNA
probe. 0.7-, 1.3-, 1.9-, 2.1-, and 5.8-kb CD59 mRNAs produced by
alternative polyadenylation are indicated.
CD59 promoter activity in HeLa, Jurkat, and Raji cells
View larger version (64K):
[in a new window]
Fig. 4.
Cell line-specific DNase I hypersensitivity
upstream of exon 2 of the CD59 gene. A,
EcoRI (E), XhoI (X), and
SacI (S) sites are indicated on the partial
restriction map of the CD59 gene. An introduced
SacI site (to isolate a 1.4-kb fragment from the gene) is
indicated by parentheses. Exons are indicated by black
boxes. DNase I-hypersensitive sites detected using probes E1 and
E2 are indicated by arrows. Positions of probe-binding
regions are shown by solid lines under the maps.
B, DNase I hypersensitivity assays were performed using
probe E2 on EcoRI-digested DNA from HeLa, Jurkat, and Raji
nuclei treated with increasing concentrations of DNase I. Two DNase
I-hypersensitive fragments detected in HeLa cells are indicated by
arrows. C, DNA filter used for probe E2
(B) was washed and rehybridized with probe E1. A cluster of
DNase I-hypersensitive fragments detected in HeLa, Jurkat, and Raji
cells are indicated by an arrow.
291 to +17) as CD59
promoter (promoter activity of this fragment was similar to that of the
2-kb fragment observed in Table I) in both orientations (Fig.
5A). The resulting plasmids
CD59 P/5 and CD59 P/13 (Fig. 5A) were transfected into HeLa
(which expresses CD59 at high level), Jurkat (intermediate level), and
Raji (low level) cells, and generated luciferase activities were
compared with that generated using plasmid CD59 P. In HeLa cells, CD59
promoter activity was up-regulated 2-fold by insertion of the 1.4-kb
fragment in both orientations. Surprisingly, this fragment suppressed
40 and 55% of CD59 promoter activities in Jurkat and Raji cells,
respectively (Fig. 5B). The activities of the cell
line-specific enhancer and the negative transcriptional regulatory
elements were also investigated using the TK promoter in pBLCAT2 (Fig.
6). A 7-fold enhancement of TK promoter
activity was detected in HeLa cells, and suppression of promoter
activity was observed in Jurkat and Raji cells by insertion of the
1.4-kb fragment upstream of the TK promoter in both orientations.
View larger version (27K):
[in a new window]
Fig. 5.
Enhancer activity of the 1.4-kb
SacI fragment located upstream of exon 2 of the CD59
gene. A, 1.4-kb SacI fragment upstream
of exon 2 was introduced downstream of the luciferase gene in plasmid
CD59 P containing the CD59 promoter. Structures of reporter plasmid
pGL3-Basic Vector (Basic), CD59 P, CD59 P/5, and CD59 P/13 are
indicated, and orientation of the promoter and the 1.4-kb fragment are
indicated by arrows. B, luciferase activities
generated using pGL3-Basic Vector, CD59 P/5, and CD59 P/13 were
compared with that generated using CD59 P in HeLa, Jurkat, and Raji
cells. Luciferase assays were repeated more than three times.
View larger version (22K):
[in a new window]
Fig. 6.
Enhancer activity of the 1.4-kb
SacI fragment located upstream of exon 2 of the CD59
gene using the TK promoter. A, 1.4-kb
SacI fragment upstream of exon 2 was introduced upstream of
the TK promoter (TK P) in pBLCAT2 (18). Structures of
reporter plasmids are indicated, and orientation of the promoter and
the 1.4-kb fragment are indicated by arrows. B,
CAT activities generated using pBLCAT3, pCTK7, and pCTK9 were compared
with that generated using pBLCAT2 in HeLa, Jurkat, and Raji
cells.
1155 and
888
upstream of the 5'-end of exon 2). Although enhancer activity was
absent in P/5 d2, this mutant seemed to suppress the CD59 promoter
activities in Jurkat and Raji cells, suggesting that the negative
transcriptional regulatory elements may be located in the 3'-flanking
region of the enhancer. It is difficult to identify the silencer
activity using this result because the reduction of luciferase activity
was not great. However, an increase in luciferase activity was caused
by a deletion of the 3' region from P/5 d1 fragment in HeLa cells (Fig.
7, P/5 d1, P/5 d5). Taken together, CD59 gene expression
might also be regulated by negative regulatory elements.
View larger version (25K):
[in a new window]
Fig. 7.
Location of the CD59 enhancer upstream of
exon 2 of the CD59 gene. A, the 1.4-kb
SacI fragment and the deletion mutants were introduced
downstream of luciferase gene in plasmid CD59 P. The structures of the
reporter plasmids are indicated. B, luciferase activities
generated using plasmids Basic (negative control plasmid pGL-3 Basic
Vector), CD59 P/5, P/5 d1, P/5 d2, P/5 d3, P/5 d4, and P/5 d5 were
compared with that using plasmid CD59 P in HeLa, Jurkat, and Raji
cells. Luciferase assays were repeated more than three times.
View larger version (40K):
[in a new window]
Fig. 8.
1.4-kb nucleotide sequence upstream of exon 2 of the CD59 gene. Nucleotide sequence of the 1.4-kb
SacI fragment was determined. The 5'-end of exon 2 is
defined as position +1. Positions of the 5'-end of the deletion mutants
and the 3'-end of P/5 d5 (Fig. 7) are indicated by arrows.
Potential transcription factor recognition sequences were screened
using the transcription factor data base (21), and some potential
recognition sequences are indicated by underlines.
1155 and
888 might be regulated
by cell type-specific nuclear factors. To investigate this, a gel
mobility band shift assay was performed using six 62-bp probes (probe
1,
1170 to
1109; probe 2,
1123 to
1062; probe 3,
1076 to
1015; probe 4,
1029 to
968; probe 5,
982 to
921; and probe 6,
935 to
874) with nuclear extracts from HeLa, Jurkat, and Raji cells
(Fig. 9). To confirm
DNA-dependent complex formation, a competition assay was
also performed with a 100-fold excess of unlabeled probe (Fig. 9,
lane 1). HeLa cell-specific or enriched complexes (C1 to C7)
were detected with probe 1, probe 3, and probe 6 and are shown in Fig.
9 (lane 2). The enhancer activity observed here might be
regulated by these nuclear factors binding to the
1170 to
1109
(probe 1),
1076 to
1015 (probe 3), and
935 to
874 (probe 6)
regions. Band A was detected both with and without competitors (Fig. 9,
lanes 1 and 2), suggesting that this band was an
artifact of the procedure (i.e. DNA-independent). Potential
transcription recognition sequences were screened using the
transcription factor data base (21), and some recognition sequences are
shown in Fig. 8.
View larger version (51K):
[in a new window]
Fig. 9.
Binding of nuclear factors to the CD59
enhancer. Binding of nuclear factors to the CD59 enhancer was
analyzed by a gel mobility band shift assay using probe 1 ( 1170 to
1109), probe 3 (
1076 to
1015), and probe 6 (
935 to
874), and
nuclear extracts from HeLa (lane 2), Jurkat (lane
3), and Raji (lane 4) cells. Binding of nuclear factors
from HeLa cells was competed with 100-fold excess of unlabeled probes
(lane 1). HeLa cell-specific or enriched complexes C1 to C7
are indicated by arrows. DNA-independent (an artifact)
band A is also indicated by an arrow.
View larger version (71K):
[in a new window]
Fig. 10.
Expression of CD59 mRNA in transgenic
mice generated using CD59 minigene-2. 10 µg of RNA
from liver, heart, and kidney of the CD59 minigene-2
transgenic mice were hybridized with the human CD59 cDNA. To
indicate amount of the loading RNA, ethidium bromide staining of the
RNA gel is shown below the Northern blot. 0.7-, 1.3-, 1.9-, and 2.1-kb CD59 mRNAs produced by alternative polyadenylation are
indicated. The copy number of the CD59 minigene in these transgenic
mice is indicated above the line number.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1155 to
888 upstream of exon 2. Since exon 1 of the CD59 gene is an untranslated exon, the
first ATG is present in exon 2. Therefore, if this enhancer also
functions as a promoter, functional CD59 mRNA may be transcribed from a site upstream of exon 2. In order to investigate the promoter activity of the enhancer, a 1.4-kb SacI fragment containing
the enhancer was introduced upstream of the luciferase gene in the pGL3-Basic Vector. Promoter activity was detected in this fragment (data not shown). We have demonstrated that some mRNA might be transcribed from approximately 50 bp upstream of the 5'-end of exon 2 by primer extension assay using total MRC5 RNA. Processing of these
mRNA may, however, be inefficient because the primer extension
products using poly(A)+ RNA are difficult to identify (data
not shown). These results suggest that this region might function
primarily as an enhancer in vivo.
1170 to
1109,
1076 to
1015, and
935 to
874). We have identified AP2 (
1150 to
1153
and
887 to
880), PEA3 (
1115 to
1110 and
923 to
918), and
AP1 (
1049 ando
1043) recognition sequences in these areas. However,
it is difficult to explain cell line-specific CD59 enhancer activity in
terms of just these factors. Therefore, this enhancer activity might be
regulated by further unknown transcription factors.
![]() |
ACKNOWLEDGEMENTS |
---|
We are most grateful to Vanessa Correa, Jeannine Okabe, and Mark Frewin for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by the Medical Research Council, The Wellcome Foundation, and The Gilman Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ010311.
§ To whom correspondence should be addressed: Sir William Dunn School of Pathology, University of Oxford, South Parks Rd., Oxford, OX1 3RE, UK. Tel.: 1865-275506; Fax: 1865-275501; E-mail: mtone{at}molbiol.ox.ac.uk.
The abbreviations used are: kb, kilobase pairs; TK, thymidine kinase; CAT, chloramphenicol acetyltransferase; HSs, hypersensitive sites; bp, base pairs.
2 M. Tone, L. E. Diamond, L. A. Walsh, Y. Tone, S. A. J. Thompson, E. M. Shanahan, J. S. Logan, and H. Waldmann, unpublished observation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|