From the
Elements of the mouse Immunoglobulin
In both early and late stage B cells, transcription termination
in the mouse immunoglobulin (Ig)
DNA constructs consisting of
various segments from the
Individual experiments were normalized
to the
See Fig. 6A for the positions of the gpt/Ig
run-on probes. Probe 9, pSV-KH3, contains the 325-nt HindIII
to KpnI fragment of the gpt gene of pSV2gpt. Probe
10, pFT13-X1(
The insertion of the
750-bp region in which
To test the potential role of the
GA repeat motifs in specifying transcription termination, we inserted
into the
The results from the run-on assays, performed
on nuclei of cells which had been transfected with the indicated
constructs, are shown in Fig. 6, B and C, and
summarized in . Relative hybridization per base pair to an
immobilized target within the gpt coding region was set as
1.0. Hybridization to probe 12 is decreased significantly in construct
AX2, indicating at least 65% termination of transcription in that
region. The run-on probe 12 spans the region from +988 to
+1228 downstream of the inserted membrane-specific poly(A) site.
From the analysis of the results in , we conclude that
the regions upstream of and including the membrane poly(A) site can
function in the gpt gene, driven by the SV40 promoter, to
cause transcription termination. We observe that deletion of the
poly(A) site, as in AX2
Since little transcription
termination was occurring just beyond the second poly(A) site, we had
expected to see only the longer gpt/Ig RNA (2.8 kb) species in
the transfectants with SphX. Those are observed, but, in addition, we
saw a species whose size (2.2 kb) is consistent with the use of the
first (membrane) poly(A) site. We conclude that the ``first come
first served'' model of poly(A) site choice
(38) is
operating in this construct.
Disruption of the
GA/CT potential stem loop in the
The ability of the
When we compare the termination in probe 6 for
constructs labeled D, G, F, and C we see a progressive increase in
termination efficiency (from 40-56% to 76-97%, see
). This progression seems to parallel an increase in the
``strength'' of the poly(A) sites involved. We previously
measured the amount of steady state cytoplasmic RNA in J558L cells
transfected with different constructs in which the membrane or
secretory-specific poly(A) regions were put in a cis-competition with
an identical downstream SV40 early poly(A) site
(35) . The
myeloma cells containing the
We thank Sharon Harrold and Makiko Hartman for superb
technical assistance. We are very grateful to Drs. Justus Cohen and
Gretchen Edwalds-Gilbert for helpful comments.
2a gene, near the
membrane-specific poly(A) addition site, were inserted into a
heterologous location in either a synthetic mouse
2b gene or a
gpt/SV40 chimeric gene and then assayed for their ability to
terminate RNA polymerase II transcription in isolated nuclei of
transfected myeloma cells. The intact
2a membrane-specific
3`-untranslated region, with its potential stem loop forming sequences
and poly(A) site, is able to efficiently terminate transcription in the
absence of the downstream region in which transcription normally
terminates (term). Termination efficiency in the presence of the
termination fragment decreases either when sequences specifying a
potential stem/loop, upstream of the poly(A) region, are interrupted or
when the stronger membrane poly(A) site is substituted with a weaker,
secretory-specific poly(A) site. We therefore conclude that the
2a
membrane-specific untranslated region plays a major role in specifying
downstream termination. We further conclude that the immunoglobulin
2a, membrane-specific, 3`-untranslated region can function in the
context of the gpt gene, driven by an SV40 promoter, to
terminate transcription in a poly(A) site dependent fashion.
(
)
2b and
2a heavy chain genes occurs in DNA sequences (term) which span the
region 500-1200 bp downstream of the respective promoter-distal
membrane poly(A) sites. The termination sites of the intact gene are
the same regardless of the developmental stage of the B
cell
(1) . Differential transcription termination upstream of the
membrane poly(A) site therefore is not involved in Ig
2b and
-
2a heavy chain developmental regulation as it may be in the µ
gene
(2, 3, 4, 5, 6, 7, 8) .
The 3`-UT and downstream regions of the mouse Ig
2a gene contains
many unusual sequence elements
(9) . There are two tandem
membrane-specific poly(A) sites separated by 77 nt. Starting about 1000
nt downstream of the M2 coding sequences, and 5` of the AAUAAA's,
there is a region containing (GAA)
, (GA)
,
(GGAA)
, (GGA)
, and (GA)
elements,
followed by an unremarkable 90 bp, followed by (CT)
,
followed by (GT)
. Then come the two A
UA
elements (see Fig. 1B). The GA and (CT)
repeats could intrastrand-base-pair to form a stem and loop
structure approximately 95 nt upstream of the first
2a membrane
poly(A) site with a
G = -114 kcal/mol.
Extensively repeated elements like those present in the
2a
membrane 3`-untranslated region are not present in the membrane
3`-untranslated regions of murine µ,
3 or
2b
genes
(10, 11, 12) . Differential expression of
the various
isotypes has been observed in different mouse
strains
(13) . It is not known what regulatory role the long
untranslated region in the
2a gene plays in differential isotype
expression.
Figure 1:
Constructs used. The
functionally rearranged mouse MPC11 Ig2b heavy chain gene (on
three EcoRI fragments) was previously inserted into the unique
EcoRI site of the pSV2gpt plasmid (A) to create
pMPC11. A large deletion of the secretory-specific poly(A) site was
made to create pMPC11 Oli Sac-Kpn, into which various portions of the
2a gene membrane 3`-untranslated region and downstream elements
was cloned (see ``Materials and Methods''). This family of
constructs was termed pMPC11 2a. . . . The origin of the inserted
segments on the
2a map is shown in B. The final
pMPC11-derived constructs are shown in Figs. 2-5, above the
relevant data. Segments of the
2a gene membrane 3`-untranslated
region (B) were cloned directly into the HpaI site of
naive pSV2gpt to create the AX2, AX2
pA, XX, and SphX constructs,
see Fig. 6A. The Ig
2a gene region of GA-repeated elements
is indicated in B as a shaded box labeled (GA); the
(CT)
element is indicated as a shaded box; the
(GT)
element is shown as a stippled box; and the
region where RNA transcription terminates in the intact Ig
2a gene
is indicated by a striped box labeled
term.
Eucaryotic RNA polymerase II transcription termination
has been defined to occur at heterogeneous sites far downstream of the
mature, processed 3` end of the
transcript
(14, 15, 16, 17, 18, 19) .
There is a linkage between polyadenylation and termination in at least
some polymerase II transcribed
genes
(20, 21, 22, 23, 24, 25) .
With Igµ, a correlation between poly(A) site strength and the
efficiency of termination was recently shown (26). Little more is known
about the eucaryotic termination process except that it may occur at
regions of repeated sequence in the DNA. Elements upstream of
retrovirus poly(A) sites, some involving stem/loop structures to
maintain proper spacing, have been implicated in mediating differential
poly(A) site use
(28, 29) . We wondered if the potential
stem loop structure in the 2a gene might serve as a terminator in
eucaryotes as stem loops do in factor-independent terminators in
procaryotes (reviewed in Ref. 27).
2a heavy chain gene membrane-specific
3`-UT and 3`-flanking regions, including the region where
2a
transcription normally terminates, were inserted into a
2b heavy
chain gene which had been deleted for the sec poly(A) site. Using
nuclei of mouse myeloma cell lines stably transfected with these
constructs, we find that the
2a membrane untranslated region
itself is the most important part of the transcription termination
signal. When
2a membrane UT sequences were inserted into a
chimeric gpt/SV40 gene of pSV2gpt, transcription termination
was further shown to be induced even with a heterologous promoter and
gene, indicating that the sequences have a mode of action independent
of the Ig gene. The rate of use of the membrane site was influenced not
only by the presence of the unusual sequence elements upstream of the
poly(A) site, but also by the poly(A) site itself.
Cell Culture and Stable DNA Transfection
The
murine plasmacytoma J558L has lost endogenous heavy chain expression
through a deletion in the heavy chain locus but maintains myeloma
regulation of secretory versus membrane-specific mRNA
production
(30, 31) . J558L cells were stably transfected
with derivatives of pSV2gpt by protoplast fusion
(30) .
Mycophenolic acid-resistant clones were isolated. Only clones which met
the criteria of RNA expression and contiguous integration of a single
copy of the DNA (Southern blots, not shown) were used for subsequent
analyses.
Ig
Plasmid pMPC11 Oli
Sac-Kpn was derived from the functionally rearranged MPC11
(31, 32) Ig2a Gene Constructs
2b heavy chain gene inserted into the EcoRI
site of the mammalian expression vector pSV2gpt
(33) , see
Fig. 1
; the vector confers resistance to mycophenolic acid. An
oligonucleotide linker containing EcoRI, HindIII, and
KpnI restriction sites was inserted at the SacI site
located downstream of the splice site at the 3` end of the Ig
2b
CH3 exon. Digestion with KpnI and subsequent religation
removed an 830-bp KpnI fragment containing the
2b sec
poly(A) site and region downstream to the second KpnI site,
which is located 0.55 kb 5` of the M1 exon. This step eliminated the
endogenous Ig
2b sec poly(A) site and would allow partial
restoration of the CH3-M1 intron distance upon insertion of
0.9-2.2-kb putative
2a terminator fragments. We refer to
pMPC11 Oli Sac-Kpn as ``Construct A'' for simplicity.
Segments from the
2a gene 3` end (GenBank
accession
number M35032
(9) ) were then cloned into the multicloning region
of pGEM3 or pGEM4
(34) prior to their excision with
EcoRI and HindIII digestion and subsequent insertion
into pMPC11 Oli Sac-Kpn in an orientation-specific fashion using the
unique EcoRI and HindIII sites in the oligonucleotide
linker at the 3` end of the CH3 exon. Construct B (pMPC11
)
contains a 2.2-kb HindIII fragment from map units
22.4-22.6 kb on the 40.4-kb genome of
phage cI857 DNA.
Construct C (pMPC11 2ap(A)m(SL) + term) contains a 2.2-kb
BglII-PstI fragment encompassing: the
2a
membrane poly(A) site and 0.45 kb of upstream 3`-UT sequence with the
GA/CT repeats (SL = stem loop) and 1.25 kb of downstream
sequence, including the
2a transcription termination region
(term). Construct D (pMPC11 2a term alone) contains a 1.1-kb
BamHI-PstI fragment encompassing the previously
identified
2a transcription termination region. Construct E
(pMPC11 2ap(A)m(SL) alone) contains a 1.4-kb
BglII-PvuII fragment which includes the
2a
membrane poly(A) site and associated sequences (including upstream (SL)
repeat motif) but lacks the downstream termination region. Construct F
(pMPC11 2ap(A)m + term) contains a 1.5-kb
HindIII-PstI fragment, including the
2a membrane
poly(A) site and downstream termination region but with only the 3`
portion (the CT) of the GA/CT repeat motif and the (CT)
element. Construct G (pMPC11 2bp(A)s + term) contains both a
167-bp SacI-BalI fragment, including the
2b sec
poly(A) site and a downstream 1.1-kb BamHI-PstI
fragment containing the
2a transcription termination region.
gpt/Ig Gene Constructs
Plasmid pSV2 gpt-EH5A has
the 2.85-kb EcoRI fragment with the Ig heavy chain enhancer
cloned into the EcoRI site of pSV2gpt
(35) . The IgH
enhancer is in the opposite transcription orientation to the gpt gene. DNA fragments of the 2a membrane 3`-UT region (see
Fig. 1B) were cloned into the unique HpaI site
of pSV2 gpt-EH5A. Clone gpt-AX2 contains the 1105 nt second
AccI to second XbaI fragment with the GA, CT, and GT
elements of the
2a 3`-UT plus the mb poly(A) sites. In
gpt-AX2
pA the two mb poly(A) sites of gpt-AX2 have been removed by
deletion of the 189-nt SphI to PvuII fragment.
Plasmid gptXX (formerly called MPA+E, or
gptmb&cjs1219;SV
(35) ) contains the 780-nt XbaI to
XbaI fragment of the
2a 3`-UT and therefore has the CT
and GT elements plus the mb poly(A) sites but lacks all the GA
elements. Plasmid gptSphX (called MSL by D. Boczkowski) contains the
670-nt SphI to XbaI fragment of the
2a 3`-UT
with the mb poly(A) sites; all of the upstream dinucleotide repeat
elements are missing.
Transient Transfection of Lymphoid Cells
J558L
cells were transiently transfected using a Lipofectin (Life
Technologies, Inc.) technique with 20 µg of DNA/5 10
cells; cells were maintained in growth medium for 48 h. At that
time cytoplasmic, poly(A) containing RNA was isolated and/or nuclear
run-on analysis was performed.
Nuclear Run-on Transcription Assay
The conditions
used for nascent RNA transcript labeling (15 min) in isolated nuclei
are identical to those described previously
(1, 36) . The
linear response range of the film was determined, and autoradiograms
from exposures in that range were scanned in a Bio-Rad densitometer.
Raw hybridization intensities minus background were obtained for each
probe region. The relative hybridization per base of each probe was
calculated using the following formula (hybridization intensity length
of probe in bases = relative hybridization per base).
Overexposed films are often shown in the figures for better
photographic reproductions.
-actin (Ac) control slot. Each construct was assayed a
minimum of two times with two different clones or transient
transfections and the range of the results from the several experiments
is indicated by the error bars in some of the graphs. The cRNA targets
are calculated to be in at least a 65-fold excess to the
P-labeled heavy chain nuclear RNA, assuming 10 pg of
nuclear RNA/cell, with 2% of total RNA as poly(A) containing RNA, 6% of
poly(A) containing RNA as Ig-specific mRNA, and a 1.88-kb mRNA. In
addition, experiments in which increasing amounts of the
[
P]-labeled nuclear RNA were added showed a
parallel increase in hybridization to the filters, indicating that the
targets are in excess (data not shown). Target sequences on the filters
were chosen such that hybridization of the
P-labeled J558L
nuclear RNA was less than 3% of that seen with cells transfected with
the appropriate gene (data not shown).
Target Probes for Run-ons
Plasmids plus T7
bacteriophage RNA polymerase were used to generate microgram quantities
of H-labeled cRNA ``target'' run-on probes
immobilized on filters as described previously
(1, 36) .
All of the cRNAs were cloned into pGEM (Promega) vectors. Probe 1,
pG4MK1, yields a 1.2-kb cRNA spanning a portion of the variable region
intron after SalI linearization. Probe 2, p2aCH3, contains a
0.31-kb SacI fragment, including the
2a CH3 exon. Probe
3, pXDB1.2, contains a 0.3-kb AccI-HindIII fragment
from the
2a mb 3`-UT, including the 5`-GA portion of the GA/CT
repeat motif upstream of the
2a membrane poly(A) site. Probe 4,
pSX, contains a 0.44-kb SacI-XbaI fragment located
0.17 kb 3` of the
2a mb poly(A) site. Probe 5, pFT13-X2 contains a
0.64-kb XbaI-PstI fragment located 0.61 kb downstream
of the
2a membrane poly(A) site, including the transcription
termination region and repetitive elements as assayed by Southern blot
analysis (data not shown). Probe 6, pM1M2 contains a 1.15-kb
KpnI-PstI fragment, including the
2b membrane
exons. Probe 7, pPSac contains a 1.4-kb PstI-SacI
fragment, including the
2b mb 3`-UT and poly(A) site. Probe 8,
pDF1 contains a 0.31-kb SacI-EcoRI fragment located
0.25 kb downstream of the
2b mb poly(A) site. Probes 2`, 3`, and
6` are sense cRNA probes designed to measure opposite strand
transcription across probe regions 2, 3, and 6, respectively. Actin
cRNA (Ac) is transcribed from pG4pal41 which contains a 1.1-kb
PstI fragment of the mouse
-actin cDNA
(37) .
S
S), contains the 222-nt SphI to
SacI fragment of the Ig
2a gene
(9) , the region
near the membrane poly(A) sites. Probe 11, pSVBH contains the 238-nt
BamHI to HincII fragment of pSV2gpt. Probe 12, pSV-HH
contains the 240-nt HincII to HincII fragment of pSV2
gpt which derives originally from SV40 and begins 288 nt downstream of
the SV40 early poly(A) site of the vector and 988 nt downstream of the
inserted membrane poly(A) site in plasmids gpt-AX2, -XX, and -SphX.
Figure 6:
Transcription run-on experiments with the
gpt/2a constructs. A, segments of the Ig
2a
3`-untranslated region and downstream sequence were inserted into the
3` end of the gpt gene of pSV2gpt. The location of sequences
used as run-on probes is indicated above the map line. Regions of GA
and CT sequences are indicated as shaded areas on the linear
map of AX2. The first AAUAAA on the map line is the first membrane
poly(A) sequence of the insert, whereas the second AAUAAA is that of
the SV40 sequences in the vector. The origin of the inserted segments
from the intact
2a gene are shown below the map line and are also
shown in Fig. 1B for comparison with the pMPC11 series
constructs. Open arrows indicate vector sequences of the full
gpt/insert transcript, present in all constructs. The
solid arrow denotes the AX2 insert; the dotted arrow shows the AX2
pA insert; the diagonally striped arrow denotes the XX insert, and the vertically striped arrow shows the SphX insert. B, transient transfections were
performed on J558L with the various constructs and nuclei were labeled
for 15 min with [
P]UTP, see ``Materials and
Methods.'' Immobilized run-on probes on the filters were the
single stranded antisense probes 9 through 12, the sense strand of 9,
denoted 9`, and
-actin (Ac). Different exposures of the film were
used to preserve details. C, the relative hybridization per
base to each run-on probe was calculated from several experiments, as
described under ``Materials and Methods.'' Hybridization
intensities were normalized to 1.0 for probe
9.
RESULTS
The
The ability of RNA polymerase II to transcribe through the
various inserted putative terminator elements in the Ig2a Membrane 3`-UT and 3`-Flanking Region
Specifies Efficient Transcription Termination in the
2b
Gene
gene was
assayed in nuclear run-on experiments on stably transfected cell lines.
A
2b heavy chain gene construct deleted for its first poly(A) site
and containing no inserted putative transcription termination element,
pMPC11 Oli Sac-Kpn, construct A, supports the transcription of the
regions downstream from the inserted 24-bp oligonucleotide polylinker
at levels nearly equal to transcription of the upstream constant
region-encoding exons (Fig. 2A, 3-7% termination;
). Insertion of a 2.2-kb fragment of heterologous
phage DNA into the polylinker, in pMPC11
, construct B, only
slightly increases termination prior to transcription of the downstream
probes 6/7 region with most RNA polymerase II molecules traversing the
DNA as indicated by the strong hybridization to probes 6 and 7
(Fig. 2B, 12-17% termination). Appropriate sense
strand cRNA controls, which were designed to detect opposite strand
transcription, were included in each nuclear run-on assay (e.g.Fig. 2
, probes 2`, 3`, and 6`). In all the experiments there
was little or no opposite strand transcription as indicated by the low
or absent hybridization to the opposite orientation probes.
Hybridization of
P-labeled nuclear RNA from untransfected
J558L cells to probes 1-4 and 6-8 was less than 3% of that
seen in the cells transfected with the intact MPC11 gene (data not
shown). A
-actin
(37) antisense cRNA probe was used to
normalize transcription rates between independent experiments. A lower
hybridization signal to the probe 1 region, which includes a portion of
the VJH to CH1 region intron, than to probe 2, was consistently
observed and may reflect polymerase passing through the probe 1 region
without subsequent new initiation during preparation of nuclei or
possible nonuniform polymerase loading. Note that probes 1 and 2 are
separated by at least 6 kb. Therefore, hybridization to probe 2 was
used for the normalizations in the figures and .
Figure 2:
Determination of nascent transcript
distribution across stably transfected transcription termination
constructs. A-C show the results of representative
nuclear run-on analyses performed on Ig constructs A, B, and C,
respectively, stably transfected into J558L myeloma cells. At the
top of each panel is a schematic map of the 2b constant
region with inserted putative termination element. Below the gene are
the DNA regions used to generate filter-bound single-stranded cRNA
target probes. Representative autoradiograms showing hybridization to
slot blots of cRNA probes with
-P-labeled nuclear
transcripts are shown below the gene. A 1.1-kb cRNA probe (Ac) specific
for mouse
-actin mRNA was used as a positive control and to
normalize relative
heavy chain transcription rates between
independent experiments. Probes 2` and 3` were used in appropriate
constructs to detect opposite strand transcription across these
respective regions. Hybridization intensity was corrected for length of
the probes used. Results shown in bar graph form at the
bottom are averages of several independent determinations per
probe fragment (see ``Materials and Methods''). Plasmids used
to generate run-on cRNA probes are described under ``Materials and
Methods.'' A, J558L/pMPC11 Oli Sac-Kpn; B,
J558L/pMPC11
term; and C, J558L/pMPC11 2ap(A)m(SL) +
term.
Insertion of a 2.2-kb fragment of the 2a gene 3` end containing
the previously identified region where
2a transcription normally
terminates (term) as well as the
2a membrane poly(A) signal/site
and upstream set of stem loop-specifying GA/CT repeat elements (SL)
plus (GT)
in the 3`-UT was able to act as a very efficient
termination element in this system. As seen in Fig. 2C,
insertion of this whole element in pMPC11 p(A)m(SL) + term,
, construct C, caused a 93-97% decrease in
transcription of downstream regions as assayed in isolated transfectant
nuclei. The effect of the
2a region appears to be specific, since
the absence of an insert or the presence of a heterologous DNA insert
failed to cause termination (Fig. 2, A and B).
The probe 5 region contains repetitive DNA elements which are
responsible for the high signal obtained so it was subsequently omitted
from the analyses. Hybridization of run-on RNA from nuclei of pMPC11
2ap(A)m(SL) + term transformants to probes 6 and 7 was very
minimal, indicating that, as we had determined by sequence comparisons,
the
2a and
2b gene elements are sufficiently diverged to
allow for our analyses with these probes.
2a transcription normally terminates (term)
with no other upstream
2a sequences into the
2b gene (pMPC11
2a term alone) leads to significant read-through of the termination
region into the probe 6/7 region ( Fig. 3and ,
construct D), indicating that the region in which
2a transcription
terminates normally fails to efficiently stop Pol II on its own.
Figure 3:
Comparison of termination efficiencies of
term alone versus the 3`-untranslated regions of the 2a
gene. The top of each panel shows a schematic map of the Ig
construct with the locations of the cRNA target probes indicated below.
Representative autoradiograms of slot blots of immobilized cRNAs
hybridized to nuclear run-on
P-labeled RNA from
transfected cells are shown below the map. The results from several
independent determinations are shown in the bar graphs. Probes
2` and 6` would detect opposite strand transcription. Ac equals
transcription of
-actin sequences used as a control as described
under ``Materials and Methods.'' These two constructs are
labeled D and E, respectively, in Table I.
The
construct pMPC11 2ap(A)m(SL) alone, construct E, contains the 2a
3`-UT with stem/loop structure and mb poly(A) sites. The data in
Fig. 3
and show, somewhat surprisingly, that the
2a mb 3`-UT and poly(A) region acts as an extremely efficient
transcriptional terminator in our constructs. Indeed, transcription has
decreased by 72% over the downstream probe 6 region and by 92% over the
probe 7 region, relative to transcription of the upstream probe 2 (CH3
exon) region. The ability of the
2a membrane 3`-UT and poly(A)
region to terminate transcription on its own and the inefficiency of
the region of termination alone (term) in specifying Pol II stopping
indicates a major role for the
2a mb 3`-UT and poly(A) region in
specifying downstream termination.
2b gene a fragment starting 30 bp upstream of the
(CT)
element of the
2a membrane 3`-UT region and
containing the downstream termination region, but lacking the GA repeat
motifs upstream, creating pMPC11 2ap(A)m + term, construct F in
. As shown in Fig. 4, this construct was still able
to cause specific transcription termination, but at a reduced
efficiency (see ) in the nuclear run on assay. Therefore,
the stem/loop may be an important determinant of the efficiency with
which the membrane 3`-UT is able to halt the polymerase molecule.
Figure 4:
Comparison of termination efficiences of
two poly(A) regions. The results are displayed as described in the
legend to Fig. 3. These Ig constructs are labeled F and G,
respectively, in Table I.
The
To determine whether another poly(A) region could
act as a functional substitute and whether the strength of the poly(A)
site played a role in termination, we inserted the 2b Secretory-specific Poly(A) Region Is Less
Efficient Than the
2a Membrane Poly(A) Site in Specifying
Termination
2b sec poly(A)
region together with the downstream
2a termination region into
pMPC11 Oli Sac-Kpn to create pMPC11 2bp(A)s + term, construct G.
We have previously shown this Ig secretory-specific poly(A) site to be
almost 10 times weaker at directing polyadenylation than the
membrane-specific sites when both are placed in competition with the
same downstream element
(35, 38) . As shown in
Fig. 4
, and summarized in , this insert is able to
cause 56-64% transcription termination. So the secretory-specific
poly(A) site + term is more efficient than the
2a term region
alone, but was not able to terminate transcription as efficiently as
the intact
2a membrane poly(A) region alone or pMPC11 2a
poly(A)m(SL) + term. We conclude that stronger poly(A) sites cause
more efficient termination than weaker sites. We conclude further that
the term region has the effect of increasing termination in conjunction
with the sec poly(A) site, since the sec poly(A) site has been
previously shown to cause little or no termination on its own (
and Ref. 1).
Sizes of Poly(A) Containing mRNA Made in Ig Gene
Constructs
The expected size or sizes of the transcripts from
each of the constructs is shown in Fig. 5. Cytoplasmic, poly(A)
containing RNA was isolated, run on denaturing formaldehyde:agarose
gels, blotted to membranes, and hybridized with a
P-labeled probe for the V
domain of the Ig
gene. As shown in Fig. 5, the recipient cell line, J558L,
produces no endogenous cytoplasmic RNA capable of hybridizing with the
V
-specific probe, whereas hybridization of the same blot
with a glycerol aldehyde phosphate dehydrogenase probe shows that RNA
was present (data not shown). We therefore conclude that the sizes of
the cytoplasmic RNAs produced from cells tranfected with constructs A,
B, C, D, and E were consistent with the results we had obtained from
the transcription run-on studies. Transfectants with constructs F and G
produce primarily the shorter of the two predicted species, indicating
little use of the second poly(A) site in the transcript. We assume that
the virtual absence of the larger predicted mRNA species in construct G
(pMPC11 2b(A)
+ term) where 40% of the molecules
should read through the second poly(A) site, indicates that either
insertion of the termination region in the CH3 to M1 intron interferes
with splicing or the first poly(A) site is able to compete for the
polyadenylation machinery of the cell because of its promoter proximal
location and the large distance between the sites.
Figure 5:
Size analysis of RNA from Ig gene
constructs. The Ig constructs, poly(A) sites, and expected mRNA sizes
are diagramed on top. An autoradiogram of a Northern blot is
shown below. One microgram of cytoplasmic, poly(A)-containing RNA was
isolated from stable transfectants of constructs A-E and pMPC11,
run on a denaturing formaldehyde-agarose gel, blotted to membranes, and
hybridized with a P-labeled probe for the V
domain of the IgG MPC11 gene. Various exposures of the film, over
a 20-fold range, are shown to preserve detail. Sizes were determined
relative to 18 and 28 S rRNA run in a separate lane. J558L is the
recipient line for all the transfectants.
The amounts of
poly(A)-containing cytoplasmic mRNA detected by the V probe
varied not only from construct to construct, but also from transfectant
to transfectant with the same construct (data not shown). We have
previously shown that between individual stable transfectants, even
with the same Ig construct, the amount of exogeneous Ig heavy chain
mRNA, as a percentage of total RNA in that cell, can vary from 5 to 50%
of the level seen in a normal plasma cell (31). We assume that this
variation is a function of the position of insertion of the exogenously
added Ig gene. Therefore, conclusions about the relative abundance of
the cytoplasmic poly(A) containing RNAs between constructs is not
informative.
Termination Directed by the
Ig2a Membrane Region Can
Occur in a Heterologous Gene and Is Dependent on the Presence of a
Poly(A) Signal
2a fragments were inserted into the unique
HpaI site of pSV2gptEH5a, downstream of the gpt coding region, yet upstream of the vector, SV40 early,
polyadenylation site as shown in Fig. 1and
Fig. 6A. In these constructs the gpt gene is
driven by the SV40 promoter:enhancer elements. Construct AX2 contains
the entire GA/CT stem/loop, (GT)
element and the
membrane-specific poly(A) encoding region. Construct XX contains only
the (CT)
portion, (GT)
and poly(A) region of
AX2. In AX2
pA the SphI to PvuII fragment
containing the two membrane-specific poly(A) site was removed from AX2.
Construct SphX contains the poly(A) site and the region 3` of it from
AX2, but lacks the GA/CT and GT regions entirely. We used transient
transfections where it is possible to detect more transcription signal
from the large number of input molecules. The gpt signal in
these experiments was roughly 5% of that seen with an internal
-actin control.
pA, causes a decrease in termination
efficiency, yet the membrane-specific poly(A) site alone is
insufficient to cause termination in SphX. Therefore, the interaction
of the region upstream of the membrane poly(A) site plus the region
surrounding the A
UA
element itself are
necessary and sufficient for efficient termination and function
independently of the Ig promoter.
Sizes of Poly(A) Containing mRNA Made in gpt Gene
Constructs
Cytoplasmic poly(A)-containing RNA was isolated from
the J558L transfectants, size-fractionated on denaturing RNA gels,
blotted to membranes, and hybridized with a P-labeled,
antisense probe for the body of the gpt message, i.e. probe 9,
see Fig. 6A. The predicted sizes of the gpt
RNAs are listed in . As shown in Fig. 7, in cells
transfected with AX2 and XX, the most abundant gpt/Ig RNA
species were the ones corresponding to use of the first,
2a,
membrane poly(A) site. In accordance with the transcription termination
data, very little of the RNA ending with the downstream (SV40 early,
vector) poly(A) site was observed.
Figure 7:
Size
analysis of RNA from gpt/2a gene constructs. Cytoplasmic,
poly(A)-containing RNA was isolated from transfectants of the
constructs shown in Fig. 6A, run on a denaturing
formaldehyde-agarose gel, blotted to membranes, and hybridized with a
P-labeled probe for the 5` end of the gpt gene,
probe 9, see Fig. 6A. Sizes of the gpt/Ig RNAs were
determined relative to 18 and 28 S rRNA run in a separate lane. J558L
is the recipient line for all the transfectants and showed no RNA
hybridizable with probe 9 (data not shown). Lanes 1-4,
four different transfections with AX2; lanes 5-8, four
different transfections with AX2
pA; lanes 9-11,
three different transfections with XX; lanes 12-14,
three different transfections with SphX.
In cells transfected with
AX2pA, gpt/Ig RNA, with a size corresponding to use of
the downstream vector poly(A), was the major species observed,
consistent with the run-on data. However, there was also a
gpt/Ig RNA species of 2.15 kb. We hypothesize that this RNA
may arises from a AATAA sequence about 35 nt downstream of the start of
the AX2 insert. Some of this RNA is also observed in a few of the AX2
transfectants with an intact stem/loop and poly(A) site, see lanes
3 and 4, Fig. 7.
DISCUSSION
We show that the Ig heavy chain 2a membrane poly(A)
region, which contains an upstream GA/CT element, a (GT)
element and two tandem poly(A) signal/sites, each with their own
downstream GT-rich elements, is capable of specifying very efficient
transcription termination in two different types of constructs. We see
72-92% termination in a construct driven by the homologous Ig
promoter and 65% termination in a construct where it is driven by the
SV40 promoter. The distance of the termination event relative to the
2a membrane poly(A) site is over a range of 320-1470 nt, analogous
to most eucaryotic genes thus far studied where the poly(A) to
termination event distance is approximately 1000 nt. The Ig
2a
membrane poly(A) site could therefore serve as a moderately sized
(approximately 600 nt) cassette for gene expression studies where an
efficient poly(A) site:terminator is required.
2a membrane 3`-UT in the
construct pMPC11 2ap(A)m + term, construct F, while still allowing
fairly efficient termination, increases the level of read-through
transcription relative to constructs C or E. The same is true in the
comparison of constructs XX and AX2 in . The formation of
an RNA stem loop 95 nt upstream of the membrane poly(A) site could
enhance termination by directly increasing membrane poly(A) site
utilization through sequestering of polyadenylation factors and/or
pausing the RNA polymerase II elongation complex. In this model
polyadenylation and termination could be linked and increasing membrane
poly(A) site utilization inherently would lead to increasing
transcription termination. Alternatively, the RNA stem loop may
increase termination efficiency directly through slowing or stalling
polymerase II and/or providing a site where a putative
``antitermination factor'' is lost or a ``termination
factor'' is bound to the elongation complex. This eucaryotic
element bears a striking resemblance to the procaryotic factor
independent terminators for RNA polymerase where the polymerase stalls
at a stem loop structure in the RNA. A nuclear factor binding site has
been located just upstream
(8) of the µ membrane poly(A)
site and has been proposed as a nucleation site for termination factor
binding. The region upstream of the µ membrane poly(A) site has
also been reported to stall or pause polymerase II elongation through
interaction with DNA repetitive elements in the vicinity of the µ
membrane poly(A) site and mediate termination
(2, 3) .
The presence of a (GT)
element, also upstream of the
2a membrane poly(A) site, is intriguing, since GT-rich elements
are found downstream of many poly(A) sites. The (GT)
element may have a role in enhancing the strength of the membrane
specific poly(A) site.
2a termination region
to cause only 40% transcription termination on its own mirrors the
inefficiency of the mouse
major globin gene termination region in
specifying termination when inserted into the adenovirus E1A gene
(20) and is not surprising, given the sequence heterogeneity
present in most identified regions of polymerase II disengagement and
the demonstrated need for a functional poly(A)
site
(20, 21, 22, 23, 24, 25) .
Repetitive DNA elements have been found at or near the termination
regions of both Ig-
(8) and non-Ig-encoding genes
(17, 40) and have been postulated to play a role in termination,
possibly mediating the ``stacking up'' of polymerase II
molecules to facilitate dissociation from the DNA
template
(3, 4) . The
2a termination region may
contribute to termination by possessing a sequence which facilitates
polymerase II pausing and/or disengagement as directed by upstream
sequence elements.
2b secretory poly(A) region produced
cytoplasmic RNA in which the sec poly(A)-terminated molecules were
three times as abundant as the SV40 poly(A)-terminated molecules. Cells
transfected with
2a membrane poly(A) region constructs with only
the (CT)
half of the potential stem/loop sequences
produced RNA in which the mb-terminated molecules were 26 times as
abundant as the SV40-terminated molecules
(35) . The cells
transfected with the full GA:CT sequence element of the 2a mb
untranslated region produced RNA in which the mb-terminated molecules
were 89 times as abundant as the SV40-terminated molecules
(41) .
So, by the criteria of stable mRNA produced, the membrane poly(A) site
is much stronger than the sec site. Likewise, we and others have also
shown that the µ membrane site is stronger than the µ secretory
site as assayed in various
constructs
(2, 3, 26, 35, 38, 39) .
In the absence of any known poly(A) site, the term alone construct
(construct D), termination falls to 40%. We conclude that a poly(A)
site is required for efficient termination, and the stronger the site,
the more active the process becomes. This conclusion is confirmed by
the lack of termination in the construct AX2
pA. A functional
poly(A) site has been shown to be mandatory for termination of
polymerase II transcripts both in cells and nuclear
extracts
(20, 21, 22, 23, 24, 25) .
It is intriguing to speculate whether poly(A) site
``strength'' in other polymerase II gene products is a true
reflection of the polyadenylation/cleavage efficiency of the sequence
itself or is more a function of the ability of that site to
promote termination which then facilitates mature 3` end formation. The
stronger membrane Igµ poly(A) site terminates transcription more
efficiently than the weaker secretory-specific Igµ site in an
adenovirus hybrid construct
(26) . Transcription termination and
poly(A) site strength are clearly interrelated, perhaps not directly
through cleavage and polyadenylation itself, but through assembly or
disassembly of an elongation complex.
Table:
Relative termination efficiencies of
inserted putative termination elements in IgG gene constructs
Table:
Relative termination efficiencies of inserted
2aIg elements in gpt constructs
2a
membrane-encoding 3`-untranslated region; term, region where the
Ig
2a gene normally terminates transcription; bp, base pair; UT,
untranslated; nt, nucleotide(s); kb, kilobase(s); mb, membrane; sec,
secretory.
2a Membrane 3`-Untranslated Region. Ph.D. thesis, University of Pittsburgh, Pittsburgh, PA
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.