©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The 3`-Untranslated Region of Membrane Exon 2 from the 2a Immunoglobulin Gene Contributes to Efficient Transcription Termination (*)

John A. Flaspohler , David Boczkowski , Brenda L. Hall , Christine Milcarek (§)

From the (1) Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261-2072

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Elements of the mouse Immunoglobulin 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.


INTRODUCTION

In both early and late stage B cells, transcription termination in the mouse immunoglobulin (Ig)() 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 Ig2b 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 Ig2a 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 AUA 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, AX2pA, XX, and SphX constructs, see Fig. 6A. The Ig2a 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 Ig2a 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).

DNA constructs consisting of various segments from the 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.


MATERIALS AND METHODS

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.

Ig2a Gene Constructs

Plasmid pMPC11 Oli Sac-Kpn was derived from the functionally rearranged MPC11 (31, 32) Ig2b 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 Ig2b 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 Ig2b 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-AX2pA 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.

Individual experiments were normalized to the -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) .

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(SS), contains the 222-nt SphI to SacI fragment of the Ig2a 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 Ig2a 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 AX2pA 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 2a Membrane 3`-UT and 3`-Flanking Region Specifies Efficient Transcription Termination in the 2b Gene

The ability of RNA polymerase II to transcribe through the various inserted putative terminator elements in the Ig 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.

The insertion of the 750-bp region in which 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.

To test the potential role of the GA repeat motifs in specifying transcription termination, we inserted into the 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 2b Secretory-specific Poly(A) Region Is Less Efficient Than the 2a Membrane Poly(A) Site in Specifying Termination

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 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 2a Membrane Region Can Occur in a Heterologous Gene and Is Dependent on the Presence of a Poly(A) Signal

Ig2a 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 AX2pA 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.

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 AX2pA, 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 AUA 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 AX2pA; 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.

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.

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 Ig2a 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.

Disruption of the GA/CT potential stem loop in the 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.

The ability of the 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.

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 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 AX2pA. 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



FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM 50145 (to C. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 412-648-9098; Fax: 412-624-1401.

The abbreviations used are: Ig, immunoglobulin; m, membrane-encoding; pA, polyadenylation signal/site; s, secretory-encoding; S/L, potential stem/loop structure in Ig2a membrane-encoding 3`-untranslated region; term, region where the Ig2a gene normally terminates transcription; bp, base pair; UT, untranslated; nt, nucleotide(s); kb, kilobase(s); mb, membrane; sec, secretory.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Flaspohler, J. A., and Milcarek, C.(1990) J. Immunol. 144, 2802-2810 [Abstract/Free Full Text]
  2. Galli, G., Guise, J. W., McDevitt, M. A., Tucker, P. W., and Nevins, J. R.(1987) Genes & Dev. 1, 471-481
  3. Guise, J. W., Lim, P. L., Yuan, D., and Tucker, P. W.(1988) J. Immunol. 140, 3988-3994 [Abstract/Free Full Text]
  4. Weiss, E. A., Michael, A., and Yuan, D.(1989) J. Immunol. 143, 1046-1052 [Abstract/Free Full Text]
  5. Yuan, D., and Tucker, P. W.(1984) J. Exp. Med. 160, 564-583 [Abstract]
  6. Yuan, D., Dang, T., and Sanderson, C.(1990) J. Immunol. 145, 3491-3496 [Abstract/Free Full Text]
  7. Tisch, R., Kondo, N., and Hozumi, N.(1990) Mol. Cell. Biol. 10, 5340-5348 [Medline] [Order article via Infotrieve]
  8. Law, A., Kuwabard, M., Briskin, M., Fasel, N., Hermanson, G., Sigman, D., and Wall, R.(1987) Proc. Natl. Acad. Sci. U. S. A. 4, 9160-9164
  9. Hall, B., and Milcarek, C.(1989) Mol. Immunol. 26, 819-826 [Medline] [Order article via Infotrieve]
  10. Wels, J. A., Word, C. W, Rimm, D., Der-Balan, G. P., Martinez, H. M., Tucker, P. W., and Blattner, F. R.(1984) EMBO J. 3, 2041-2046 [Abstract]
  11. Richards, J. E., Gilliam, A. C., Shen, A., Tucker, P. W., and Blattner, F. R.(1983) Nature 306, 483-487 [Medline] [Order article via Infotrieve]
  12. Ward, S. B., and Morrison, S. L.(1992) Mol. Immunol. 29, 279-285 [Medline] [Order article via Infotrieve]
  13. Natsuume-Sakai, S., Motonishi, K., and Migita, S.(1977) Immunology 32, 861-865 [Medline] [Order article via Infotrieve]
  14. Citron, B. E., Falck-Pederson, E., Salditt-Georgieff, M., and Darnell, J. E., Jr.(1984) Nucleic Acids Res. 12, 8723-8731 [Abstract]
  15. Hagenbuchle, O., Wellauer, P. K., Cribbs, D. L., and Schibler, U. (1984) Cell 38, 737-744 [Medline] [Order article via Infotrieve]
  16. LeMeur, M. A., Galliot, B., and Gerlinger, P.(1984) EMBO J. 3, 2779-2786 [Abstract]
  17. Pribyl, T. M., and Martinson, H. G.(1988) Mol. Cell. Biol. 8, 5369-5377 [Medline] [Order article via Infotrieve]
  18. Maa, M.-C., Chinsky, J. M., Ramamurthy, V., Martin, B. D., and Kellems, R. E.(1990) J. Biol. Chem. 265, 12513-12519 [Abstract/Free Full Text]
  19. Rohrbaugh, M. L., Johnson, J. E., III, James, M. D., and Hardison, R. C.(1985) Mol. Cell. Biol. 8, 5369-5377
  20. Falck-Pedersen, E., Logan, J., Shenk, T., and Darnell, J. E., Jr. (1985) Cell 40, 897-905 [Medline] [Order article via Infotrieve]
  21. Whitelaw, E., and Proudfoot, N.(1986) EMBO J. 5, 2915-2922 [Abstract]
  22. Logan, J., Falck-Pedersen, E., Darnell, J. E., and Shenk, T.(1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8306-8310 [Abstract]
  23. Lanoix, J., and Acheson, N. H.(1988) EMBO J. 7, 2515-2522 [Abstract]
  24. Connelly, S., and Manley, J. L.(1988) Genes & Dev. 2, 440-452
  25. Miralles, V. J.(1991) Nucleic Acids Res. 19, 3593-3599 [Abstract]
  26. Edwalds-Gilbert, G., Prescott, J., and Falck-Pedersen, E.(1993) Mol. Cell. Biol. 13, 3472-3480 [Abstract]
  27. Das, A.(1993) Annu. Rev. Biochem. 62, 893-930 [CrossRef][Medline] [Order article via Infotrieve]
  28. Russnak, R., and Ganem, D.(1990) Genes & Dev. 4, 764-776
  29. Gilmartin, G. M., Fleming, E. S., and Oetjen, J.(1992) EMBO J. 11, 4419-4428 [Abstract]
  30. Morrison, S. L., and Oi, V. T.(1984) Annu. Rev. Immunol. 2, 239-256 [CrossRef][Medline] [Order article via Infotrieve]
  31. Kobrin, B. J., Milcarek, C., and Morrison, S. L.(1986) Mol. Cell. Biol. 6, 1687-1697 [Medline] [Order article via Infotrieve]
  32. Lang, R. B., Stanton, L. W., and Marcu, K. B.(1982) Nucleic Acids Res. 10, 611-630 [Abstract]
  33. Mulligan, R. C., and Berg, P.(1980) Science 209, 1422-1427 [Medline] [Order article via Infotrieve]
  34. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R.(1984) Nucleic Acids Res. 12, 7035-7056 [Abstract]
  35. Lassman, C. R., Matis, S., Hall, B. L., Toppmeyer, D. L., and Milcarek, C.(1992) J. Immunol. 148, 1251-1260 [Abstract/Free Full Text]
  36. Flaspohler, J. A., and Milcarek, C.(1992) BioTechniques 13, 68-72 [Medline] [Order article via Infotrieve]
  37. Minty, A. J., Alonso, S., Guenet, J. L., and Buckingham, M. E.(1983) J. Mol. Biol. 167, 77-101 [Medline] [Order article via Infotrieve]
  38. Lassman, C. R., and Milcarek, C.(1992) J. Immunol. 148, 2578-2585 [Abstract/Free Full Text]
  39. Peterson, M. C., and Perry, R. P.(1989) Mol. Cell. Biol. 9, 726-738 [Medline] [Order article via Infotrieve]
  40. Frayne, E. G., Leys, E. J., Crouse, G. F., Hook, A. G., and Kellems, R. E.(1984) Mol. Cell. Biol. 4, 2921-2924 [Medline] [Order article via Infotrieve]
  41. Hall, B. L.(1989) Sequence and Secondary Structure of the Murine Immunoglobulin 2a Membrane 3`-Untranslated Region. Ph.D. thesis, University of Pittsburgh, Pittsburgh, PA

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.