Differential regulation of transcription termination occurring at two different sites on the µ–{delta} gene complex

Myoung Kim, Ping Qiu, Raed Abuodeh, Jianzhu Chen1 and Dorothy Yuan

Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA
1 Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

Correspondence to: D. Yuan


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The progression of polymerases across the µ–{delta} Ig heavy chain gene complex is characterized by two termination events occurring at different sites on the transcription unit and at different times during B cell differentiation. We have utilized two mouse strains to analyze the regulatory determinants for these events in primary B cells. In the transgenic pµ.µ{delta}Ratt strain a 1160 bp intervening DNA segment (the att site) has been inverted. This mutation results in the abrogation of transcription termination that occurs in early B cells. Using a novel method that takes advantage of an internal ribosome entry site we have further restricted the size of the segment that is needed for inducing transcription termination in transfectants. This 200 bp termination-inducing sequence operates in tumor equivalents of early but not mature B cells and the activity is correlated with differential binding of nuclear proteins. To explore the regulatory basis for the change in site of transcription termination upon B cell activation we have examined the µS–/– deletion mutant strain in which the µS poly(A) site has been eliminated. The results suggest that polyadenylation at the µS site plays a dominant but not exclusive role in regulating transcription termination in activated B cells.

Keywords: B lymphocytes, transcription termination, polyadenylation, IgD, IgM, internal ribosome entry site


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the course of B cell differentiation the expression of surface Ig changes from the presence of only IgM on immature cells in the bone marrow to both IgM and IgD on the majority of mature, peripheral B lymphocytes. After B cell activation the level of IgD is down-regulated, accompanied by the production of secretory IgM. Changes in expression of these classes of Ig heavy chain have been largely attributed to alterations in synthesis of mRNA derived from the µ–{delta} gene complex. The regulation of this process is complicated by the requirement for alternative splicing of the assembled VDJ gene segment to exons encoding either µ or {delta} heavy chains (1). In addition, alternative processing is involved in the synthesis of mRNA for the secretory (µS) or membrane (µM) forms of IgM. Based on the assumption that transcription termination would pre-empt alternative processing to downstream exons, we and others have extensively analyzed the nature of the primary transcripts at various stages of B cell differentiation, deduced from run-on transcription analysis, in order to understand the regulation of these events (24). Transcription termination can be induced by invariant specific cis-regulatory sites on the DNA template (511). On the other hand, trans-regulatory factors can conceivably be induced upon cellular activation. These factors can reduce the processivity of the RNA polymerase as it transits either specific or non-specific pause sites, or interact with the nascent RNA to cause dissociation from the template (1218). In addition, there is evidence showing that processing of the primary transcript can affect transcription read-through. Polyadenylation, in particular, plays an important role in downstream polymerase termination. This relationship has been classified into poly(A)-dependent versus poly(A)-assisted events with the requirement for a cis-regulatory motif in the latter case (19 and References therein). It is therefore necessary to identify the relationship between the various polyadenylation sites that are differentially utilized during B cell differentiation and the effect of cis-regulatory sequences located within the gene complex in order to understand fully the regulatory factors for transcription termination.

In this study, we have utilized two mutant mouse strains to investigate the regulation of transcription termination within the µ–{delta} gene complex. The pµ.µ{delta}Ratt strain harbors a transgene in which a 1160 bp µ–{delta} intergenic segment, termed att, is inverted. This segment contains the putative cis-regulatory region necessary for transcription termination upstream of the {delta} exons (20,21) in immature B cells. In the µS–/– strain the µS exon, along with its polyadenylation site, has been deleted in the genomic DNA resulting in a complete deficit of secretory IgM production (22). The deletion does not include the region containing a conotical binding site for MAZ, a zinc finger protein originally isolated by virtue of its binding to a site within the c-myc P2 promoter. Occupancy of this site has been postulated to cause DNA bending resulting in polymerase pausing (23) and therefore may participate in the regulation of termination within the µ–{delta} gene complex. The use of primary B cells provides the opportunity for examination of the possible additional effect of changes in extent of polymerase initiation during different stages of B cell differentiation. This difference (24,25), not detectable in tumor cells (26,27), may alter the ability of polymerases to recognize pause/termination sites. By transcription analysis of the µ–{delta} gene complex in these two strains we show that whereas both polyadenylation as well as cis-regulatory sequences are important for transcription termination, the mode of action of the sequences depends on the stage of B cell differentiation.

Because run-on transcription analyses of transfectants are cumbersome and sometimes results are difficult to interpret, we designed a novel method to assess transcription read-through in an attempt to further limit the att site sequences. Using this method, we have identified a 200 bp segment from the att site that induces transcription termination as effectively as the entire full-length segment in early B lineage cells but not in mature B cells. Correspondingly we have also demonstrated B cell stage-specific proteins that bind to this gene segment.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Constructs
The pµ.µ{delta} Ratt construct was derived from the Pµ (27) vector which contains an Igµ gene rearranged to the 17.2.25 V region, extending up to the XhoI site within the µ–{delta} intron, the polyoma virus early region and a modified histone gene. Into the XhoI site was ligated, in the reverse orientation, a 1160 bp XhoI–EcoRV fragment derived from the genomic intronic region between µMII and C1 followed by a 4600 bp EcoRV–EcoRV fragment derived from pLLn5R (20). This fragment contains genomic sequences starting from the remaining µ–{delta} intron up to and including the CH to C3 intron, and cDNA sequences from C3, {delta}MI and {delta}MII (a allotype). The fusion of the cDNA segment eliminates ~6 kb from the genomic sequence including the {delta}X and {delta}S exons.

Animals
A SalII–BsaAI fragment (see Fig. 2AGo) isolated from the pµ.µ{delta}Ratt construct, devoid of plasmid sequences, was microinjected into mouse oocytes of the CD-1 strain (Jackson Laboratories, Bar Harbor, ME) by the Microinjection Services of UTSW Medical Center. Offspring were typed for transgene insertion by Southern blot analysis of tail DNA utilizing a {delta}M specific probe. Because of the insertion of cDNA sequences in the construct, genomic hybridizing fragments could be distinguished from fragments containing the transgene. Two of these animals, lines 4393 and 4399, successfully transmitted the gene to the offspring. B cells in these two lines displayed similar phenotypes. The founders were mated with C57BL/6 (Jackson Laboratories). Further typing of transgene in offspring of founder mice was performed by PCR amplification utilizing primers containing sequences from {delta}MI (5'-GTGACAGCTACATG) and {delta}MII (5'-AGACCACAGCATGCTT) which amplified a 380 bp fragment from the transgene and a 600 bp fragment from the endogenous gene. The transgenic mouse line carrying a rearranged Ig heavy chain gene with µ and {delta} encoding regions in the genomic context (MD-3, 29) was obtained from Dr Christopher Goodnow (John Curtin School of Medical Research, Canberra, Australia) and bred to C57BL/6 mice in our facilities. Mice carrying the transgene were typed by FACS staining with {delta}a and µa reagents as described below. The generation of the µS–/– mouse line (22) has been previously described.



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 2. The effect of inversion of the att site on transcription read-through. (A) Map of the construct. The Pµ.µ{delta}Ratt construct differs from an endogenous rearranged µ–{delta} transcription unit in that the 3' end has been replaced by a C3–{delta}MI and {delta}MII from cDNA. In addition the 1160 bp XhoI–EcoRV fragment located within the µ–{delta} intronic region has been reinserted in the original site but in the opposite orientation. (B–D) Transcription profiles of total bone marrow cells from either the 4399 strain harboring the Pµ.µ{delta}Ratt transgene or the MD-3 strain (B), spleen cells from 4399 animals (C) and spleen cells from the same animal activated with LPS for 4 days (D). RNA extracted from 2xl07 nuclei which had been pulse-labeled with [{alpha}-32P]UTP was hybridized with a panel of DNA probes immobilized on nitrocellulose. The width of each bar represents the region covered by each probe identified by number indicated to scale in the map below. #11 denotes the GAPDH probe and k denotes the {kappa} probe. After subtraction of background hybridization to control DNA (KS vector, indicated by `c') the relative hybridization of each RNA was first normalized to the size of the probe and then to the value obtained for the Cµ probe (#2). Total incorporation varied in the range 2–10x106 c.p.m. Except for the profile obtained from MD-3, which was only performed once, the other results are representative of two completely independent experiments.

 
Cell preparation and cell lines
Red blood cells in tail vein bleeds were lysed to prepare peripheral blood lymphocytes. T cells were depleted from splenocytes as previously described (25). Preparation of bone marrow cells, splenic B cells and activation with lipopolysaccharide (LPS) were performed as described previously (21). The TD-1.1 cell line is one of several clones that arose spontaneously in our laboratory from Whitlock–Witte long-term bone marrow cultures (30). It has the phenotype of a pre-B cell in that the cells expressed B220 as well as µ mRNA, but neither intracellular nor cell surface IgM expression was detectable (T. Dang and D. Yuan, unpublished observations). Bine 4.8 is a pre-B cell tumor (31). M12.4 cells is a mature B cells lymphoma (32); however, very low levels of {delta} mRNA is made in transfectants of unmutated µ–{delta} constructs (D. Yuan and P. W. Tucker, unpublished observations). J558L (33) is a plasmacytoma that can secrete copious amounts of IgM or IgG from transfected constructs.

In vitro `run-on' analysis
Nuclei were prepared from cells and nascent RNA was labeled for 15 min at 30°C as previously described (2) using 100 µCi of [{alpha}-32P]UTP (3000 Ci/mol; ICN Radiochemicals, Irvine, CA) per sample. Following the labeling, RNA was extracted and hybridized at 42°C for 3 days to 5 µg of double-stranded DNA probes immobilized on nitrocellulose. Subsequently, the filters were washed, treated with RNase as previously described, and exposed to a PhosphorImager screen and quantified using the Imagequant software package (Molecular Dynamics, Sunnyvale, CA). After subtraction of background hybridization to vector alone, relative hybridization to each probe was divided by the size of the probe to assess relative polymerase loading on each gene segment.

Probes used for run-on analysis
Plasmid DNA containing gene fragments as previously described (25) and listed below were used for run-on analysis of bone marrow cells. This is possible because the DNA fragments introduced into mouse eggs contained no plasmid sequences, as evidenced by low hybridization to the control KS plasmid with no insert. (#1) The VD probe (21) is a 400 bp PCR product generated from the Pµ. vector and cloned into pBluescript II (KS, Stratagene, La Jolla, CA). (#2) The 5' Cµ probe is a 550 bp TagI–PstI fragment from cDNA of MOPC 104 µ mRNA (34) containing part of CH2, all of CH3 and part of CH4 subcloned into pGEM4 (Promega, Madison, WI). (#3)The 3' Cµ probe is the adjoining 440 bp cDNA fragment containing part of CH4, the µS exon up to the end of the µS processed mRNA subcloned into pGEM4. (#4) The µS–µM intronic probes is a 346 bp KpnI–HaeIII genomic fragment subcloned into pGem2. (#5) The µMI probe is the adjoining 385 bp HaeIII–Pst1 genomic fragment subcloned into pGem 2. (#6) The µMII probe is the adjoining 330 bp PstII–HincII fragment genomic containing the µMII exon and all of the 3' untranslated region of µM mRNA subcloned into pGEM4. Because the µS–/– transgene contains a deletion of the intronic region between µS and µM and between the two µM exons, the value of the probe size used for normalization was reduced accordingly. (#7) The 5' att probe is a 345 bp XhoI–Pst1 genomic fragment, located in the µ–{delta} intron, subcloned into pGEM4. (#8) The 3' att probe is the adjoining 550 bp PstI–EcoRI genomic fragment subcloned into pGem 4. (#9) The C1-3 probe is a 764 bp fragment subcloned from TEPC 1017 cDNA into pUC 8 containing C1, {delta}H and C3. (#10). The {delta}M probe is a 710 bp HincII–BglII genomic fragment containing {delta}M1 and {delta}MII, subcloned into pGEM-2. Because the pµ.µ{delta}Ratt transgene has a deletion of the MI–MII intron, the value of the probe size used for normalization was reduced accordingly. (#11) The GAPDH probe is a 1233 bp complete rat cDNA (35) cloned into KS. The C{kappa} probe is a 482 bp HpaI–BglII genomic fragment including the C{kappa} exon subcloned into pUC 8.

FACS
Peripheral blood lymphocytes or bone marrow cells were analyzed on the FACScan flow cytometer (Becton Dickinson, San Jose, CA) using reagents previously described (21).

Plasmid construction for luciferase assay
pGLIRES
This vector was created by modification of the pGL3-basic (Promega, Madison, WI). The SalI site at 2010 was first destroyed before insertion of the internal ribosomal entry sites (IRES) from EMCV virus into the BglII–NcoI site. The IRES sequence (36,37) was amplified from pCIN4 (Dr Stephen Rees, Glaxo Wellcome) with the primers 5'-CGAGCATAGATCTAGGGCGGCGAATTCG-3', incorporating BglII and EcoRI sites (underlined) and 5'-GGGGTTGTGCCATGGTTATCATCGTGTT-3' containing NcoI sites (underlined).

pGLIgµ
A 1084 bp DNA segment containing 460 bp 5' of µM I and extending 140 bp 5' of the µMII poly(A) signal was amplified from a vector containing the genomic sequences using the primers 5'-ACGCACGGGAATTCTGATCAAGAAAGT-3', containing EcoRI site (underlined) and 5'-CATCAGAATTCACTGGTCGACTTCCAGT-3' incorporating EcoRI and SalI sites (underlined). The PCR product was digested with EcoRI and cloned into EcoRI site of PGLIRES to generate PGL3µM. The rearranged VDJ segment and IgH promoter region was then amplified from pµPoly (27) with the primers 5'-GGCGTATCAGAGGCCCT-3' which anneals to upstream of IgH promoter and 5'-GGTCAGTCTAGAAAGAAGACCATC-3' which anneals within µMII. The PCR product was digested with SalI and BclI and blunt-ended by Klenow fill-in and cloned into SmaI site of pGL3µM to generate pGLIgµ.

pIgµfIVS or pIgµrIVS
A1200 bp gene segment containing the µ–{delta} intronic sequences spanning the PstI site 3' of µMII up to 25 bp 5' of the C1 acceptor splice site was excised from the pµMSUSIR+ vector (20) and ligated into the SalI site of pGLIgµ in both orientations. Vectors containing subfragments of the `IVS' region (pGL5'IVS, pGL3'IVS, pGL3'IVSx and pGL3'IVSy) were all derived by subcloning the fragments as shown in Fig. 4Go(B) into the SalI site of pGLIgµ.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 4. Use of a IRES–luciferase construct to determine induction of transcription termination. (A) Comparison of luciferase activities of transfectants of pGL3p control versus pGL3pIRES. Two days after transient transfection of each plasmid together with pSVß-galactosidase vector into J558L cells luciferase activity of each was determined and normalized to the ß-galactosidase activity from each transfectant. Error bars indicate the SEM for three experiments. (B) Map of luciferase constructs. A number of restriction enzyme digested fragments isolated from subclones of genomic DNA were cloned into a unique SalI site located just upstream of the IRES sequence in the pGLIgµ vector. Locations of the IgH promoter, enhancer and µM exons as well as the poly(A) site are indicated to scale. The shaded area indicates the minimal DNA region for which differential binding activity was found. (C and D) Induction of transcription termination by the entire `IVS' region and subfragments thereof. TD1.1 or J558L cells were transfected with each of the indicated constructs together with the pSVß-galactosidase vector. Luciferase activity determined 1–2 days after transfection was first normalized to the ß-galactosidase level for each transfectant, then to the relative luciferase activity of pGLIgµ determined in the same experiment. Error bars represent SEM obtained from at least three experiments for each construct.

 
Transfection
Samples of 20 µg of test plasmid and 15 µg of pSVß-galactosidase control plasmid were transient transfected together as previously described (38). Transfected cells were assayed 24–48 h later.

Luciferase and ß-galactosidase assays
Luciferase activity was determined by using the Luciferase Assay Kit from Promega (Madison, WI) according to the manufacturer's instructions and measured with Optocomp luminometer (MGM Instrument). For the ß-galactosidase assay, 100 µl of cell extract was diluted in 50 µl of 1xCell Lysis Buffer (Promega) and 150 µl of 2xAssay Buffer (0.2 M sodium phosphate buffer, pH 7.3, 2 mM MgCl2, 0.1 M ß-mercaptoethanol, 1.33 mg/ml ONPG) was then added. Reactions were incubated at 37°C until the absorbance could be read at 420 nm. Luciferase activity was normalized to ß-galactosidase activity in each transfection to correct for transfection efficiency.

Nuclear extract preparation
Nuclear extracts from TD1.1 or J558L cells were prepared by slight modifications of previously described methods (39).

Gel electrophoresis mobility shift assays
DNA probes were either generated by restriction enzyme digestions of subclones of the `IVS' region as indicated in Fig. 4Go(B) or by PCR amplification from the plasmid template containing the relevant sequences. Oligonucleotides used to generate the PCR fragment for the EcoRI–AccI region were: forward primer 5'-CAGGGAAAGAACAGAATTCTG and reverse primer: 5'-ATAAGTGTAGACCTGTGACAC. These primers were in turn used to generate the F105 and F88 fragment by using two additional oligonucleotides located in the middle of the segment. These are: forward primer 5'-AATTGGATGATGAACCCTGA and reverse primer: 5'-CCAGAGTCACCTCTATGT. Products of the appropriate size were isolated by agarose gel electrophoresis and 5'-end-labeled with [{gamma}-32P]ATP (4500 Ci/mmol) and T4 polynucleotide kinase. Binding reactions contained 20 mM HEPES (pH 7.9), 0.2 mM EDTA, 20% glycerol, 100 mM KCl, indicated amount of poly(dI)–poly(dC) heteropolymer (Pharmacia), 5 mM DTT and 10,000–100,000 c.p.m. of end-labeled probe. Reactions initiated by the addition of protein were incubated for 30 min at 23°C. Protein–DNA complexes were resolved by PAGE containing 90 mM Tris–borate (pH 8.3), 2 mM EDTA and 2% glycerol. The running buffer was the same as the gel buffer except that glycerol was omitted. Gels were dried and exposed to phosphorimaging screens.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of the Pµ.µ{delta}Ratt transgene
Previously, we had generated a mouse strain harboring a transgene in which an intervening segment termed att, for attenuation of transcription, located between the µM and C exons was deleted (21). This deletion effectively allowed transcription read-through to the C genes in immature B cells which would otherwise have terminated (21). However, in this line the expression of the transgenic µ gene was compromised for reasons which are still under investigation. The lack of allelic exclusion of endogenous Ig gene due to the absence of transgenic µ gene expression complicated transcription analysis of the transgene. Therefore we have now generated a second transgenic line in which the att region was replaced in the opposite orientation. Based on previous studies in transfected cell lines showing that the effect of the att region is mediated in an orientation-dependent manner (20) transcription termination should be also alleviated. The 4399 line, harboring the pµ.µ{delta}Ratt construct, expresses transgenic IgM and therefore allelically excludes endogenous gene rearrangement (Fig. 1Go) as evidenced by the absence of expression of IgM bearing the b allotype when it is crossed to C57BL/6 mice. Interestingly, however, the level of transgenic IgD expression was lower than that exhibited by non-transgenic littermates or a control transgenic line, MD-3, which harbors an unmutated µ–{delta} transgene (29). In addition IgD expression cannot be detected in transgenic bone marrow cells. These results suggest that inversion of the att site may have adversely affected transcription read-through and/or processing to the {delta} exons.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1. Cell surface expression of IgD and IgM. (A) Peripheral blood lymphocytes from 6-week-old 4399 mice and age-matched MD-3 mice were stained with phycoerythrin (PE)–anti-µa either together with FITC–anti-{delta}a or with FITC–anti-µb. B. Bone marrow cells from 6-week-old 4399 mice and littermate controls were stained with PE–anti-B220 together with FITC–anti-{delta}a.

 
Transcription analysis of the Pµ.µ{delta}Ratt transgenic line
To determine if insertion of the att sequences in the opposite orientation eliminated the transcription termination that usually occurs in immature B cells, bone marrow B cells from the transgenic animals were examined by run-on transcription analysis (Fig. 2BGo). Nuclei prepared from these cells were pulse labeled with [32P]UTP for 15 min and the labeled RNA was hybridized to a series of probes spanning the µ–{delta} region. Because the transgene excluded gene rearrangement (Fig. 1Go) transcription can be assessed in the absence of endogenous gene expression. There is, therefore, a relatively high level of hybridization to the transgene-specific V region probe although the level is still lower than the extent of transcription across the Cµ gene because of contributions from sterile transcription of non-rearranged genes in the pro-B and pre-B cell compartment (40,41). It is also possible that the sterile transcription may persists to variable extents in mature B cells of transgenic mice with extensive allelic exclusion. By normalizing the extent of hybridization to each probe to that of the Cµ probe, it is possible to compare the relative level of hybridization to the C exons in this strain with that from the MD-3 strain which does not differ significantly from non-transgenic bone marrow cells (21). Clearly in the 4399 line transcription termination was alleviated to a great extent, although not completely. These findings confirm in primary B cells our earlier data obtained from transfected tumor cells (20) showing that this intergenic segment has to be present in the appropriate orientation for transcription termination upstream of the C exons to occur.

The µS polyadenylation site induces transcription termination
We had previously observed that in activated B cells transcription termination occurs at a region significantly more upstream of that occurring in early B cells. The transcription profile obtained from day 4 LPS-activated splenic B cells from 4399 mice (Fig. 2CGo) shows that this alteration of the site of transcription termination is not compromised by the inversion of the att site. Termination of the majority of the transcripts in activated B cells occurs within the µMII exon. There also appears to be pausing of polymerases in regions immediately preceding this termination site. This profile does not differ from that found for LPS-activated non-transgenic B cells (Fig. 3CGo). We had previously observed that the site of termination in activated B cells is variable depending on the state of activation (25). Because of the preferential usage of the µS poly(A) site in activated B cells, however, it is not clear whether the termination is a consequence of this usage or whether activation of termination-inducing trans-regulatory factors, such as, for example, the MAZ protein, results in the use of the upstream poly(A) site by default. To distinguish between these possibilities, we sought to determine whether termination in the µM region occurs in B cells obtained from the µS–/– mouse strain which cannot utilize the µS poly(A) site because it has been deleted. It should be noted that the µM poly(A) site and immediate downstream regions were not disturbed, therefore the binding of possible termination factors to the template or nascent RNA that extends through this segment should not be affected. The transcription profile of non-stimulated splenocytes from these µS–/– mutant animals was similar to that of normal littermates (Fig. 3BGo and data not shown). Upon LPS activation the relative level of hybridization between the µMII probe (#6) versus the Cµ probe (#2) did not change. In contrast, in LPS-activated cells from littermate controls there was a significant decrease in this ratio. Thus, the termination usually found within this region was much alleviated. Interestingly, the pronounced pausing of polymerases in the region defined by the µMI probe often found in non-transgenic B cells also occurred in the µS–/– strain despite the deletion of the µMI–µMII intron. Significantly, although polymerases fail to exit in the region defined by probe #6, the majority of the newly induced transcripts in the mutant still terminates in a more upstream region than that in resting B cells.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3. Run-on transcription analysis of B cells from the µS–/– strain. (A) Map of the gene deletion carried by the µS–/– strain. Note that whereas the µS–µMI as well as the µMI–µMII intron were deleted, the MAZ binding site as well as the att sites were kept intact. (B) RNA extracted from 2x107 nuclei from either resting or day 4 LPS-activated spleen cells were labeled as in Fig. 2Go and hybridized to a panel of probes. The width of each bar represents the region covered by each probe indicated to scale in the map below. After subtraction of background hybridization (`c', pGEM4 vector) and normalization to the probe size, each value was normalized to that obtained for the GAPDH probe (#11). (C) The same profile obtained from activated cells presented in B was compared to the pattern of hybridization from day 4 LPS-activated cells from +/+ littermates. In this case the two profiles were normlized to the value obtained for the Cµ probe (#2). These results are representative of two completely independent experiments.

 
The increase in relative hybrization to the Cµ probe when the transcription profiles are normalized to the level of hybridization to the GAPDH probe provides an estimate of the extent of increase in polymerase initiation induced by B cell activation (Fig. 3BGo). This increase was found to be the same for activated B cells from the µS–/– mutants and control animals (probe 11; Fig. 3CGo) therefore the diminished level of termination in the region of the µMII exon cannot be attributed to a lower response of the mutant cells to LPS activation.

Luciferase assay to measure transcription termination efficiency
Despite the alleviation of transcription termination in bone marrow cells of animals harboring the Pµ.µ{delta}Ratt transgene to a level that is approximately the same as in mature spleen cells (Fig. 2CGo) the extent of processing of the transcripts to the C exons is not increased. Further analysis indicated that splicing of VDJ to the C1 exon is diminished even in mature B cells (manuscript in preparation). Correspondingly, no IgD+ cells can be detected in the bone marrow (Fig. 1BGo) and a significant fraction of peripheral B cells express only IgM in the absence of IgD (see Fig. 1AGo). The alteration in processing cannot be attributed to deletion of part of the {delta} gene in the 3' end (Fig. 2AGo) because this phenotype was not observed in the strain harboring the pµ.µ{delta}{Delta}att transgene which has an identical 3' configuration (21). The disruption of downstream processing appears to be due to the mutation of the att insert, therefore it is necessary to further refine the limits of this cis-regulatory element so that a change therein can be targeted exclusively to transcription termination.

IRES have been employed to produce polycistronic mRNAs in retroviral-mediated gene transfer systems (37). Here we use IRES from the encephalomyocarditis virus genome, a member of the picornaviridae family of viruses (36,37), for efficient expression independent of the 5' cap site of transcripts. The strategy is to construct a series of transfection vectors that contain putative termination sites located upstream of an IRES element such that only read-through transcripts that extend into the luciferase gene can be translated by ribosomes that enter at the internal ribosome binding sites and produce luciferase protein. In the absence of the luciferase promoter transcripts that terminate before the IRES will not extend to the coding sequence for the luciferase gene and no enzyme can be produced. In order to first test if the presence of IRES itself affects luciferase expression, we compared luciferase activity derived from the pGL3p control and the pGL3pIRES vector which has an IRES sequence inserted between the SV40 promoter and luciferase coding region. Figure 4Go(A) shows that in the presence of IRES (pGL3pIRES) luciferase expression in a plasmacytoma cell line (J558L) was decreased by 45–60% of that generated by direct transcription/translation from the SV40 promoter (pGL3p control). This level of decrease is not dissimilar to the effect of IRES shown previously in other systems (42,43) and the signal intensity is still sufficient for use as a reporter system. We then substituted, for the SV40 promoter in pGL3pIRES, a gene segment that includes the Ig heavy chain promoter together with rearranged VDJ sequences as well as the µM coding region with intact poly(A) signal (pGLIgµ; Fig. 4BGo).

To ascertain that the level of transcription read-through as measured by this method is similar to that measured by run-on transcription analysis we first tested the effect of inserting the entire 1200 bp `IVS' sequence previously shown to induce transcription termination (20). The pIgµfIVS vector was transfected in parallel with control plasmid pGLIgµ and the relative luciferase activity derived from each plasmid was compared. Two cell lines representing two extreme ends of the B cell differentiation spectrum were transfected. The TD1.1 lymphoma cell line is derived from a pre-B cell culture and J558L is a plasmacytoma. Figure 4Go(C) shows that in TD1.1 cells luciferase activity derived from pIgµfIVS was <40% of that derived from the control plasmid, indicating that more than half of the polymerases that transited the µM region failed to reach the IRES and luciferase gene. However, in J558L cells the same fragment showed little effect on the abilities of polymerases to read through IRES and the luciferase gene. Moreover, transfection of pIgµrIVS, in which the 1200 bp `IVS' segment was inserted in the reverse orientation, into either TD1.1 or J558L cells demonstrated comparable luciferase activity to that derived from the control plasmid in both cell lines showing that the termination efficiency of this segment is orientation dependent as described previously. These results are comparable to those from previous in vitro run-on analyses and confirm both the validity of the assay and the functionality of the terminator present in the µ–{delta} intronic region in the context of the IRES–luciferase construct.

Deletion analysis of the µ–{delta} intergenic region
In order to further delineate the elements required for regulation of termination, various fragments in the `IVS' region were generated by restriction enzyme digestion and inserted into pGLIgµ and the effect of each fragment was assessed for termination efficiency. Figure 4Go(D) shows that luciferase expression from pIgµ3'IVS which contains the 3' half of `IVS' region was decreased to 50% of that from pGLIgµ, while luciferase expression from pIgµ5'IVS which contains the 5' half of `IVS' region did not differ from the control vector. These results suggest that the EcoRI–BglII fragment within the `IVS' region plays an important role in transcription termination. This fragment was further bisected and cloned separately. Analysis of these two constructs, pGLIgµ3'X and pGLIgµ3'Y, showed that the 5' half of active EcoRI–BglII fragment decreased luciferase activity as effectively as the entire fragment, whereas the 3' half did not significantly affect termination. Thus, these results demonstrate that essential elements for termination reside within the 200 bp fragment cloned into pGLIgµ3'IVS.

Protein binding to the fragment required for termination
To understand the mechanism by which termination may be dictated by the 182 bp EcoRI–AccI fragment within the pGLIgµ3'IVS we assessed whether proteins could bind to it in a B cell stage-specific manner. This fragment was 5'-end-labeled with [{gamma}-32P]ATP and binding reactions were performed by incubating the probe with either TD1.1 or J558L nuclear extracts followed by analysis of DNA–protein complexes on a non-denaturing polyacrylamide gel. Complexes with two different mobilities were formed by nuclear extracts from TD1.1 cells but not by those from J558L cells (Fig. 5AGo), whereas a control fragment derived from a gene segment located more upstream but still within the `IVS' region yielded complexes of similar mobility when bound to nuclear extracts from either TD1.1 or J558L cells (Fig. 5BGo). The complex C1 with slower mobility was derived from specific binding because of its susceptibility to competition by the specific competitor consisting of the unlabeled probe but not by a non-specific competitor (Fig. 5CGo), whereas the complex with the faster mobility, C2, which appeared with greater variability, was competed away by both specific and non-specific competitors. Other representative cell lines at similar developmental stages were also screened for the binding activity. As shown in Fig. 5Go(D), Bine 4.8, another pre-B cell line, demonstrated a similar binding activity as that found for TD1.1 cells, whereas a lower level of binding was found in extracts from M12.4, a lymphoma line representing an intermediate stage of B cell differentiation.



View larger version (79K):
[in this window]
[in a new window]
 
Fig. 5. Differential binding activity of nuclear extracts from immature versus mature tumor cells. (A) Increasing concentrations (20, 40 or 60 µg) of nuclear proteins from TD1.1 or J558L cells were incubated with a 193 bp 32P-end-labeled PCR generated fragment encompassing the EcoRI–AccI fragment contained within pGL3'IVSx. (B) Either extract (40 µg) was incubated with a 47 bp AluI fragment from the pGL5'IVS region. (C) Increasing concentrations of the unlabeled EcoRI–AccI fragment or with the non-specific AluI fragment used in (B) was added during incubation of 40 µg of TD1.1 extract to the probe. (D) Increasing concentrations of nuclear protein (5, 10 and 20 µg) from either Bine 4.8, TD1.1, J558L or M12.4 cells were incubated with the 193 bp probe. Complexes were resolved on 4% non-denaturing polyacrylamide gels and exposed to the PhosphorImager screen.

 
In addition we have screened a number of fragments contained within most of the µ–{delta} intervening region. (data not shown). Although complexes were detected with some of the fragments no outstanding differences were detectable between extracts from the TD1.1 versus that from J558L plasmacytoma cells. A number of other fragments yielded no detectable binding for either cell type.

In an effort to further localize the protein binding site we bisected the EcoRI–AccI fragment by PCR amplification using two sets of primers resulting in adjacent 5' 105 bp and 3' 88 bp fragments (F88 and F105). When each of these fragments was separately assessed, F88 fragment formed complexes resulting in four bands with extracts from TD1.1 cells. Only the band with the slowest mobility was concentration dependent and showed reproducible binding (Fig. 6AGo) and could be effectively competed by the homologous unlabeled fragment (Fig. 6BGo) but to a much lesser extent by F105. The faster mobility bands were competed by both homologous and heterologous fragments. In contrast, labeled F105 exhibited much lower levels of binding activity for the same extract. Whether the strong binding to the 88 bp fragment can account for the total termination activity of the pGL3'IVSx vector still awaits further functional confirmation.



View larger version (90K):
[in this window]
[in a new window]
 
Fig. 6. Differential binding of nuclear extracts to sub-fragments of pGL3'IVSx. (A) Increasing concentrations (5, 10 and 15 µg) of TD1.1 cells were incubated with either a 5' 32P-end-labeled 88 bp fragment (3' half of the 193 bp EcoRI–AccI fragment in Fig. 5Go) or with the 32P-end-labeled 105 bp fragment (5' half). (B) Approximately 10-, 50-, 100- or 200-fold excess of unlabeled F88 or F105 fragment was added during binding to 15 µg of TD1.1 extract with the labeled F88 fragment.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The steps of RNA synthesis and subsequent processing are now viewed as occuring as part of a complex unit in which all the factors needed for 5' capping, splicing as well as polyadenylation continue to be associated with the polymerase until it is disengaged from the template at the termination site (4446). In this context, analysis of the expression of the murine µ–{delta} gene complex is not only complicated by the close proximity of the two genes expressed differentially at different stages of B cell development but also influenced by the differential usage of multiple polyadenylation sites during B cell development and subsequent activation. Analysis of constructs containing restricted segments of this gene complex has been further complicated by the tendency of many B cell tumors to utilize the more proximal µS poly(A) site regardless of the differentiation stage (20,4749) and/or terminate transcription in an apparently deregulated manner. The utilization of non-transformed B cells from transgenic mice has allowed us to overcome some of these difficulties. Analysis of LPS-activated B cells from the µS–/– mouse strain, in which the µS poly(A) site has been deleted, shows that a substantial degree of transcription termination within the µM region in activated B cells is alleviated. It is important to note that this change occurred in spite of the fact that the extent of B cell activation is not affected by the mutation; therefore any putative trans-regulatory factor(s) necessary for altering upstream termination must have been induced.

This observation is consistent with recent findings showing that an increase in abundance of the 64 kDa subunit of the cleavage stimulation factor, CstF64, in activated B cells may be an important (50) but not sole driving force (51) for the selection of the µS poly(A) site. Interestingly, the site of termination upon increased µS polyadenylation is located >2 kb downstream of the cleavage site. Therefore, despite the dependence on polyadenylation, this process cannot be classified as poly(A) directed (19). In contrast, in repeated run-on transcription assays (25; see also Fig. 2DGo), we have noted large variations in the extent of polymerase loading, which can often be much higher than that in the Cµ region, in the probes encompassing the region upstream of the µM poly(A) site. Therefore it appears that there is a polymerase pause site just downstream of this region that causes variations in the extent of polymerase entrapment. The best candidate for this site is the conotical MAZ binding site (23) located 200 bp downstream of the µM poly(A) site within a region previously documented to be a polymerase unloading site in tumor cells (4,52). The termination of transcription close to a pause site located some distance from the polyadenylation site is consistent with a model whereby polymerase activates polyadenylation (46) by continued association with these processing factors (45). The presence of the MAZ site may provide the specificity needed for targeting of the increased amount of functional CstF64 to the IgH gene.

Regardless of the possible involvement of the MAZ site in activated B cells, clearly this site is bypassed in mature, resting B cells so that the {delta} exons are transcribed. In contrast, analysis of the pµ.µ{delta}Ratt strain shows that in the presence of the att site, which is located further downstream, in the appropriate orientation, is necessary for transcription termination in immature B cells. Much of the polymerase downloading is effectively prevented by the insertion of this intergenic segment in the wrong orientation. The data presented herein does not address the question of whether the µM poly(A) site is necessary for termination at this site in primary B cells although previous data in transfectants indicated a necessity for the site. However, recent studies in a new gene knockout mutant offer some insight into this question. The {delta} polypeptide chain has been shown to have the ability to completely replace µ chain function during early B cell development in the IgM–/– mouse (53) which harbors a deletion of the entire µ gene as well as the att sequence located upstream of the {delta} exons. Interestingly, in the µMT–/– mouse strain (54) in which only the µMI exon was replaced by a neo gene, the presence of the downstream {delta} gene does not replace µ gene function in early B cells. It appears, therefore, that the retention of the att sequence in this case effectively prevented transcription progression to the {delta} exons despite the presence of the strong poly(A) site used by the inserted neo gene transcript. IgD expression from the mutated chromosome can be found in mature B cells of heterozygous animals, showing that the effect of the termination site can be overcome upon B cell maturation. Hence even in the presence of a heterologous polyadenylation signal the att sequence can function as an attenuation site.

Using a novel assay for assessing transcription termination we have defined the minimal cis-regulatory sequences within the att region that mediate termination. This assay is much more sensitive than run-on transcription analysis because it does not rely upon measurement of hybridization signals derived from labeled nascent transcripts. By this means we have defined a 200 bp segment within the att region that can induce transcription termination which, in the genomic context, is located 1650 bp downstream of the µMII poly(A) site and 650 bp 5' of C1. Of special significance is the apparent differentiation stage specificity of the ability of this segment to dictate transcription termination. Thus, termination is registered only in tumor cells that represent the equivalent of immature B cells, whereas in the plasmacytoma line, J558L, representing more mature B cells, the segment does not affect polymerase transit. Because termination activity is based on comparison with relative read-through in the parental vector that also contains the µM poly(A) site, any termination directed by this site should not affect the read-out. For all of the vectors analyzed, the level of termination detected by this method is, however, still incomplete for reasons that are unclear. It is possible that there are further upstream as well as downstream elements not included in the construct that exert additional modulatory effects. Nevertheless, these results establish the sequences contained within the 200 bp EcoRI–AccI fragment as a minimum requirement for induction of termination.

The mechanism underlying the termination-inducing activity of the EcoRI–AccI fragment remains unclear. One clue is derived from the correlation of protein binding with the functional activity of the fragment. We have shown that nuclear extracts from transformed counterparts of early B cells can bind to this fragment, whereas much less activity is contained with extracts from more mature B cell equivalents. Furthermore, specific binding can be restricted further to a 88 bp sequence. A simplistic model would predict that the protein binding serves as a block to transcription read-through and this binding is altered upon B cell activation. Further characterization, including footprint analysis, will be necessary in order to show whether there is preferential binding to the coding strand. However, the orientation dependence of the effect on transcription suggests that the att sequence does not function by bending DNA as has been shown for MAZ (23). Furthermore, from the transcription profile there is no evidence for the region to act as a strong pause site for polymerase. Finally, it is always important to bear in mind that in vitro assays performed in transformed B cells do not always correlate completely with in vivo mechanisms occuring in normal B cells.

In conclusion, the findings reported here allow us to present a comprehensive view of transcription across the µ–{delta} gene complex. In early B cells, the µM poly(A) site is preferentially utilized to produce cells expressing exclusively membrane IgM. Usage of this site does not induce significant transcription termination despite the presence of the MAZ site located immediately downstream. It is not until the att site located some 1650 bp downstream is encountered that polymerase downloading occurs. We had previously designated this region as an attenuation site (21) because, in contrast to a termination site, the recognition of the att site is reversibly regulated by B cell maturation such that to allow read-through to downstream C exons, the site must be bypassed. We have now further refined the limits of this att site. At the other end of the differentiation spectrum occurring upon B cell activation, transcription initiation of the IgH gene complex is increased. The corresponding increased loading of polymerases may augment the effectiveness of the MAZ site resulting in the accumulation of polymerases in the immediate upstream region. The enhanced cleavage of the newly induced transcripts at the µS poly(A) site due to the increase in the polyadenylation factor CstF64 must destablize the polymerases to an extent that results in the dissociation of the majority of the transcripts when they reach a relatively discrete site within the µM 3'-untranslated region.


    Acknowledgments
 
We thank E. S. Rees, Glaxo Research and Development, Middlesex, UK. for the gift of the pCIN IRES vector. We thank Tam Dang and Rula Bibi for expert technical assistance. This work was supported by NIH grant no. GM37743.


    Abbreviations
 
IRESinternal ribosome entry site
LPSlipopolysaccharide
µSsecretory form of IgM
µMmembrane form of IgM
PEphycoerythrin

    Notes
 
Transmitting editor: K. Knight

Received 4 November 1998, accepted 2 February 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Knapp, M., Liu, C. P., Newell, N., Ward, R. B., Tucker, P. W., Strober, S. and Blattner, F. 1982. Simultaneous expression of immunoglobulin mu and delta heavy chains by a cloned B-cell lymphoma: a single copy of the VH gene is shared by two adjacent CH genes. Proc. Natl Acad. Sci. USA 79:2996[Abstract]
  2. Yuan, D. and Tucker, P. W. 1984. Transcriptional regulation of the µ–{delta} gene in normal murine B lymphocytes. J. Exp. Med. 160:564.[Abstract]
  3. Mather, E. L., Nelson, K. J., Haimovich J. and Perry, R. P. 1984. Mode of regulation of immunoglobulin mu- and delta-chain expression varies during B-lymphocyte maturation. Cell 36:329[ISI][Medline]
  4. Tisch, R., Kondo, N. and Hozumi, N. 1990. Parameters that govern the regulation of immunoglobulin {delta} heavy-chain gene expression. Mol. Cell. Biol. 10:5340[ISI][Medline]
  5. Baek, K.-H., Sato, K., Ito R. and Agarwal, K. 1986. RNA polymerase II transcription terminates at a specific DNA sequence in a HeLa cell-free reaction. Proc. Natl Acad. Sci. USA 83:7623.[Abstract]
  6. Kuhn, A. and Grummt, I. 1989. 3'-End formation of mouse pre-rRNA involves both transcription termination and a specific processing reaction. Genes Dev. 3:224[Abstract]
  7. Reines, D., Chamberlin, M. J. and Kane, C. M. 1989. Transcription Elongation Factor SII (TFIIS) enables RNA polymerase II to elongate through a block to transcription in a human gene in vitro. J. Biol. Chem. 264:10799[Abstract/Free Full Text]
  8. Kerppola, T. K. and Kane, C. M. 1990. Analysis of the signals for transcription termination by purified RNA polymerase II. Biochemistry 29:269.[ISI][Medline]
  9. Enriquez-Harris, P., Levitt, N., Briggs, D. and Proudfoot, N. J. 1991. A pause site for RNA polymerase II is associated with termination of transcription. EMBO J. 10:1833.[Abstract]
  10. Fernandezsilva, P., Martinezazorin, F., Micol, V. and Attardi, G. 1997. The human mitochondrial transcription termination factor (MTERF) is a multizipper protein but binds to DNA as a monomer, with evidence pointing to intramolecular leucine zipper interactions. EMBO J. 16:1066.[Abstract/Free Full Text]
  11. Langst, G., Blank, T. A., Becker P. B. and Grummt, I. 1997. RNA polymerase I transcription on nucleosomal templates—the transcription termination factor TTF-I induces chromatin remodeling and relieves transcriptional repression. EMBO J. 16:760[Abstract/Free Full Text]
  12. Gottlieb, E. and Steitz, J. A. 1989. Function of the mammalian La protein: evidence for its action in transcription termination by RNA polymerase III. EMBO J. 8:851.[Abstract]
  13. Lee, D. N., Phung, L., Stewart, J. and Landick, R. 1990. Transcription pausing by Escherichia coli RNA polymerase is modulated by downstream DNA sequences. J. Biol. Chem. 265:15145.[Abstract/Free Full Text]
  14. Resnekov, O., Pruzan, R. and Aloni, Y. 1991. Elements involved in an in vitro block to transcription elongation at the end of the L1 mRNA family of adenovirus 2. Nucleic Acids Res. 19:1783[Abstract]
  15. Flaspohler, J. A., Boczkowski, D., Hall, B. L. and Milcarek, C. 1995. The 3'-untranslated region of membrane exon 2 from the gamma 2a immunoglobulin gene contributes to efficient transcription termination. J. Biol. Chem. 270:11903.[Abstract/Free Full Text]
  16. Deng, L. and Shuman, S. 1997. Transcription termination by vaccinia RNA polymerase entails recognition of specific phosphates in the nascent RNA. J. Biol. Chem. 272:695[Abstract/Free Full Text]
  17. Steinmetz, E. J. and Brow, D. A. 1996. Repression of gene expression by an exogeneous sequence element acting in concert with a heterogeneous nuclear ribonucleoprotein-like protein, Nrd1, and the putative helicase, Sen1. Mol. Cell. Biol. 16:6993.[Abstract]
  18. Aso, T., Haque, D., Barstead, R. J., Conaway, R. C. and Conaway, J. W. 1996. The inducible elongin A elongation activation domain: structure, function and interaction with the elongin BC complex. EMBO J. 15:5557[Abstract]
  19. Yeung, G., Choi, L. M., Chao, L. C., Park, N. J., Liu, D., Jamil, A. and Martinson, H. G. 1998. Poly(A)-driven and poly(A)-assisted termination: two different modes of poly(A)-dependent transcription termination. Mol. Cell. Biol. 18:276.[Abstract/Free Full Text]
  20. Moore, B. B., Tan, J., Lim, P.-L., Tucker, P. W. and Yuan, D. 1998. Regulatory elements necessary for termination of transcription within the Ig heavy chain gene locus. Nucleic Acids Res. 21:1481.[Abstract]
  21. Yuan, D., Witte, P. L., Tan, J., Hawley, J. and Dang, T. 1996. Regulation of IgM and IgD heavy chain gene expression. effect of abrogation of intergenic transcriptional termination. J. Immunol. 157:2073[Abstract]
  22. Boes, M., Esau, C., Fischer, M. B., Schmidt, T., Carroll, M. and Chen, J. 1998. Enhanced B-1 cell development, but impaired IgG antibody responses in mice deficient in secreted IgM. J. Immunol. 160:4776[Abstract/Free Full Text]
  23. Ashfield, R., Patel, A. J., Bossone, S. A., Brown, H., Campbell, R. D., Marcu, K. B. and Proudfoot, N. J. 1994. MAZ-dependent termination between closely spaced human complement genes. EMBO J. 13:5656[Abstract]
  24. Chen-Bettecken, U., Wecker, E. and Schimpl, A. 1985. IgM RNA switch from membrane to secretory form is prevented by adding anti-receptor antibody to bacterial lipopolysaccharide-stimulated murine primary B cell cultures. Proc. Natl Acad. Sci. USA 82:7384.[Abstract]
  25. Weiss, E. A., Michel, A. and Yuan, D. l989. Role of transcriptional termination in the regulation of and mRNA expression in B lymphocytes. J. Immunol. 142:1095.[Abstract/Free Full Text]
  26. Gerster, T., Picard D. and Schaffner, W. 1986. During B-cell differentiation enhancer activity and transcription rate of immunoglobulin heavy chain genes are high before mRNA accumulation. Cell 45:45.[ISI][Medline]
  27. Yuan, D. and Dang. T. l989. Regulation of µM vs µS mRNA expression in an inducible B cell line. Mol. Immunol. 26:l059
  28. Grosschedl, R. and Baltimore, D. 1985. Cell-type specificity of immunoglobulin expression is regulated by at least three DNA elements. Cell 41:885.[ISI][Medline]
  29. Goodnow, C., Crosbie, J., Adelstein, S., Lavoie, T., Smith-Gill, S., Brink, R., Pritchard-Briscoe, H., Wotherspoon, J., Loblay, R., Raphael, K., Trent, R. and Basten, A. 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334:676[ISI][Medline]
  30. Yuan, D. and Witte, P. L. 1988. Transcriptional regulation of µ and {delta} gene expression in bone marrow pre-B and B lymphocytes. J. Immunol. 140:2808[Abstract/Free Full Text]
  31. Burrows, P., Bech-Engeser, G. B. and Wabl, M. 1983. Immunoglobulin heavy-chain class switching in a pre-B cell line is accompanied by DNA rearrangement. Nature 306:243.[ISI][Medline]
  32. Laskov, R., Kim, J. K., Woods, V. L., McKeever, P. E. and Asofsky, R. 1981. Membrane immunoglobulins of spontaneous B lymphomas of aged BALB/c mice. Eur. J. Immunol. 11:462[ISI][Medline]
  33. Oi, V. T., Morrison, S. L., Herzenberg, L. A. and Berg, P. 1983. Immunoglobulin gene expression in transformed lymphoid cells. Proc. Natl Acad. Sci. USA 80:825[Abstract]
  34. Rogers, J., Early, P., Carter, C., Calame, K., Bond, M., Hood, L. E. and Wall, R. 1980. Two mRNAs with different 3' ends encode membrane-bound and secreted forms of immunoglobulin mu chain. Cell 20:303[ISI][Medline]
  35. Fort, P., Marty, L., Piechaczyk, M., Sabrouty, S. E., Dani, C., Jeanteur, P. and Blanchard, J. M. 1985. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigene family. Nucleic Acids Res. 13:1431.[Abstract]
  36. Jang, S. K., Krausslich, H.-G., Nicklin, M. J. H., Duke, G. M., Palmenberg, A. C. and Wimmer, E. 1988. A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J. Virol. 62:2636.[ISI][Medline]
  37. Kaufman, R. J., Davies, M. V., Wasley, L. C. and Michnick, D. 1991. Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus. Nucleic Acids Res. 19:4485.[Abstract]
  38. Sherman, P. A., Basta, P. V., Heguy, A., Wloch, M. K., Roeder, R. G. and Ting, J. P.-Y. 1989. The octamer motif is a B-lymphocyte-specific regulatory element of the HLA-DR alpha gene promoter. Proc. Natl Acad. Sci. USA. 86:6739.[Abstract]
  39. Dignam, J. D., Lebovitz, R. M. and Roeder, R. G. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract isolated from mammalian nuclei. Nucleic Acids Res. 11:1475.[Abstract]
  40. Reth, M. G. and Alt, F. W. 1984. Novel immunoglobulin heavy chains are produced from DJH gene segment rearrangement in lymphoid cells. Nature 312:418[ISI][Medline]
  41. Lennon, G. G. and Perry, R. P. 1985. Cµ-containing transcripts initiate heterogeneously within the IgH enhancer region and contain a novel 5'-nontranslatable exon. Nature 318:475.[ISI][Medline]
  42. Morgan, R. A., Couture, L., Elroy-Stein, O., Ragheb, J., Moss, B. and Anderson, W. F. 1992. Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy. Nucleic Acids Res. 20:1293.[Abstract]
  43. Rees, S., Coote, J., Stables, J., Goodson, S., Harris, S. and Lee, M. G. 1996. Bicistronic vector for the creation of stable mammalian cell lines that predisposes all antibiotic-resistant cells to express recombinant protein. BioTechniques 20:102[ISI][Medline]
  44. Flaherty, S. M., Fortes, P., Isaurralde, E., Mataj, I. W. and Gilmartin, G. M. 1997. Participation of the nuclear CAP binding complex in pre-mRNA 3' processing. Proc. Natl Acad. Sci. USA 94:11893[Abstract/Free Full Text]
  45. McCracken, S., Fong, N., Yankulov, K., Ballantyne, S., Pan, G., Greenblatt, J., Patterson, S. D., Wickens, M. and Bentley, D. L. 1997. The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature 385:357[ISI][Medline]
  46. Hirose, Y. and Manley, J. L. 1998. RNA polymerase II is an essential mRNA polyadenylation factor. Nature 395:93.[ISI][Medline]
  47. Alt, F. W., Yancopoulos, G. D., Blackwell, T. K., Wood, C., Thomas, E., Boss, M., Coffman, R., Rosenberg, N., Tonegawa, S. and Baltimore, D. 1984. Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO J. 3:1209[Abstract]
  48. Yuan, D., Gilliam A. C. and Tucker, P. W. 1985. Regulation of expression of immunoglobulins M and D in murine B cells. Fedn Proc. 44:2652
  49. Kelly, D. E. and Perry, R. P. 1986. Transcriptional and post transcriptional control of immunoglobulin mRNA production during B lymphocyte development. Nucleic Acids Res. 14:5431[Abstract]
  50. Takagaki, Y., Seipelt, R. L., Peterson, M. L. and Manley, J. L. 1997. The polyadenylation factor CstF-64 regulates alternative processing of IgM heavy chain pre-mRNA during B cell differentiation. Cell 87:941[ISI]
  51. Martincic, K., Campbell, R. Elwalds-Gilbert, G. Souan, L. Lotze, M. T. and Milcarek, C. 1998. Increase in the 64-kdA subunit of the polyadenylation/cleavage stimulatory factor during the G(0) to S phase transition. Proc. Natl Acad. Sci. USA 95:11095[Abstract/Free Full Text]
  52. Law, R., Kuwabara, M. D., Briskin, M., Fasel, N., Hermanson, G., Sigman, D. S. and Wall, R. 1987. Protein-binding site at the immunoglobulin mu membrane polyadenylation signal: possible role in transcription termination. Proc. Natl Acad. Sci. USA 84:9160[Abstract]
  53. Lutz, C., Ledermann, B., Kosco-Vilbois, M. H., Ochsenbein, A. F., Zinkernagel, R. M., Kohler, G. and Brombacher, F. 1998. IgD can largely substitute for loss of IgM function in B cells. Nature 393:797[ISI][Medline]
  54. Kitamura, D. and Rajewsky, K. 1992. Targeted disruption of µ chain membrane exon causes loss of heavy-chain allelic exclusion. Nature 356:154[ISI][Medline]




This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Request Permissions
Google Scholar
Articles by Kim, M.
Articles by Yuan, D.
PubMed
PubMed Citation
Articles by Kim, M.
Articles by Yuan, D.