Jun 30, 1994 - This is best illustrated by the analysis of multiple. K light chain transgene (LK) copies present in hybridomas from hyperimmunized animals ...
The EMBO Journal vol.13 no. 19 pp.4617-4622, 1994
Low cytoplasmic mRNA levels of immunoglobulin light chain genes containing nonsense codons correlate with inefficient splicing K
Francisco Lozano1'2, Bart Maertzdorf, Richard Pannell and Cesar Milstein Medical Research Council Centre, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK 'Present address: Servicio de Immunologia, Hospital Clfnico y Provincial de Barcelona, Villarroel 170, Barcelona 08036, Spain
2Corresponding author Communicated by C.Milstein
We have previously reported down-regulation of mRNA expression of some of the K light chain transgenes in a hybridoma derived from a secondary immune response. Of the five heavily mutated transgene copies present in that hybridoma, three included premature stop codons and were poorly represented at the mRNA level. Here we show that the nonsense mutations are the cause of the low mRNA levels. While we found no evidence that the reduction in mRNA abundance was attributable to an increased rate of cytoplasmic mRNA decay, the amount of cytoplasmic mRNA correlated with the accumulation of unspliced transcripts in the nucleus. Similar results were obtained with a chimeric immunoglobulin gene containing a premature chain termination codon in the variable gene segment. We suggest that inhibition of splicing induced by in-frame premature stop codons is an important mechanism for down-regulation of undesirable immunoglobulin transcripts. Key words: immunoglobulins/nonsense codons/RNA splicing/transcription
Introduction The immunoglobulin genes have been extensively used for the analysis of eukaryotic gene expression. The regulation of immunoglobulin gene transcription in mammalian cells involves multiple elements that act together to ensure high levels of gene expression. In addition, posttranscriptional and post-translational events have important implications in normal and abnormal immunoglobulin expression (Klausner and Sitia, 1990). In particular, somatic mutations during B cell development can lead to abnormalities in sorting and secretion of immunoglobulin chains. This is best illustrated by the analysis of multiple K light chain transgene (LK) copies present in hybridomas from hyperimmunized animals (Lozano et al., 1993). We found that mutations that improved affinity in one transgene copy favour selection for down-regulation of other copies, both at the post-transcriptional and the posttranslational level. More specifically, in a hybridoma containing five heavily mutated copies of the transgene, only one copy giving improved affinity for antigen gave © Oxford University Press
high levels of secreted products. Of the other four, one gave a light chain which was degraded intracellularly, while the other three were very poorly expressed at the mRNA level. All three, among other mutations, contained nonsense codons. Baumann et al. (1985) selected a number of frame-shift mutants producing truncated g chains due to premature in-frame termination codons in the C,u region. They observed that in those mutants the mRNA pool was seriously depleted and suggested that this was due to increased mRNA degradation. The correlation of premature termination codons and mRNA down-regulation has been emphasized on several occasions (Maquat, 1991; Sachs, 1993). Indeed, there have been a number of instances in which nonsense mutations lead to a decrease in specific mRNA pools by increasing the rate of mRNA decay (Losson and Lacroute, 1979; Maquat et al., 1981; Graves et al., 1987; Barker and Beemon, 1991; Lim et al., 1992). Furthermore, it has been shown that the trans-acting factor encoded by the yeast UPFJ gene is a component of a degradative pathway that acts specifically on mRNAs containing premature stop codons (Leeds et al., 1991). However, more recently it has been suggested that other post-transcriptional events leading to the final expression of cytoplasmic mRNA may also be involved in controlling mRNA pools. A nonsense codon-mediated effect on nuclear RNA metabolism has been suggested for dihydrofolate reductase (DHFR) (Urlaub et al., 1989), triosephosphate isomerase (TPI) (Cheng and Maquat, 1993), human f-globin (Baserga and Benz, 1992) and minute virus of mice (MVM) (Naeger et al., 1992). Although in most cases the exact mechanism has not been elucidated, for MVM it has been shown that nonsense mutations can affect nuclear RNA processing events by inhibiting splicing (Naeger et al., 1992). In this paper, we demonstrate the correlation between nonsense mutations and low cytoplasmic mRNA levels of transgene copies present in an antigen-specific hybridoma. We found accumulation of incompletely spliced RNA products in mutants containing nonsense codons, suggesting that mRNA down-regulation is controlled at the level of splicing.
Results Down-regulation of immunoglobulin K light chain transgenes is induced by nonsense mutations Hybridoma NQT17/10 was derived after secondary immunization of a transgenic LK6 mouse. The LK construct codes for an immunoglobulin K light chain in which the variable (V) region is the mouse VKOx1-JK5, which is the dominant gene combination in the response to the phenyloxazolone hapten. All five transgene copies present in the hybridoma are heavily mutated. Only light chains encoded by copy A are successfully secreted. Light chains 461 7
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encoded by copy B are degraded intracellularly and those encoded by copies C, D and E are not translated due to the presence of premature nonsense codons in their V segments (Lozano et al., 1993). To establish an unequivocal correlation between low mRNA levels and nonsense codons, we prepared new versions of the LK construct containing the mutation pattern of the relevant transgene copies. In order to simplify cloning procedures, the constructs made included only the nucleotide changes present upstream of the PflMI site located at Lys45, the downstream sequences being those of the wild-type transgene. Alternative versions were made which differ from the above in single base substitutions, removing the stop codons in variants D* and E* and re-introducing the stop codon in variant C (Figure 1). All six constructs were cloned into the mammalian expression vector pSV2gpt and used to electroporate the mouse myeloma NSO cell line. Transfectant cells successfully surviving the selection procedures were pooled (to reduce interference due to the integration position and the number of integrated copies) and cytoplasmic RNA was isolated. The measurement of mRNA levels in cytoplasmic fractions revealed a lowmRNA phenotype only for those cells transfected with constructs retaining nonsense codons (variants C*, D* and E*) (Figure 2). It should be noted that the decrease in mRNA pools was significantly more marked in C* and D* than in E*, and this was a consistent observation. We estimated that mRNA levels for variants C* and D* were 1/10 of the well-expressed variants, while for variant E* there was a 1/3 reduction (Figure 3). The results obtained with variants D and E showed that nucleotide changes downstream of the PftMI site were irrelevant in establishing the low-mRNA phenotype of the original copies. The variants D* and E* allowed us to identify the stop codons at positions 35 (TAA) and -2 (TGA), respectively, as the only nucleotide changes upstream of the PftMI site responsible for the low-
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Fig. 3. Decay of LK mRNA in NSO transfectants. Actinomycin D was added to pools of NSO cells transfected with the indicated LK variants. At the indicated times, samples of cells were removed and cytoplasmic RNA isolated for Northern blot analysis with Lic- and actin-specific radiolabelled probes. The intensity of the bands was determined by densitometry and the LK/actin ratio plotted.
mRNA phenotype of the original copies in the NQT17/10 hybridoma. Similarly, the removal of all the nucleotide changes downstrean of the PflMI site (which include the nonsense codon) gave rise to normal cytoplasmic RNA levels for the variant C, equivalent to those seen in cells transfected with a control construct (A). In the variant C* construct the presence of a TAA codon at position 86 was the only difference from C. Therefore, of all the nucleotide changes present in the LK copies, only those introducing nonsense codons were responsible for the relatively lowmRNA phenotype in the NSO transfectants and, presumably, in the original NQTl7/l0 hybridoma. We analysed another set of NSO transfectants which had been electroporated with a chimeric immunoglobulin K light chain gene, which were prepared in two slightly different versions {LK-Vneo(*) and LK-Vneo(*)A[XS]i}. For that purpose, the V region of the LK construct was disrupted by in-frame introduction of a nearly complete neo gene instead of sequences between the beginning of the CDR2 and the end of the CDR3 regions (Figure 1). Versions of both constructs were also made which differed only in the presence of a nonsense codon (TAG) at position His34 (CAC) {LK-Vneo* and LK-Vneo*A[XS]i). As shown in Figure 2, a dramatic drop in cytoplasmic mRNA
Nonsense codons, mRNA pool and abnormal splicing
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Fig. 4. Comparison of LKc transcript levels by RT-PCR assays. Equal amounts (5 ig) of nuclear and cytoplasmic RNA were used for cDNA synthesis. cDNA samples were subjected to PCR amplification for the indicated number of cycles. The RNA imput was controlled by the amount of a 372 bp product obtained with actin-specific primers. (A) For the Lic variants (A, C, C*, D, D*, E and E*) the VkOxBACK and Vk212 primers were used to amplify both mature (342 bp) and immature (513 bp) transcripts. (B) For the LK-Vneo variants (1, LKc-Vneo*; 2, LK-Vneo; 3, LcVneo*A[XS]i; 4, LK-VneoA[XS]i) the VkOxBACK and NEOUPBAM primers were used to amplify both mature (268 bp) and immature (436 bp) transcripts. Samples from cells cultured in the presence (+) or absence (-) of cycloheximide (CHX) are also included.
levels was found for cells containing nonsense codons. It is worth mentioning that there was a lower mRNA level expressed by the constructs containing a deletion of -700 bp between the J segment and the matrix attachment region. The presence of regulatory regions in this segment has recently been described (Lichtenstein et al., 1994). This, however, seems to be irrelevant in the induction of a lowmRNA phenotype by the two single nucleotide changes introducing a premature termination codon. The low level of cytoplasmic L'K mRNA in the NSO transfectants could have been caused by an increased rate of cytoplasmic degradation of the mature mRNA. If this was the case, we would expect to detect degradation following inhibition of RNA polymerase II-catalysed transcription with actinomycin D. These experiments were performed on pools of transfectants representative of each of the normal (variants A and C), intermediate (variant E*) and low (variant D*) cytoplasmic mRNA phenotypes. Pooled transfectants were exposed to actinomycin D for different lengths of time and then cytoplasmic RNA fractions subjected to Northern blot analysis. L'K mRNA levels were normalized to actin mRNA levels. Our results showed that no significant decay could be detected after 6 h follow-up (Figure 3). Poor cell viability did not allow longer incubation times. Although the results do not allow measurements of half-life, they suggest that instability of LK mRNA variants was neither the only nor the main cause of their low steady-state level.
Nonsense codons induce accumulation of RNA splicing intermediates If mRNA stability was not the origin of low mRNA pool levels, it seemed likely that low levels of mRNA would also be detectable in the nucleus. A Northern blot analysis
was performed with nuclear RNA preparations from cells transfected with both the LK variant and the LK-Vneo series of constructs. Care was taken to minimize contamination of the nuclear RNA with cytoplasmic material. No high molecular weight rRNA precursors were found in cytoplasmic fractions, but high molecular weight rRNA was the prominent band in the nuclear fraction. In agreement with our previous data for cytoplasmic RNA fractions, the presence of premature stop codons also resulted in similar reductions in the level of mRNA that fractionated with the nuclear fractions (data not shown). The results above suggest that the low-mRNA phenotype could have resulted from deficient nuclear metabolism. To study these events further we utilized RT-PCR analysis, which is an extremely sensitive method for detecting low abundance RNA molecules. cDNA was made with identical amounts (5 jig) of nuclear and cytoplasmic RNA fractions from cells transfected with the LK variants. cDNA samples were used as a template for PCR amplification with different pairs of primers. By using actin-specific primers, which amplify a 372 bp product, it was shown that all the samples gave reproducible amounts of amplified material (Figure 4). Samples taken at the end of different numbers of PCR cycles were analysed during the phase of exponential accumulation of products. Using the VkOxBACK and CkRATFORWARD primers (which amplify from the beginning of the leader region to the end of the CK region), a 680 bp fragment was amplified which corresponded to the predicted size of mature mRNA molecules. The amount of amplified material obtained with cytoplasmic and nuclear fractions confirmed the results of the Northern blots. In addition, in some samples containing nonsense mutations there was a hint of larger nuclear components, in spite of the fact that we did not expect detection of immature mRNA
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molecules due to the inefficient amplification of large products (data not shown). In order to analyse splicing efficiency better, we performed new RT-PCR assays with the VkOxBACK and Vk212 primers, so that the resulting products were smaller and so that mature and immature transcripts could be easily fractionated on suitable agarose gels. Those primers prime at the beginning of the leader region and at the end of the CDR2 region, respectively. In that way, PCR products of 438 bp and 268 bp should be expected from the unspliced and spliced leader intron forms, respectively. Lower amounts of mature mRNA transcripts were consistently detected in the nucleus from transfectants containing nonsense codons (Figure 4A). Once again, the phenomenon was less evident for variant E* than for C* and D*. However, the relative amount of immature mRNA transcripts (438 bp band) was consistently higher in those transfectants with nonsense codons (C*, D* and E*). The experiment shown in Figure 4A represents an example in which the unspliced transcript was undetected in wildtype and barely detectable in E*. We performed similar experiments with the LK-Vneo(*) and LK-Vneo(*)A[XS]i constructs. For that purpose, both nuclear and cytoplasmic RNA fractions were amplified with the VkOxBACK and NEOUPBAM primers, which prime at the beginning of the leader region and the proximal part of the neo gene, respectively. Two bands were amplified, one corresponding to immature RNA molecules containing 513 bp, which include the leader intron, and another corresponding to mature RNA transcripts (342 bp). The results obtained with nuclear fractions showed that the immature component is consistently enriched in cells containing nonsense codons (Figure 4B). These results were in full agreement with those described above, suggesting that nonsense codons interfere with the correct nuclear RNA processing. The protein synthesis inhibitor cycloheximide had some effect, consistent with the suggestion that it increases the rate of splicing (Qian et al., 1993a,b). In our case, however, the effect was rather small and not very reproducible. Interestingly, when the same RT-PCR assays were performed with cytoplasmic RNA fractions (Figure 4B), we observed the appearance of the 513 bp band only in the cells transfected with constructs containing nonsense codons, a phenomenon which was also observed with construct C*, but only after a higher number of PCR cycles. Similar observations were never made with any of the other constructs or indeed with the control samples (not shown). Therefore, the increased ratio of accumulated immature to mature RNA molecules induced by nonsense codons was seen not only in nuclear fractions, but in some cases also in cytoplasmic fractions. The immature RNA molecules were predominant in the nucleus, but only a minor component in the cytoplasm.
Discussion The results of this paper show that chain termination mutants produced during somatic hypermutation not only disrupt protein synthesis, but also deplete the mRNA pool. We have previously suggested that during affinity maturation of responses in animals containing multiple copies of the appropriate transgene, the antigen selects
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deleterious mutations of redundant copies, which would otherwise compete with copies containing mutations which improve affinity (Lozano et al., 1993). A considerable proportion of deleterious mutants appeared to express a depleted mRNA pool, particularly those including nonsense mutations. In this paper we show that this was a property of the premature termination codon, and not related to other nucleotide substitutions. The results raise the question of whether cells contain a quality control mechanism to either mop out or down-regulate mRNA species coding for such non-functional truncated polypeptides. While this is an issue of general interest in cell biology, in the immune system such down-regulation is of direct physiological relevance. Premature termination is common when out-of-frame events take place during V(D)J recombination and allelic exclusion. The excluded allele is usually expressed, leading to the production of unwanted mRNA capable of directing the synthesis of potentially harmful peptide fragments. Our results suggest that the premature termination of protein synthesis also affects normal splicing, thus leading to accumulation of splicing intermediates and depletion of mRNA pools. It is relevant that the previously described accumulation of splicing intermediates of TCR-1 transcripts in a murine lymphoma (Qian et al., 1993a) and in murine thymus (Qian et al., 1 993b) may correlate with out-of-frame rearrangements of the TCR-0 genes. Thus premature chain termination in non-productive rearrangements of both TCR and immunoglobulin genes may result in the downregulation of 'junk' mRNA pools. Until recently, depletion of the pool of mRNA species containing stop codons was thought to be a consequence of the loss of protection afforded by ribosomes during protein synthesis (Losson and Lacroute, 1979; Maquat et al., 1981; Graves et al., 1987; Barker and Beemon, 1991; Lim et al., 1992). The recent demonstration of an enzyme in yeast which specifically degrades such mRNAs (Leeds et al., 1991), suggests one mechanism for such quality control. This does not seem to be the way in which depletion of the mRNA pools described in this paper is achieved. The depletion of the mRNA pool does not correlate with a faster degradation of cytoplasmic mRNA. While the experiments do not show that the half-life of LK variants containing nonsense codons is the same as their normal counterparts, a 10-fold decay in the mRNA pool (as we observe in variant D*) would require a corresponding change in mRNA half-life. The half-life of K light chain mRNA has been estimated to be 14 h (Cowan and Milstein, 1974) and such an increase in the rate of decay should have been easily detected in our experiments. While we cannot exclude increased mRNA degradation as a contributory factor, the very small amounts of mature mRNA found in the nuclear fraction, and the corresponding increase in unspliced intermediates, supports the view that the down-regulation of the mutated transgenes is a nuclear event. Our results suggest that the step at which downregulation occurs is at the level of splicing. The ratio between the unspliced intermediate and the mature mRNA increases considerably in the mutants. The weakness of this parameter is that it could be distorted by cytoplasmic contamination. We took great care to avoid this, and
Nonsense codons, mRNA pool and abnormal splicing
the analysis of the nuclear ribosomal fractions, with a predominance of the large 45S component, indicated that such contamination could not have been substantial. Fortunately, in our case we also found that the unspliced intermediates are present in larger amounts in the nuclear fractions of missense mutants than in their normal counterparts. It also follows that a decrease in transcription rate or increase in the rate of degradation of the primary transcript is unlikely to be the origin of the mRNA downregulation. RT-PCR has been used before to compare mRNA expression between related cell lines or during development and in a variety of other studies (Cheng and Maquat, 1993; Higgins and Hames, 1994). While the method has many pitfalls, we did not rely on small differences and the results were observed repeatedly and were consistently observed with the different samples. There are other reports in the literature suggesting that nonsense mutations may affect RNA processing and/ or transport, hence down-regulating cytoplasmic mRNA (Baserga and Benz, 1988; Daar and Maquat, 1988; Urlaub et al., 1989; Cheng and Maquat, 1993). However, even within the nucleus there may be more than one way to achieve down-regulation of mRNA. A number of studies suggest that the splicing and nuclear export of pre-mRNA molecules are competing processes (Maquat, 1991). In order to be efficiently processed, a pre-mRNA molecule has to be stably associated into a spliceosome, otherwise it enters the export pathway and moves to the cytoplasm (Legrain and Rosbash, 1989). The 'translation/translocation' model linking premature termination of translation to inhibition of translocation of the spliced RNA to the cytoplasm (Urlaub et al., 1989) predicts that the nonsense mutation inhibits removal of downstream introns. In this paper all our experiments refer to the inhibition of an upstream intron. In addition, with the LK-Vneo constructs we detected significant amounts of unspliced intermediate in the cytoplasm. This does not appear to be due to impurities from nuclear debris, since no ribosomal RNA precursor was detectable. The presence of such unspliced material in the cytoplasm suggests that inhibition of splicing does not prevent the intermediate moving into the cytoplasm, where it is likely to be degraded. However, the relative amounts of mature and unspliced intermediate in the nucleus and in the cytoplasm (Figure 4B) are consistent with preferential transport of the spliced form (Legrain and Rosbash, 1989). Our results thus suggest that nonsense codons are somehow recognized in the nucleus, to prevent association with or favour dissociation of spliceosomes from immature transcripts. The nuclear exon scanning model (Urlaub et al., 1989) is attractive in the context of a proposed scanning model of exon definition (Robberson et al., 1990). Thus, it has been suggested that definition of exons and introns might include scanning of ORFs (Naeger et al., 1992). In triosephosphate isomerase (TPI) no accumulation of unspliced intermediates was observed (Cheng and Maquat, 1993), while in the parvovirus minute virus of mice (MVM) nonsense mutations correlate with alterations in the ratio of splicing (Naeger et al., 1992). Ignoring possible complications resulting from the overlapping nature of the transcription unit, these are the closest to our results. However, Naeger et al. found that the abnormal splicing
ratio decreased as the distance of the initiation codon from the nonsense mutations increased. We find that the reduction in cytoplasmic mRNA levels is less marked in LK variant E* than in any of the others we analysed, even though the nonsense codon present in that variant is the nearest to the initiation methionine. Furthermore, there was no significant drop in the cytoplasmic mRNA levels in a previously reported LK variant (LK[p5S']G), which contains a premature stop codon further upstream, in the preceding exon leader (Betz et al., 1994). A larger database will be required to clarify these disparities. The downregulation of the K light chain mRNA, because it is so pronounced in some of the examples we describe, constitutes an excellent system to perform this type of study.
Materials and methods Plasmid construction The gene constructs described here are modifications of LK (Meyer et al., 1989). The LK construct is a 14.5 kb EcoRI-EcoRI fragment containing a VKOx 1 -JK5 mouse V segment and a rat CK region, together with all the mouse regulatory elements necessary to be properly expressed. Basically, the modifications introduced are either (i) the presence of point mutations in the V region, as well as in the upstream segments (LK variants A, C, C*, D, D*, E and E*), (ii) replacement of part of the VKOx1 gene by the neomycin resistance gene neo (LK-Vneo, LK-Vneo*, LK-VneoA[XS]i and LK-Vneo*A[XS]i). The point mutations present in the LK variant constructs are those previously reported upstream of the PflMI site of the transgene copies found in the NQT17/ 10 hybridoma (Lozano et al., 1993). For plasmid construction, genomic DNA from the NQT17/10 hybridoma was amplified with the 5PRIMAOxBACK and Jk5FORBAM primers, which prime near the 5' end and the JK5 segment of LK, respectively (Lozano et al., 1993). The PCR products were cloned into EcoRI- and BamHI-digested Ml3mpl8 and sequenced (Sequenase kit, USB) using the JkSFOR, VkOx3O and LPOx primers (Lozano et al., 1993). Once identified as A, C, D and E, EcoRI- and PflMI-digested fragments were cloned into an appropriately digested pUC19 vector containing the EcoRI-HindIII fragment of Lwc. Finally, EcoRI-SaclI fragments from these intermediate constructs were cloned into a modified version of LK cloned into pSV2gpt (Mulligan et al., 1981). For this purpose, both the unique BamHI and 3' EcoRI sites of pSV2gpt and LK, respectively, were replaced by NotI. This gave LK constructs containing all the nucleotide changes characteristic of copies A, C, D and E upstream of the PflMI site, but completely unmutated downstream. As a result, variant C lacked a stop codon at
position Tyr86. By oligonucleotide-directed mutagenesis (Sambrook et al., 1989), new versions were made in which the stop codons at positions Trp35 and Arg(-2) of copies D and E were substituted by the nucleotides present in the original LK construct by using the 5'CTTCTGCTGGTACCAGTGCATGTAA-3' and 5'-CAATTTGTCCTCTGGATATTATGAC-3' mutagenic primers, respectively. Similarly, the stop codon characteristic of copy C at position Tyr86 was restored by using the 5'-CTGCTGGCAGTATTAAGTGGCAGCA-3' primer. The LK-Vneo constructs were made by replacing part of the VKOx1 gene (from the beginning of the CDR2 region to the end of the CDR3 region) with almost the entire coding sequence of the neo gene (Beck et al., 1982). The EcoRI-PflMI fragment of LK (which includes the leader intron and part of the VKOx1 sequence) was fused in-frame with a 1.9 kb EagI-BamHI fragment from pSV2neo (Southern and Berg, 1982). The fragment lacks the first 35 bp of the N-terminal coding sequence of the neo gene and its expression is dependent on the upsteam immunoglobulin sequences. To fuse in-frame the 3' end of the neo gene to the JK5 region, the small intron and the SV40 polyadenylation site present in the neo fragment was removed by AsuII and ApaI digestion and further looping-out mutagenesis (Sambrook et al., 1989) with the 5'CCCAGCACCGAACGTGAGGTCCCGCTCA-3' oligonucleotide. The final construct was obtained as for the LK variants. In LK-Vneo* and LK-Vneo*A[XS]i, codon His34 (CAC) at the last position of the CDRI region was converted into a termination codon (TAG) by site-directed mutagenesis (Sambrook et al., 1989). A 0.7 kb XbaI-SacII fragment in the J-C intron region was removed in the LK-VneoA[XS]i and LK-
Vneo*A[XS]i
constructs. Plasmid DNA was
prepared by standard
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FLozano et al. methods and purified by centrifugation in CsCI-ethidium bromide (Sambrook et al., 1989).
Electroporation and cell selection procedures The non-secretory mouse myeloma NSO cells (Galfre and Milstein, 1981) were electroporated with Sfil-linearized plasmid DNA (10-20 ,ug) and plated in non-selective Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum in 24-well plates. Selection was carried out 48 h later with medium supplemented with hypoxanthine (20 tg/ml), xanthine (250 tg/ml) and mycophenolic acid (2.5-5 ,ug/ml). In order to obtain a representative transfectant population, no less than 20 wells, each containing five or more clones, were individually expanded and then pooled. In some cases, pooled cells were exposed to either 10 tg/ml cycloheximide for 6 h or 5 tg/ml actinomycin D for 1, 3 or 6 h, before RNA extraction.
RNA preparation and Northern blot analysis Cytoplasmic RNA was prepared by cell resuspension in cold lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 30 U/mI RNAsin, 0.5% Nonidet P-40) and further phenol-chloroform extraction and ethanol precipitation of the soluble fraction. Nuclear RNA was obtained from pelleted nuclei of the previous fractionation step and resuspension in lysis buffer containing 1% SDS (instead of NP-40). RNA concentration was measured by A260 and integrity of the 28S and 18S rRNAs assessed by agarose gel electrophoresis. For Northern blot analysis, 5 .tg RNA samples were suspended with glyoxal (Fluka) in 10 mM sodium phosphate buffer and denatured at 50°C for I h. Samples were electrophoresed in a 1% agarose gel and transferred to a nylon membrane (Hybond-N, Amersham) by capillary blotting with 20XSSC. Filters were UV-crosslinked and then baked at 80°C for I h. Hybridization with LK- and actin-specific cDNA probes which had been 32P-labelled by random priming (Oligolabelling kit, Pharmacia) was performed by overnight incubation at 65°C in 3XSSC solution. The LK cDNA probe was obtained by PCR amplification with the VkOxBACK and CkRATFORWARD primers (Sharpe et al., 1990) and the actin cDNA probe was amplified using the 5'-TAGGAATCCATGGCCACTGCCGCATCCTCTTCC-3' and 5'-CACGATGGAGGGGCCGGACTCATC-3' primers. After two 30 min washes at 65°C with 0.1 xSSC plus 0.1 % SDS, filters were autoradiographed for 4-24 h. Signal intensity was quantified with a Computing Densitometer Model 300A (Molecular Dynamics). RT- PCR assays cDNA was made from 5 tg RNA by using 20 U Super RT (HT Biotechnology) and 0.5 gg oligo(dT) (Collaborative) at 42°C for 60 min (in 50 mM Tris-HCI, pH 8.3, 100 mM KCI, 10 mM MgCI2, 5 mM DTT, 500 ,uM each dNTP). Then, 1/10 of the reaction was used to specifically amplify LK and actin transcripts using 5 U Taq DNA polymerase (Promega), 5 mM each dNTP and 25 pmol primers. The amplification conditions were 1 min at 94°C for denaturation, 1 min at 55°C for annealing and 2 min at 72°C for extension. The fully spliced LK transcripts were amplified with the VkOxBACK and CkRATFORWARD primers. For amplification of transcripts containing the intron leader region, the VkOxBACK primer was used in conjunction with either the Vk2l2 (5'-GAGTAAGAGGTCCCAGACCCACT-3') or the NEOUPBAM (5'-GCCGGATCCTGCAGTTGATTCAGGG-3') primers for the analysis of the LK variant and the LK-Vneo constructs, respectively.
Acknowledgements We thank Y.Loon for help and advice during the generation of the LKVneo constructs and C.Rada for her collaboration in the preparation of illustrations. F.Lozano is the recipient of a Celltech Fellowship.
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Received on April 25, 1994; revised on June 30, 1994
