Alternative splicing of transcripts encoding the [alpha]- and [beta]-subunits of mouse glucosidase II in T lymphocytes

Christopher W. Arendt, Wojciech Dawicki and Hanne L. Ostergaard1

Department of Medical Microbiology and Immunology, University of Alberta, Edmonton T6G 2H7, Canada

Received on May 26, 1998; accepted on July 31, 1998

Glucosidase II is a processing enzyme of the endoplasmic reticulum that functions to hydrolyze two glucose residues in immature N-linked oligosaccharides attached to newly synthesized polypeptides. We previously reported the cDNA cloning of the [alpha]- and [beta]-subunits of mouse glucosidase II from T cells following copurification of these proteins with the highly glycosylated transmembrane protein-tyrosine phosphatase CD45. Subsequent examination of additional cDNA clones, coupled with partial genomic DNA sequencing, has revealed that both subunits are encoded by gene products that undergo alternative splicing in T lymphocytes. The catalytic [alpha]-subunit possesses two variably expressed segments, box [Agr]1, consisting of 22 amino acids located proximal to the amino-terminus, and box [Agr]2, composed of 9 amino acids situated between the amino-terminus and the putative catalytic site in the central region of the molecule. Box [Bgr]1, a variably expressed 7 amino acid segment in the [beta]-subunit of glucosidase II, is located immediately downstream of an acidic stretch near the carboxyl-terminus. Screening of reverse transcribed RNA by polymerase chain reaction confirms the variable inclusion of each of these segments in transcripts obtained from a panel of T-lymphocyte cell lines. Thus, distinct isoforms of glucosidase II exist that may perform specialized functions.

Key words: ER quality control mechanism/glucosidase II/glycoprotein processing/N-glycosylation

Introduction

Glycosylation of transmembrane and secreted proteins is a multistep process that begins in the endoplasmic reticulum (ER) and continues as proteins transit the Golgi apparatus. In the ER, carbohydrate processing is closely coupled to the process of protein folding (Helenius, 1994). N-glycosylation is initiated on nascent polypeptides by en bloc addition of a precursor oligosaccharide moiety, Glc3Man9GlcNAc2, containing a tri-glucose extension (Liu et al., 1979). Cleavage of the terminal glucose residue is catalyzed by glucosidase I, a type II membrane protein of the ER (Kalz-Fuller et al., 1995). Glucosidase II, a soluble enzyme, then cleaves the middle glucose residue generating a monoglucosylated oligosaccharide product that is recognized by a lectin domain of the membrane-bound chaperone calnexin (Hammond et al., 1994; Hebert et al., 1995; Ware et al., 1995; Zapun et al., 1997; Vassilakos et al., 1998) or its lumenal homolog calreticulin (Peterson et al., 1995; Vassilakos et al., 1998). For most proteins, the process of folding does not proceed to completion immediately (Ganan et al., 1991), necessitating transient dissociation from calnexin/calreticulin by glucosidase II-induced deglucosylation, followed by initiation of a subsequent folding cycle upon reattachment of the monoglucose moiety by UDP-glucose:glycoprotein transferase (Helenius et al., 1997). Inhibition of glucosidase II activity after the first cycle of folding has been shown to inhibit protein folding in vitro (Hebert et al., 1996; Wada et al., 1997). Oligosaccharide processing by glucosidase II is therefore likely to play a central role in priming multiple stages of protein folding in the ER.

The importance of glucosidase II in functioning at the nexus of oligosaccharide processing and protein 'quality control" is supported by a number of in vivo studies where glucosidase II activity has been ablated by mutagenesis, as in the case of the BW-derived PHAR2.7 T-lymphoma line (Reitman et al., 1982), or by treatment with chemical inhibitors such as castanospermine, bromoconduritol, or 1-deoxynojirimycin. Glycoproteins from cells deficient in glucosidase II activity, in addition to exhibiting decreased levels of complex-type oligosaccharides (Datema et al., 1982; Reitman et al., 1982), are unable to associate with calnexin/calreticulin (Hammond et al., 1994; Kearse et al., 1994; Hebert et al., 1995; Ora and Helenius, 1995). This undermining of the quality control apparatus of the ER is manifested in reduced rates of protein folding (Tector and Salter, 1995), accelerated protein degradation (Kearse et al., 1994), reduced expression of cell-surface proteins (Trowbridge and Hyman, 1978; Arakaki et al., 1987; Edwards et al., 1989; Balow et al., 1995), and defects in protein secretion (Lodish and Kong, 1984; Yeo et al., 1989). Interestingly, in most cases these effects are observed for specific subgroups of proteins but not others, and no major effects on cell proliferation or viability have been reported. The lack of a more severe phenotype in glucosidase II deficient cells may be attributed to activation of alternative chaperone and processing pathways (Moore and Spiro, 1992; Balow et al., 1995).

Despite considerable progress in the characterization of the catalytic activity and biological functions of glucosidase II, its molecular identity has only recently been elucidated. Trombetta et al. were the first to convincingly demonstrate that glucosidase II purified from rat liver microsomes exists as a highly stable complex of two protein subunits, termed GII[alpha] (glucosidase II [alpha]-subunit) and GII[beta] (glucosidase II [beta]-subunit) (Trombetta et al., 1996). Extensive microsequence information was obtained on these proteins, allowing identification of a GII[alpha] gene homolog in Saccharomyces cerevisiae that, when disrupted, ablates glucosidase II activity (Trombetta et al., 1996). Further evidence that GII[alpha] encodes the catalytic subunit of glucosidase II was obtained by Flura et al., who isolated the cDNA encoding this protein from a pig liver library and performed a series of expression studies (Flura et al., 1997). Contemporaneous to this work, we carried out the cDNA cloning of mouse GII[alpha] and GII[beta] after copurifying these proteins with the highly glycosylated transmembrane protein-tyrosine phosphatase CD45 (Arendt and Ostergaard, 1997). Interestingly, we observed that GII[alpha] and GII[beta] exhibit high evolutionary conservation, with 90% and 86% amino acid sequence identity, respectively, between mouse and human. The significance of the [beta]-subunit to glucosidase II function has not yet been experimentally addressed. However, it is likely that a conserved carboxyl-terminal HDEL motif in GII[beta] acts to prevent secretion of the soluble enzyme complex by coupling to the retrieval mechanism of the KDEL receptor (Wilson et al., 1993). Now that the molecular identities of GII[alpha] and GII[beta] have been solved, more detailed characterization of this important processing enzyme is possible.


Figure 1. Distribution of alternatively spliced boxes A1, A2, and B1 in mouse glucosidase II. Schematic representation of the [alpha]- and [beta]-subunits of glucosidase II showing the sequences encoded by the variably expressed box A1, A2, and B1 segments. Amino acids presented in standard single-letter code are numbered according to the longest coding sequence for each protein, with position 1 assigned to the first residue following leader peptide cleavage. The putative catalytic site (CS) of GII[alpha] and the Glu-rich acidic stretch (AS) of GII[beta] are labeled.

Interestingly, while characterizing mouse cDNA clones of glucosidase II, we have discovered that sequence heterogeneity exists at two sites in GII[alpha], designated boxes [Agr]1 and [Agr]2, and at one site in GII[beta], termed box [Bgr]1. We have sequenced the genomic DNA flanking each of these three regions and have identified splice junctions at the predicted sites. Moreover, we have obtained evidence from RT-PCR screening of transcripts derived from various T-cell lines that alternative splicing of the box sequences gives rise to multiple isoforms of glucosidase II. Herein, we provide the first report that the two subunits of glucosidase II are encoded by alternatively spliced gene products and speculate as to the possible biological significance of this phenomenon.

The nucleotide sequences reported in this article have been submitted to the GenBank/EBI Data Bank with accession numbers AF066057-AF066061.

Results

Heterogeneity in GIIa and GIIb cDNA clones

While cataloguing partial cDNAs encoding the [alpha]- and [beta]- subunits of glucosidase II (Arendt and Ostergaard, 1997), we identified three regions variably expressed among the panel of clones (Figure 1). The first of these, termed box [Agr]1, is located proximal to the amino terminus of the [alpha]-subunit of mouse glucosidase II and encodes a segment of 22 amino acids bearing no homology to known sequences. The nucleotide sequence encoding this segment was previously reported in our cloning of full-length GII[alpha] (Arendt and Ostergaard, 1997), however cDNA clones have since been identified that lack the box A1 residues. Of five cDNA clones examined, four were missing the box A1 element (Table I). We hypothesize that a differentially spliced exon segment encodes this 22 amino acid insert. Interestingly, this segment is not present in two independently reported cDNA clones of human GII[alpha] (Nagase et al., 1995; EMBL database clone AJ000332) or in a cDNA clone of pig GII[alpha] (Flura et al., 1997).

The second variable region that has been identified, designated box A2, is located in the [alpha]-subunit of glucosidase II at a position 145 amino acids downstream of box [Agr]1 (Figure 1). Box [Agr]2 encodes a segment of 9 amino acids absent in the previously reported sequence of mouse GII[alpha] (Arendt and Ostergaard, 1997). Out of the five mouse GII[alpha] cDNA clones surveyed, one was found to contain the box A2 segment (Table I). Within this small pool of cDNA clones, three isoforms of GII[alpha] could therefore be distinguished: Form 1 (previously reported, box A1+, box A2-), Form 2 (box A1-, box A2+), and Form 3 (box A1-, box A2-). The existence of Form 4 GII[alpha] (box A1+, box A2+) has not yet been confirmed. Like the box A1 segment, box A2 is also absent in both the pig and human cDNA clones of GII[alpha]; however, its variable representation among our panel of mouse cDNA clones suggests that it represents a second site of alternative splicing within the GII[alpha] transcript.

The third and final variable region discovered, box [Bgr]1, is positioned immediately downstream of an acidic stretch near the carboxyl terminus of GII[beta](Figure 1). Box [Bgr]1 codes for a 7 amino acid segment that has been identified in 3 out of 10 mouse GII[beta] cDNA clones derived by various methods (Table II). Interestingly, an unpublished bovine GII[beta] cDNA sequence of unknown fidelity (GenBank accession number U49178) contains an element of identical location and similar sequence to the mouse box A2 VQGEQPK segment, although the bovine element (DSQGEQPKD) is slightly longer. The human cDNA sequence reported for a single GII[beta] clone (Sakai et al., 1989), in contrast, encodes at this position a segment (DSEE) with identity to the first two amino acids in the bovine sequence but no identity to the mouse box B1 element. We postulate, therefore, that variable expression of the mouse box B2 element can be ascribed to alternative mRNA splicing, a process that may also account for heterogeneity at the corresponding region of GII[beta] in other mammals expressing this highly conserved protein.

Table I. Summary of box A1 and A2 utilization in GII[alpha] cDNA clones
cDNA clone Size (kb) Cloning method Cell Box A1 Box A2 Designation
116FL.A 3.1 PCR SAKR - - Form 3
116FL.Ba 3.1 PCR SAKR + - Form 1
116FL.G 3.1 PCR SAKR - - Form 3
6R5-13 1.6 5[prime] PCR-RACE SAKR - - Form 3
6R5-14 1.6 5[prime] PCR-RACE SAKR - + Form 2
aThe sequence of this cDNA clone has been previously reported (Arendt and Ostergaard, 1997).

Table II. Summary of box B1 utilization in GII[beta] cDNA clones
cDNA clone Size (kb) Cloning method Cell Box B1 Designation
80-1 1.5 cDNA library EL4 - Form 2
80-2 1.0 cDNA library EL4 + Form 1
80-3 1.3 cDNA library EL4 - Form 2
80-4 1.5 cDNA library EL4 + Form 1
80-5a 1.8 cDNA library EL4 - Form 2
80FL.I 1.7 PCR SAKR + Form 1
8R3-5 1.5 3[prime] PCR-RACE SAKR - Form 2
8R3-12 1.5 3[prime] PCR-RACE SAKR - Form 2
8R3-17 1.5 3[prime] PCR-RACE SAKR - Form 2
aThe sequence of this cDNA clone has been previously reported (Arendt and Ostergaard, 1997).

Genomic sequencing of the regions encompassing boxes [Agr]1, [Agr]2, and [Bgr]1

To verify that the three variably expressed exon segments identified in mouse glucosidase II are susceptible to alternative exon splicing, genomic sequence information was sought that would allow exon/intron boundaries to be located and the presence of donor and acceptor splice sites to be confirmed. Genomic DNA was purified from the SAKR T-cell lymphoma cell line from which the full-length cDNAs encoding GII[alpha] and GII[beta] were originally isolated (Arendt and Ostergaard, 1997). Oligonucleotide primer pairs flanking boxes [Agr]1, [Agr]2, and [Bgr]1 were employed to amplify these regions of genomic DNA by PCR. PCR reaction products were directly sequenced to exclude the possibility that the variably expressed segments are encoded by two independent alleles differing only at the variable box sites. Fidelity of genomic sequences was further confirmed by parallel cloning of the PCR products and sequencing of plasmid DNA. Consistent with the earlier analysis of GII[alpha] cDNA clones, box [Agr]1 was found to be encoded by an internal exon located between a 456 bp 5[prime]-flanking intron (fully sequenced) and a ~0.9 kb 3[prime]-flanking intron (partially sequenced, Figure 2A). The presence of donor and acceptor splice sites at the predicted sites of the intron/exon boundaries supports a model whereby the box [Agr]1 exon is either included or excluded from GII[alpha] transcripts by a mechanism of differential mRNA splicing.


Figure 2. Partial genomic sequences of mouse glucosidase II [alpha]- and [beta]-subunits. Mouse genomic DNA flanking the box A1 (A), box A2 (B), and box B1 (C) elements was sequenced to identify donor (gt) and acceptor (ag) splice sites. Partial intronic sequences are presented in lowercase letters, and the approximate sizes of the introns are indicated. The entire sequences obtained have been submitted to the GenBank database (AF066057-AF066061).

Unlike box [Agr]1, box [Agr]2 is not located adjacent to a 5[prime]-flanking intron, although a ~0.5 kb 3[prime]-flanking intron (partially sequenced) is present (Figure 2B). Rather, the first two nucleotides encoding box [Agr]2 specify a GT-donor splice site while a second GT-donor splice sequence is present at the 5[prime] boundary of the downstream flanking intron. At the 3[prime] boundary of this same intron is an AG-acceptor splice site, allowing for a splicing reaction that either omits or includes box [Agr]2. Together with our cDNA sequence data, this suggests that box [Agr]2 comprises a second alternatively spliced exon cassette in GII[alpha].

Similar to box A2, box B1 in GII[beta] exists as an exon cassette juxtaposed by a single downstream flanking intron which, in this case, consists of only 77 nucleotides (Figure 2C). The pattern of donor and acceptor splice sites mirrors that of box A2, suggesting that a similar mechanism of differential mRNA splicing is operative. In the sequencing of the mouse genomic DNA in this region, we also located a 184 nucleotide intron (fully sequenced and submitted to GenBank) between amino acid residues 269 and 270, upstream of box B1 (data not shown). The available cDNA sequence data, however, indicate that splicing at this second intron is not coupled to variable expression of GII[beta] coding sequences.

PCR analysis of utilization of boxes A1, A2, and B1

The flanking primers employed to amplify the regions of genomic DNA encompassing boxes A1, A2, and B1 were also used to examine the representation of these variably expressed segments within reverse-transcribed RNA prepared from various T-cell lines. Amplification of the region containing box A1 generated two DNA fragments corresponding in size to the two different splice forms of GII[alpha] produced by variable inclusion of the 66 bp box A1 segment (Figure 3A). Both splice forms could be detected in the transformed T-cell lines SAKR and BW as well as the non-transformed cytotoxic T-lymphocyte clone CTL AB.1. A slight bias favoring amplification of box A1-negative DNA fragments was consistently observed under our PCR conditions. Similar results were obtained regardless of whether the cDNA template was prepared using oligo(dT) or random primers. Interestingly, these two splice forms of GII[alpha] could also be detected, albeit with lower efficiency in the PHAR2.7 cell line, a glucosidase II-deficient derivative of BW (Reitman et al., 1982). This mutant T-lymphoma cell has been exploited in a variety of experimental systems (Gabel and Kornfeld, 1982; Moore and Spiro, 1992; Suh et al., 1992; Kearse et al., 1994; Balow et al., 1995; Ora and Helenius, 1995; Wiest et al., 1995) and has recently been shown to be deficient in GII[alpha] mRNA by Northern blot analysis (Flura et al., 1997). Using the present PCR-based assay, it is clear that the total levels of GII[alpha] mRNA are dramatically reduced compared to BW parental cells (Figure 3A and data not shown), owing possibly to reduced transcription of this gene and/or decreased message stability. Moreover, we failed to detect enzymatically active glucosidase II in PHAR2.7 cell extracts (data not shown), consistent with the notion that functional GII[alpha] protein is not expressed by this cell line (Reitman et al., 1982; Flura et al., 1997).


Figure 3. RT-PCR analysis of isoform distribution.Oligo(dT) or random (R) primed cDNA prepared from RNA extracted from various T-cell lines was amplified using primers flanking the box A1 (A), box A2 (B), or box B1 (C) elements. PCR reaction products were visualized by ethidium bromide staining. Sizes of known standards are indicated in base pairs.

Examination of PCR products generated during amplification of the box A2 region allowed us to distinguish between GII[alpha] isoforms containing or lacking this 27 bp segment (Figure 3B). Interestingly, in all cells examined it was observed that amplification of the isoform containing box A2 occurred with considerably greater efficiency than did the isoform lacking this element. In fact, our ability to detect the lower-molecular-weight isoform in this assay appeared to be operating near a threshold, as evidenced by inconsistent amplification of the smaller DNA fragment in the two reactions primed from CTL AB.1 cDNA. It is not known whether isoform preferences detected in this assay reflect differences in protein expression in vivo. Amplification of box A2- isoforms was not improved by altering the method of reverse transcription priming. The box A2- cDNA species could not be detected in PHAR2.7 cells; however, this may reflect limitations in assay sensitivity, given the diminished level of GII[alpha] transcripts in this cell. Taken together, these data confirm that alternative splicing of the box A2 region can be detected in various cellular populations and raise the possibility that utilization of the downstream donor splice site at the 3[prime] boundary of box A2 (Figure 2B) may be favored.

Finally, the PCR assay was used to examine GII[beta] isoform representation in a several T-cell lines. DNA species amplified in this experiment migrated as two closely spaced bands on agarose gels, consistent with there being only a 21bp difference between isoforms exhibiting variable inclusion of the box B1 segment (Figure 2C). The large and small fragments corresponding to the two splice forms of GII[beta] were amplified with similar efficiency in all cells examined. Notably, both isoforms could be amplified from PHAR2.7 with an efficiency comparable to that observed in wild-type cells, including the parental BW line. This result is consistent with expression of normal levels of GII[beta] protein by PHAR2.7 cells, as revealed by Western blot analysis (data not shown). Interestingly, the [beta]-subunit protein has been observed to migrate as a doublet on polyacrylamide gels (Arendt and Ostergaard, 1997), a phenomenon that may relate to alternative splicing of the 7 amino acid box B1.

Discussion

We previously employed several strategies of cDNA cloning to determine the coding sequences of the [alpha]- and [beta]-subunits of glucosidase II (Arendt and Ostergaard, 1997). Further examination of the clones we derived has revealed the presence of two variably expressed segments in GII[alpha], termed boxes A1 and A2, and one variably expressed segment in GII[beta], designated box B1. By identifying donor and acceptor splice sites in the respective genes, we have confirmed that these differentially utilized sequences are derived from alternative splicing of GII[alpha] and GII[beta] transcripts. Finally, we have utilized RT-PCR to verify that alternative splicing of these regions occurs in a variety of T-cell lines. It will be of interest to determine whether these patterns of splicing have been retained during evolution, since the coding sequences of GII[alpha] and GII[beta] are otherwise highly conserved among mammals. A sequence similar to the mouse box B2 element has been discovered in a bovine GII[beta] cDNA clone (GenBank accession number U49178); however, nothing comparable to the box A1 and box A2 segments exists in the limited cDNA clones of GII[alpha] reported for other mammalian species.

At present, it is only possible to speculate as to the potential biological relevance of the polypeptide regions encoded by the alternatively spliced segments. One possibility is that these regions function to modulate protein-protein interactions, such as those mediating the association between GII[alpha] and GII[beta]. A variety of empirical approaches indicate that GII[alpha] and GII[beta] isolated from rat liver microsomes exist as noncovalently linked heterodimers that are highly refractory to dissociation (Trombetta et al., 1996). In the context of the present study, it will be of interest to assess the binding kinetics of each of the four potential isoforms of GII[alpha] with the two different isoforms of GII[beta] to determine whether the variably expressed segments influence enzyme dimerization. We are currently addressing the possible effects of the variably expressed segments on enzyme dimerization by utilizing recombinant proteins to map association domains within the [alpha]- and [beta]-subunits of glucosidase II.

It is also possible that alternative splicing of glucosidase II influences interactions between the enzyme and other resident ER proteins or substrates. The functions of glucosidase II closely intersect with those of other ER proteins including glucosidase I, calnexin, calreticulin, and UDP-glucose:glycoprotein glucosyltransferase (Helenius et al., 1997). Interestingly, we initially purified glucosidase II by virtue of its highly stable interaction with the transmembrane protein-tyrosine phosphatase CD45 in T-lymphoma cells. This physical association between glucosidase II and CD45 is unusual in that glucosidase II does not stably interact with other abundant glycoproteins, such as class I major histocompatibility complex molecules (Arendt and Ostergaard, 1997). Previously obtained microsequence information indicates that CD45-associated glucosidase II contains the box A1 element (Arendt and Ostergaard, 1997); however, it is unclear whether glucosidase II lacking this element is also capable of associating with CD45. Experiments are currently underway to address the possible contributions of boxes A1, A2, and B1 to the binding interaction between glucosidase II and CD45 in comparison to other substrates.

Finally, it is necessary to consider that alternative splicing of glucosidase II may alter its intrinsic activity or subcellular localization. Although the putative catalytic site of GII[alpha] is located distal to boxes A1 and A2, nothing is known about how the activity of the enzyme is regulated. In the evolutionarily related lysosomal [alpha]-glucosidase; however, alterations in a single amino acid residue located well outside the catalytic site have been shown to ablate enzyme activity (Lin and Shieh, 1995). It is also possible that alternative splicing of glucosidase II might have repercussions on as yet unexamined retention and/or retrieval mechanisms operating to maintain the enzyme in the ER. In this context it is interesting that an isoform of the human invariant chain protein has been identified that lacks an ER retention signal due to alternative initiation of translation (Schutze et al., 1994). While glucosidase II has been shown to localize primarily to the ER in rat and pig hepatocytes (Grinna and Robbins, 1979; Brada and Dubach, 1984; Lucocq et al., 1986), there is also evidence to indicate that in some cell types GII[alpha] (Brada et al., 1990) and GII[beta] (Li et al., 1996) are capable of trafficking to the cell surface. Studies focused on the functions of the alternatively spliced domains of glucosidase II may thus reveal additional levels of biological complexity stemming from isoform heterogeneity.

Materials and methods

cDNA and genomic DNA sequencing

cDNA clones of mouse GII[alpha] and GII[beta], obtained previously (Arendt and Ostergaard, 1997), were subjected to automated sequencing by the dideoxy chain termination method. 3[prime] PCR-RACE clones of GII[beta] were derived using the gene-specific primer 5[prime]-CTCATTGAGGAGTGGAAGACAGCC and reaction conditions identical to those used to obtain 5[prime] PCR-RACE clones (Arendt and Ostergaard, 1997). Genomic DNA was extracted from 2 × 107 SAKRTLS 12.1 (SAKR) mouse T-lymphoma cells (Arendt and Ostergaard, 1997) using the QIAamp Tissue Kit (QIAGEN). Genomic DNA at 2-4 µg/ml was amplified in PCR reactions catalyzed by 50 U/ml Taq polymerase (Promega) in the presence of 1.5 mM Mg2+, 50 mM KCl, 0.1% Triton X-100, 10 mM Tris-HCl (pH 9), 0.2 mM each dNTP, and 0.4 µM each forward and reverse primer. A 1.4 kb genomic DNA fragment containing box [Agr]1 was amplified from SAKR DNA using the primer pair 5[prime]-TCCTGCTCAGTGTCAATGCCCG (forward) and 5[prime]-CACCATCCCCAGGTGTTGCTTC (reverse) and reaction parameters of 20 s at 94°C, 90 s at 65°C, and 100 s at 68°C (5 cycles), followed by 20 s at 94°C, 90 s at 60°C, and 100 s at 68°C (25 cycles), with a final 10-min extension at 68°C. A 1.1 kb genomic stretch containing box A2 was amplified for 20 s at 94°C and 90 s at 68°C (25 cycles), followed by a 10 min extension at 68°C, using primer pairs 5[prime]-GCTCCTGGCACACAGCTTTCATCG (forward) and 5[prime]-TGTCTGTCTGTGGAGTCTCCCCAG (reverse). A 0.6-kb genomic fragment containing box B1 was amplified with the primer pair 5[prime]-AGACTGACACCACCTCCTTCTATG (forward) and 5[prime]-TCTCCTCATCATAGGGCGGCATC (reverse) for 30 s at 94°C, 90 s at 61°C, and 30 s at 68°C (5 cycles), followed by 30 s at 94°C, 90 s at 58°C, and 30 s at 68°C (25 cycles), with a final 10 min extension at 68°C. PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN), followed by ethanol precipitation. Automated sequencing was performed using primers flanking the putative splice junctions. To confirm the results of this analysis and clarify any sequence ambiguities, the PCR amplicons were additionally cloned with the TOPO TA Cloning Kit (Invitrogen) to allow for sequencing of plasmid DNA.

Polymerase chain reaction analysis of isoform distribution

The RNeasy Kit (QIAGEN) was used to extract high quality RNA from fully viable cultures of the following cell lines, maintained as described previously (Arendt et al., 1995): SAKR, BW5147 (BW), and clone CTL AB.1. RNA was also extracted from PHAR2.7, a glucosidase II-deficient derivative of BW generously provided by Dr. Ian Trowbridge (Salk Institute, La Jolla, CA). Total cellular RNA was reverse transcribed by Super-script II (Life Technologies) using oligo(dT) (Life Technologies) or random (Stratagene) oligonucleotide primers. Following the RT reaction, RNA was digested by incubation with 2 U of RNase H (Promega). cDNA derived from 475 ng of total RNA was analyzed for GII[alpha] and GII[beta] isoform usage by PCR in 50 µl reactions employing buffer conditions and primer pairs identical to those employed in the genomic amplifications. For amplification of boxes A1 and A2, the cycling parameters were set to 30 s at 94°C and 120 s at 68°C (5 cycles), followed by 30 s at 94°C, 90 s at 64°C, and 30 s at 68°C (5 cycles), then 30 s at 94°C, 90 s at 60°C, and 30 s at 68°C (20 cycles), with a final 10 min extension at 68°C. Box [Bgr]1 was amplified under similar conditions, but using annealing temperatures of 58°C, 56°C, and 54°C. PCR reaction products were visualized by ethidium bromide staining of gels containing 2% MetaPhor Agarose (FMC BioProducts) or 1.5% Ultrapure Agarose (Life Technologies).

Acknowledgments

We thank Dr. Ian Trowbridge for providing the PHAR2.7 mutant and gratefully acknowledge the excellent sequencing work of the University of Alberta Department of Biochemistry DNA Sequencing Facility. This work was supported by the Medical Research Council of Canada. C.W.A. is supported by a studentship from the Alberta Heritage Foundation for Medical Research. H.L.O. is a Senior Scholar of the Alberta Heritage Foundation for Medical Research.

Abbreviations

ER, endoplasmic reticulum; GII, glucosidase II; kb, kilobase pair(s); PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase.

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