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Genomic organization of the murine polysialyltransferase gene ST8SiaIV (PST-1)
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
Genomic organization of the murine polysialyltransferase gene ST8SiaIV (PST-1)
Introduction
Polysialic acid (PSA) is a differentially expressed posttranslational modification of the neural cell adhesion molecule (NCAM). PSA-expression is highly regulated during development (Seki and Arai, 1993; Rutishauser, 1996) and changes in the amount of PSA seem to be important for plastic processes in the central nervous system (Kiss and Rougon, 1997). Recently, it has been demonstrated that PSA is necessary for the two paradigms used to determine activity-dependent synaptic plasticity, i.e., long term potentiation and long-term depression (Becker et al., 1996; Muller et al., 1996). PSA modulates the neurite outgrowth-promoting effect of NCAM (Doherty et al., 1990) and is believed to be essentially involved in axon fasciculation (Cremer et al., 1997).
Two enzymes, ST8SiaIV (PST-1) (Eckhardt et al., 1995; Nakayama et al., 1995; Yoshida et al., 1995) and ST8SiaII (STX) (Livingston and Paulson, 1993; Kojima et al., 1995a; Scheidegger et al., 1995), are able to synthesize PSA (for a recent review, see Kiss and Rougon, 1997). The acceptor structure recognized by both enzymes is NCAM that carries terminal sialic acid linked [alpha]2,3 or [alpha]2,6 to galactose (Kojima et al., 1995b; Mühlenhoff et al., 1996a,b). Additional enzymatic activities are not required to polysialylate NCAM under in vitro conditions (Kojima et al., 1995b; Mühlenhoff et al., 1996b; Nakayama and Fukuda, 1996).
The expression patterns of the polysialyltransferases closely parallel that of polysialylated NCAM (Kurosawa et al., 1997; Phillips et al., 1997). However, in the developing rat brain some neuroepithelial areas were found to be PSA-negative although mRNAs for both enzymes and NCAM were present (Phillips et al., 1997; Wood et al., 1997). In early developmental phases the two enzymes are expressed in the brain and in many non-neural tissues (Angata et al., 1997; Phillips et al., 1997). The level of ST8SiaII mRNA is thereby multiple times higher than the level of ST8SiaIV mRNA (Angata et al., 1997). In the adult, polysialyltransferases become restricted to discrete areas in the brain (Phillips et al., 1997). Northern blot and in situ hybridization studies were carried out to analyze the tissue-specific and developmental expression of these genes (Angata et al., 1997; Kurosawa et al., 1997; Phillips et al., 1997). Results arising from these studies are in part contradicting, especially with respect to the relative expression levels of the two genes. However, a direct comparison of the results is difficult, since different species were analyzed (human, mouse, and rat, respectively) and differences might be due to species specific expression patterns. Nevertheless, all studies clearly show that in PSA-positive tissues the amount of PSA correlates with the level of polysialyltransferase mRNAs expressed. Furthermore, the expression levels correlate with polysialyltransferase activities determined in different cell lines and during differentiation of P19 cells (Mühlenhoff, 1996; Kojima et al., 1996).
In contrast, data concerning the expression level of the proteins are not available. This fact together with the observation that the polysialyltransferases exhibit strong differences in their enzymatic activity in vitro (Kojima et al., 1997; Mühlenhoff et al., 1997), does not allow an evaluation to which extent an individual polysialyltransferase contributes to the polysialylation of NCAM. The situation is further complicated by the fact that very little information exists on the signal transduction pathways involved in the regulation and activity of polysialyltransferases. A recent study suggests regulation via an increase in the intracellular calcium concentration and activation of protein kinase C (Rafuse and Landmesser, 1996). To get further insight into the developmental and tissue-specific regulation of polysialyltransferases, the structural organization of the genes and the identification of regulatory elements is required. Yoshida et al. (1996b) reported the structure of the murine ST8SiaII gene and demonstrated that the basal promoter of 158 bp upstream from the transcription start site is sufficient to promote cell-type specific expression of ST8SiaII. In the present report, we describe the genomic structure of the murine ST8SiaIV gene and demonstrate considerable similarity to the organization of the ST8SiaII gene.
Results
Isolation and characterization of the ST8SiaIV gene
A genomic DNA bacteriophage [lambda]FIXII library of mouse strain 129/Sv was screened by hybridization with a digoxigenin labeled RNA probe complementary to the 2 kb hamster ST8SiaIV cDNA described previously (Eckhardt et al., 1995). Approximately 106 plaques were screened and five different lambda phages containing the 5[prime]-region, including the first three exons, and the 3[prime]-end of the gene were obtained. In order to identify the missing part of the gene, a digoxigenin labeled DNA probe of exon 4 was generated by PCR, and the phage library was rescreened. One positive clone was identified which was found to contain exon 4 and adjacent intronic sequences.
Phage clones were subjected to restriction mapping and Southern blot analysis. Fragments encoding exon sequences were subcloned into the vector pBluescript and the exact size of exons and the sequences of exon-intron boundaries were determined by sequencing. From these data the organization of the ST8SiaIV gene could be deduced as shown in Figure
Figure 1. Genomic structure of the mouse ST8SiaIV gene. A schematic representation of the exon/intron organization is shown at the top. Boxes represent exons; solid boxes correspond to sequences encoding amino acids of ST8SiaIV, open boxes indicate the noncoding regions at the 5[prime]- and 3[prime]-end. Restriction sites recognized by XbaI (X) and SacI (S) are shown. The size of intron 3 could be estimated by PCR to be approximately 23 kb. Based on Southern blot analysis of genomic DNA, the size of intron 4 is beyond 10 kb. The positions of genomic phage clones are indicated below.
The ST8SiaIV gene is divided into five exons, spanning over more than 55 kb. The exon-intron boundaries (Table I) were found to be consistent with the splice acceptor and donor consensus sequences (Breathnach and Chambon, 1981) and correspond to the GT-AG rule (Mount, 1982). All introns interrupt exons after the second nucleotide of a codon. Exon 1 and exon 2 encode the cytosolic amino-terminus and the transmembrane domain, respectively. The catalytic domain of ST8SiaIV is encoded by exons 3 to 5 (Table II). Exons 3 and 4 harbor the sialyl motif L, which is involved in CMP-sialic acid binding (Datta and Paulson, 1995). Exon 5 contains the sialyl motif S and the recently identified sialyl motif VS (Geremia et al., 1997).
Northern blot analysis of murine tissues gives an hybridization signal of approximately 5.5 kb (Eckhardt et al., 1995; Yoshida et al., 1995), suggesting a long 3[prime]-untranslated region, which is not contained in the published mouse ST8SiaIV cDNA sequence (Yoshida et al., 1995). To identify the polyadenylation site of the gene, 3[prime]-RACE was carried out using a primer deduced from the known sequence of exon 5 (corresponds to nucleotides +3467 to +3489). A single 1.6 kb PCR product was obtained and characterized by sequencing. The polyadenylation site was found to be located 3995 bp downstream of the stop codon. Polyadenylation occurs 18 nucleotides downstream of an AATAAA sequence (data not shown).
Table I. 
Exon
Size (bp)
Amino acids
Features
1
437
1-38
5[prime]-UTR, transmembrane domain
2
132
39-82
Stem region
3
258
83-168
Catalytic domain/L-motif
4
294
169-266
Catalytic domain/L-motif
5
4278
267-359
Catalytic domain/S-motif, 3[prime]-UTR
Identification of an alternative polyadenylation signal
Northern blot analysis of the murine cell line AtT20 using an RNA probe transcribed from the complete cDNA of hamster ST8SiaIV revealed not only the 5.5 kb mRNA normally found in murine tissues, but an additional 1.4 kb band (Figure
Figure 2. Identification of an alternative polyadenylation site in the ST8SiaIV gene. (A) Poly(A)+ RNA of neonatal mouse brain and the cell line AtT20 was resolved in a 1% agarose/formaldehyde gel and transferred to nylon membrane. ST8SiaIV mRNAs were detected by hybridization with a digoxigenin-labeled antisense RNA probe of hamster ST8SiaIV cDNA. While only a single band of 5.5 kb is detectable in mouse brain, two specific transcripts of 5.5 and 1.4 kb were observed in the murine cell line AtT20. The 1.4 kb mRNA results from usage of an alternative polyadenylation signal present in intron 4, shown in (B). (B) Partial sequence of exon 4 and intron 4 which is identical to the 3[prime]-end of a short ST8SiaIV cDNA isolated from AtT20 cells. This probably corresponds to the 1.4 kb mRNA. The poly(A)-tail was found to be added 13 nucleotides downstream of a typical polyadenylation site (underlined).
Figure 3. Mapping of the transcription start site by 5[prime]-RACE. (A) Poly(A)+ RNA from AtT20 cells was reverse transcribed and subjected to 5[prime]-RACE as described in Materials and methods. Nested PCR was performed using one of the three gene specific primers P1, P2, and P3. (B) Sequencing of the PCR products shown in (A) revealed two transcription start sites 324 and 204 nucleotides upstream of the ATG start codon. Positions of the primers used for reverse transcription (RT) and nested PCR (P1-P3) are indicated. Identification of the transcription initiation site and sequence analysis of the promoter region
To identify the transcription start site of the gene, we used the 5[prime]-RACE technique. mRNA from AtT20 cells was reverse transcribed and subjected to two consecutive PCRs as described in Materials and methods (Figure
Figure 4. Nucleotide sequence of the promoter region and exon 1 of mouse ST8SiaIV. The sequence was numbered relative to the translation start site. The two transcription start sites are indicated by arrows. Restriction sites and the position of primer ME119 used for the construction of the reporter gene plasmids are shown. The purine- and pyrimidine-rich regions are marked by dotted lines. Putative binding sites for Sp1, AP1, AP2, and PEA3 are underlined.
The promoter region (Figure
Table II. Demonstration of promoter activity
Murine and hamster cell lines with the phenotype NCAM+/PSA+ (CHO (Eckhardt et al., 1995) and AtT20 (Alcaraz and Goridis, 1991)) and NCAM+/PSA- (NIH-3T3 (Eckhardt et al., 1995) and a PSA-negative subline of AtT20 (unpublished observations)) were used to determine activity of the ST8SiaIV promoter. In Northern blot analysis the PSA-negative cell lines were shown not to express ST8SiaIV (Eckhardt et al., 1995, and data not shown). For the promoter studies a 1.4 kb sequence covering nucleotides -1609 to -162 of the ST8SiaIV gene was cloned into plasmid pGL2Basic in front of the luciferase gene. The resulting construct pGL2PST2 together with the ß-galactosidase expressing vector pCMVlacZ was transiently transfected into the cell lines mentioned above. Luciferase activity was determined 48 h later and normalized to [beta]-galactosidase activity. All cell lines tested showed significant promoter activity irrespective of whether they express PSA or not (Figure
Figure 5. Activity of the mouse ST8SiaIV gene promoter in different cell lines. Promoter/luciferase gene constructs are shown on the left. At the top, restriction sites used to generate the 5[prime]- and 3[prime]-truncated promoter fragments (see Materials and methods) and the transcription start site at -324 are indicated. CHO (solid bars), NIH-3T3 (open bars), PSA-positive AtT20 (striped bars), and PSA-negative AtT20 cells (gray bars) were transiently cotransfected with the plasmids indicated and pCMVlacZ. Luciferase activity was determined 48 h later and normalized to the [beta]-galactosidase activity. Results are expressed as the fold induction in luciferase activity compared to the activity obtained by transfecting the promoterless plasmid pGL2Basic. The data are representative of three independent experiments, each performed in duplicate.
Sialyltransferases form a family of at least 15 different members that transfer sialic acid in [alpha]2,3-, [alpha]2,6-, or [alpha]2,8-linkage to galactose or sialic acid (for review, see Tsuji, 1996). The genomic structure has been elucidated for six sialyltransferases (Svensson et al., 1990; Wang et al., 1990; Chang et al., 1995; Kitagawa et al., 1996; Kurosawa et al., 1996; Yoshida et al., 1996a,b). All are divided into several (4-10) exons, spanning over up to 100 kb. A related structural composition has been found for the polysialyltransferase genes ST8SiaII (Yoshida et al., 1996b) and ST8SiaIV (this study). ST8SiaIV and ST8SiaII are unique in comparison with other sialyltransferases because of their ability to polymerize sialic acid and because of their restricted substrate specificity. The only identified cellular substrate recognized by both polysialyltransferases is the neural cell adhesion molecule NCAM. Furthermore, in contrast to other sialyltransferases, where primary sequence similarity is restricted to the sialyl motifs L, S (Drickamer, 1993), and VS (Geremia, 1997), ST8SiaIV and ST8SiaII exhibit more than 60% identity throughout the catalytic domain (Eckhardt et al., 1995). Despite these similarities in structure and substrate specificity, the polysialyltransferases are differentially expressed with respect to cell types and developmental stages (Angata et al., 1997; Kurosawa et al., 1997; Phillips et al., 1997), suggesting separate signal transduction pathways and transcription factors to be involved in the regulation of these genes.
In addition to their high amino acid sequence homology, which probably reflects a close evolutionary relationship, the genomic structures of ST8SiaIV and ST8SiaII are very similar. Exon-intron boundaries between exons 2-5 of ST8SiaIV and exons 3-6 of ST8SiaII are located at identical sites and the sequences around the splice donor and acceptor sites show a high degree of similarity. Differences are found in the 5[prime]-region of the genes. While transmembrane and stem region of ST8SiaIV are encoded by a single exon, the corresponding part of ST8SiaII is split by a very long intron resulting in 6 exons for ST8SiaII (Yoshida et al., 1996b). As is the case for ST8SiaII, no TATA or CAAT boxes were found in the promoter region of the ST8SiaIV gene. The region around the transcription initiation sites has a high GC content (60%) and is enriched in CpG dinucleotides, i.e., shows the characteristics of CpG islands. Taken together, the promoter of ST8SiaIV exhibits the structural characteristics of other sialyltransferase genes including ST8SiaII (Yoshida et al., 1996b, and references therein).
Analysis of AtT20 cells using the 5[prime]-RACE technique revealed the presence of two transcriptional start sites at positions -324 and -204 relative to the translation start. This result is consistent with the observation that many promoters lacking a TATA box have multiple initiation sites (Geng and Johnson, 1993; Haun et al., 1993). Luciferase gene assays clearly demonstrated that the region from nucleotide -443 to -162 is sufficient to initiate transcription in PSA-positive CHO and AtT20 cells. However, the minimal promoter is also able to promote gene expression in the PSA-negative AtT20 subclone and in NIH-3T3 cells, albeit at a lower level. These results strongly suggest the existence of regulatory elements outside the 1.4 kb fragment tested. However, it seems worthwhile to mention that the artificial systems used to determine promoter activities do not respect the role of the chromatin structure (Smith and Hager, 1997). Promoter activity observed in ST8SiaIV negative cells may therefore result form the artificial assay system. In contrast, Yoshida et al. (1996b) recently demonstrated that the ST8SiaII basal promoter is sufficient for the differentiation specific regulation of ST8SiaII in P19 cells. Moreover, activity of the minimal promoter was undetectable in NIH-3T3 cells, which are negative for ST8SiaII and ST8SiaIV (Yoshida et al., 1996b).
Two functional Sp1 sites have been identified in the ST8SiaII minimal promoter (Yoshida et al., 1996b). Putative binding sites in the basal promoter of ST8SiaIV that could be involved in the expression of the gene also include one Sp1 site. Further support for the involvement of Sp1 sites in the regulation of polysialyltransferases comes from the observation that Sp1 expression in many tissues parallels the expression of the enzymes. Sp1 expression is high in lung, thymus, and fetal cells, but low in several adult organs, e.g., liver and kidney (Saffer et al., 1991). ST8SiaII and ST8SiaIV are highly expressed in different fetal tissues (Angata et al., 1997; Phillips et al., 1997) as well as in adult lung and thymus, but are low or undetectable in adult liver and kidney (Yoshida et al., 1995; Angata et al., 1997). Thus, the different expression levels of ST8SiaIV in these organs may in part be due to changes in the Sp1 level. However, the identification of the factors involved in the highly restricted cell-type-specific and developmental regulation of ST8SiaIV requires the analysis of further upstream elements and the application of analytical methods which allow to determine the promoter activity in its “In Vivo Environment.”
The sequence data reported in this paper have been submitted to the EMBL/GenBank data bank under accession numbers AJ223955 and AJ223956. Isolation and characterization of genomic and cDNA clones of ST8SiaIV
A [lambda]Fix II phage genomic library of mouse strain 129/Sv (Stratagene) was screened by hybridization with a digoxigenin-labeled RNA probe transcribed from the cloned hamster ST8SiaIV cDNA (Eckhardt et al., 1995). Hybridizations were performed overnight at 60°C in 5× SSC, 50% formamide, 7% SDS, 50 mM sodium phosphate, 1% blocking reagent (Boehringer Mannheim). Filters were then washed twice in 2× SSC, 0.1% SDS at room temperature and twice in 0.5× SSC, 0.1% SDS for 20 min at 65°C. Bound probes were detected by incubation with anti-digoxigenin Ig-alkaline phosphate conjugate (Boehringer Mannheim) and chemiluminescence detection using di-sodium-3-(4-methoxyspiro{1,2-dioxetane-3,2[prime]-(5[prime]-chloro)tricyclo-[3.3.1.13,7]decane}-4-yl)phenylphosphate (CSPD; Boehringer Mannheim) as a substrate. The phage library was rescreened with an exon 4 specific DNA probe, which was generated by PCR using the primers ME87 (5[prime]-CTCCTGTGGTGGAGTTCGCTGCTGATGTG-3[prime]) and ME88 (5[prime]-TCTGACTGCATGAATAAGTCTAAGTGATGGATAG-3[prime]) and a PCR DIG probe synthesis kit (Boehringer Mannheim). Hybridization with the DNA probe was performed under identical conditions as described above, except that the hybridization temperature was decreased to 37°C and stringency washes were performed at 55°C. Positive phages were plaque purified until all plaques gave positive hybridization signals. Phage DNA from positive clones was isolated from plate lysates using a lambda phage DNA preparation kit (Qiagen), and mapped by restriction analysis using restriction enzymes BamHI, EcoRI, XbaI, and SacI. Fragments containing exon sequences were identified by Southern blotting with digoxigenin labeled DNA probes, and subcloned into pBluescript SK(-) (Stratagene) for sequence analysis. Sequencing was done by the dideoxy chain termination method (Sanger et al., 1977) using a T7 DNA polymerase sequencing kit (Pharmacia). A [lambda]ZAP cDNA library of the murine cell line AtT20 was screened essentially as described for the genomic DNA library using a digoxigenin labeled ST8SiaIV hamster antisense RNA probe. To determine the length of intron 3 and 4 long range PCR was performed using the long template PCR system of Boehringer Mannheim. Primers used were: ME83 (5[prime]-GATTGACAGCCACAACTTTGTAATAAGGTGAGC-3[prime]) and ME84 (5[prime]-ACATCAGCAGCGAACTCCACCACAGGAGC-3[prime]) to amplify intron 3, and ME85 (5[prime]-TCCATCACTTAGACTTATTCATGCAGTCAGAGG-3[prime]) and ME86 (5[prime]-GTGAATTTCATCACAGAATCTGGTGGCAAGTG-3[prime]) to amplify intron 4. Southern blot analysis
Genomic DNA was isolated from AtT20 cells by proteinase K digestion and phenol/chloroform extraction (Sambrook et al., 1989). DNA was digested with restriction endonucleases BamHI, EcoRI, SacI, and XbaI. Samples (10 µg per lane) were electrophoresed in a 0.8% agarose gel and transferred to a nylon membrane using standard procedures (Sambrook et al., 1989). The membrane was hybridized with digoxigenin labeled DNA probes, prepared by PCR using exon specific primers. Hybridization and probe detection procedure was the same as described above for the phage library screening. Northern blot analysis
Total RNA was isolated from CHO cells by CsCl gradient centrifugation of guanidinium isothiocyanate lysates (Sambrook et al., 1989). RNA (5 µg) was electrophoresed in a 1% agarose/1 M formaldehyde gel in 20 mM MOPS (pH 7.0), 10 mM sodium acetate, 1 mM EDTA and transferred to a nylon membrane (Qiagen). Nylon filters were hybridized overnight at 65°C in 5× SSC, 50% formamide, 50 mM sodium phosphate, 7% SDS, 1% blocking reagent (Boehringer Mannheim) to a digoxigenin-labeled antisense RNA probe of the hamster ST8SiaIV cDNA (Eckhardt et al., 1995). After hybridization the filters were washed twice in 2× SSC, 0.1% SDS at room temperature for 5 min and twice in 0.1× SSC, 0.1% SDS at 65°C for 20 min. Bound probes were detected by chemiluminescence detection as described above. Rapid amplification of cDNA ends (RACE)
RNA was isolated from AtT20 cells and enriched for poly(A)+ RNA as described (Eckhardt et al., 1996). The RACE protocol follows the procedure of Frohman (1993). To amplify the 5[prime]-end of ST8SiaIV 1 µg mRNA was reverse transcript for 1 h at 42°C using the antisense primer RT (5[prime]-CTTATAGTGCAGATGGTCC-3[prime]; corresponds to nucleotides +41 to +23; +1 = A of the start codon) and Superscript II RNase H- reverse transcriptase (Gibco). Thereafter, primers were removed passing the sample through a Glassmax spin column (Gibco). The cDNA was tailed using dATP and terminal transferase (Boehringer Mannheim). Second strand synthesis was done using the primer ME44 (5[prime]-GCGGATCCTCGAGTCGACTTTTTTTTTTTTTTTTT-3[prime]) and Superscript II RNase H- reverse transcriptase (Gibco) for 30 min at 42°C. Again, excess primers were removed by using Glassmax spin column. The cDNA was subjected to PCR (Saiki et al., 1988) using Taq DNA polymerase, the forward primer ME45 (5[prime]-GCGGATCCTCGAGTCGAC-3[prime]), and the gene specific primers ME29 (5[prime]-CGTTTTCTAATTGAGCGCATC-3[prime]; complementary to nucleotides +20 to -1). Reaction conditions were: initial denaturation for 3 min at 94°C, followed by 36 cycles of 30 s at 68°C, 1 min at 72°C, 30 s at 94°C. One microliter of the PCR product was further amplified using the same forward primer (ME45) and the gene specific primers P1 (5[prime]-TGCGAAGGGAGCGTGGAGCCG-3[prime]; corresponds to nucleotides -102 to -122), P2 (5[prime]-GGCGGTTTCTTCTGCAGCTG-3[prime]; corresponds to nucleotides -289 to -308), and P3 (5[prime]-CCCTTTCCTCGCCGTAGCAG-3[prime]; corresponds to nucleotides -328 to -347), respectively. PCR products were gel purified using Qiaquick gel purification kit (Qiagen) and sequenced.
3[prime]-RACE was performed similar to the 5[prime]-RACE protocol. mRNA (1 µg) was reverse transcribed with the primer ME44 and primers were removed by centrifugation through a Glassmax spin column (Gibco). The cDNA was amplified using primer ME45 and the ST8SiaIV specific primer ME109 (5[prime]-ATCACTGCTCAATCCCTATCACG-3[prime], corresponds to nucleotides +3467 to +3489) for 40 cycles of 30 s at 68°C, 90 s at 72°C, 30 s at 94°C. The PCR product was digested with BamHI and XbaI and subcloned into pBluescript for sequencing. Construction of luciferase gene plasmids
To generate the luciferase reporter gene construct pGL2PST1, the 1.16 kb XbaI-SacI fragment of phage [lambda]14.1, which is located 120 bp upstream of the transcription start site was subcloned into the SmaI and SacI sites of pGL2Basic (Promega). A fragment containing sequence between nucleotide -848 and -161 was amplified by PCR using the primers ME75 (5-GGCGAGATCAGCTCATCAGC-3[prime]) and ME119 (5[prime]-CGAAGCTTGCTAGCTCTCCCGGTTCTCCAG-3[prime]), introducing NheI and HindIII restriction sites (underlined) to facilitate subcloning. The 280 bp SacI-NheI fragment of the PCR product was subcloned into the SacI and NheI sites of pGL2Basic to generate construct pGL2PST4, and into the same sites of pGL2PST1 to create plasmid pGL2PST2. Furthermore, plasmid pGL2PST3 was prepared by subcloning the 500 bp XhoI-HindIII fragment of the PCR product into the XhoI and HindIII sites of pGL2Basic. All constructs were confirmed by sequencing. Transfection and luciferase assay
Chinese hamster ovary (CHO) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) Nut Mix F12 (Gibco) supplemented with 5% fetal calf serum (FCS), 2 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin. AtT20(+) and AtT20(-), showing the phenotype NCAM+/PSA+ and NCAM+/PSA-, respectively, and NIH-3T3 cells were grown in DMEM with Glutamax (Gibco) supplemented with 5% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. All cells were maintained at 37°C in a 5% CO2 incubator. Approximately 2 × 105 cells were seeded on 35 mm cell culture dishes and transfected 24 h later using Lipofectamine (Gibco) following the instructions of the manufacturer. Cells were transfected with equimolar amounts of the luciferase gene plasmids constructs (~0.5 µg DNA) and 0.5 µg pCMVlacZ. After 2 days cells were lysed in 25 mM Tris-H3PO4 (pH 7.8), 2 mM EDTA, 2 mM DTT, 10% glycerol, and 1% NP-40. Aliquots of the lysate were analyzed for luciferase activity in 25 mM glycylglycine (pH 7.8), 15 mM MgSO4, 2 mM ATP, 250 µM coenzyme A, and 45 µM luciferine (Sigma) using a Berthold Multi-biolumat LB 9505C. To measure [beta]-galactosidase activity, aliquots of the lysates were incubated in 100 mM sodium phosphate (pH 7.0), 10 mM KCl, 1 mM MgSO4, 2 mM DTT, and 1 mg/ml o-nitrophenyl-[beta]-galactopyranoside. The reaction was stopped with Na2CO3 and absorbence was measured at 405 nm.
We thank Andrea Bethe for her expert technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft and Boehringer Mannheim. M. Eckhardt was supported by a postdoctoral fellowship of the Deutsche Forschungsgemeinschaft.
NCAM, neural cell adhesion molecule; PCR, polymerase chain reaction; PSA, polysialic acid; RACE, rapid amplification of cDNA ends; ST8SiaII, CMP-Neu5Ac:(Neu5Ac)nNeu5Ac[alpha]2,3(6)Gal [alpha]2,8 sialyltransferase II; ST8SiaIV, CMP-Neu5Ac:(Neu5Ac) nNeu5Ac[alpha]2,3(6)Gal [alpha]2,8 sialyltransferase IV.
5[prime]-Exon/splice donor
Size of intron
Splice acceptor / 3[prime]-exon
CTC ATC GG gtaaatgcat
Intron 1: 6 kb
tcttttcag A GAT GGT
Leu Ile Gly(38)
Asp Gly
GAG ATA AG gtgagtttct
Intron 2: 7.5 kb
ccaatacag G AAG AAC
Glu Ile Arg(82)
Lys Asn
GTA ATA AG gtgagcatct
Intron 3: 23 kb
tttcctcag G TGC AAT
Val Ile Arg(168)
Cys Asn
GTC AGA GG gtaagtggct
Intron 4: >10 kb
gtttcttag A TAC TGG
Val Arg Gly(266)
Tyr Trp
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
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