The autosomal dominant cerebellar ataxias (ADCA) are a heterogeneous group of neurodegenerative disorders characterized by progressive degeneration of the cerebellum, brain stem and spinal cord (1). Clinically, ADCA has been divided into three groups: ADCA types I-III (1). In ADCA type I (ADCAI) progressive cerebellar ataxia is associated with ophthalmoplegia, optic atrophy, extrapyramidal signs and dementia. ADCA type II (ADCAII) always presents with pigmentary macular dystrophy but is variably associated with ophthalmoplegia, dementia or extrapyramidal signs (2,3). ADCA type III (ADCAIII) is often referred to as the `pure' cerebellar syndrome.
ADCAI is genetically heterogeneous, with five genetic loci, designated spinocerebellar ataxia (SCA) 1, 2, 3, 4 and 6, being assigned to five different chromosomes. Four ADCAI genes (SCA1, 2, 3 and 6) have been cloned and shown to contain an expanded CAG repeat in their coding sequence producing an elongated polyglutamine tract in the corresponding protein (4-7). In each case the expanded repeats are variable in size. Also, the expanded repeats are unstable and usually increase in size when transmitted to successive generations, explaining the anticipation phenomenon observed in ADCAI families. All ADCAI disorders share common features, i.e. a negative correlation between number of repeats and onset age and the appearance of symptoms above a certain repeat length specific for each disease gene. However, their pathophysiology differs depending on the location of the repeat in the gene. In contrast to ADCAI, both ADCAII and ADCAIII are most likely homogeneous disorders. The pure form ADCAIII was assigned to chromosome 11p-q11 (SCA5) but the corresponding gene has not yet been cloned (8).
Subsequent to the genetic fine mapping of SCA7 to the 12 cM region between D3S1300 and D3S1285 (12; Fig. 1), DNA diagnosis of ADCAII based on haplotype analysis was included in the genetic counseling program of members of the Belgian SCA7 family CA-1 (14). In total 16 at-risk individuals had DNA diagnosis of SCA7 and their DNA was analyzed with 12 simple tandem repeat (STR) markers located between D3S1300 and D3S1285, including four STR markers that we had not used before: D3S3631, D3S3571, D3S1228 and D3S3644 (Fig. 2). All individuals had a complete clinical work-up, which was done blind from the DNA data. Three individuals had been diagnosed previously as mildly affected (VI-27) or probably affected (V-23 and VI-29) (12,14). Their clinical work-up confirmed that they are very mild (V-23) to mildly affected (VI-27 and VI-29), which is in agreement with them carrying the risk haplotype (Fig. 2). Of the eight individuals that showed no symptoms, five did not segregate the risk haplotype (V-17, V-25, V-27, V-30 and VI-34) while the other three did (VI-31, VI-35 and VI-38). The latter three are young individuals aged 34, 28 and 24 years respectively at examination. Clinical examination of five other at-risk individuals showed that they are very mildly (V-26 and VI-33) to mildly (V-19, V-22 and V-24) affected and all five carry the at-risk haplotype.
Analysis of the haplotypes showed that the recombinant detected by D3S1285 in patient VI-27 (12) is also seen with D3S3644, reducing the candidate region further (Fig. 2). Also, a recombination has occurred between D3S1600 and D3S1287 in the asymptomatic individual V-30, mapping the SCA7 gene centromeric of D3S1600 (Fig. 2). Individual V-30 was 63 years old at examination; she had no complaints, a normal neurological examination and normal laboratory tests.
DNA from 22 yeast artificial chromosomes (YACs) selected for the region contained between D3S1300 and D3S1285 was tested by PCR amplification for the presence of the STR markers used in the genetic analysis of family CA-1 as well as for sequence tagged sites (STSs) and expressed sequence tags (ESTs) previously located in this region (15). A total of 23 markers were used and the YACs were ordered into a clone contig based on their marker content (Fig. 1). The order of the markers is in agreement with previously published YAC maps (11,15). However, our YAC contig map contains an additional four markers D3S2984, D3S3424, D3S4010 and D3S1228. With the exception of one marker, D3S3631, the order of the STR markers is also in agreement with the genetic map of this region (16). The location of D3S3631 between D3S1300 and D3S1312 has also been reported by David et al. (11).
From the YAC contig we selected two overlapping YACs, 882D9 and 830A12, for gene cloning experiments, since together they spanned the candidate region defined by the informative recombinants with D3S1312 and D3S3635 (11; Fig. 1). The size of the two selected YACs was determined by pulsed field gel electrophoresis. The size of YAC 830A12 was estimated at 1.5 Mb, which is much larger than the reported 660 kb (17). Also, in 882D9 two YACs were detected of 1.6 Mb and 840 kb respectively, which are most likely the result of co-cloning. Later we showed by hybridization that the SCA7 gene is contained within the 1.6 Mb YAC clone (data not shown).
Yeast DNA was prepared from both YACs 882D9 and 830A12, partially digested with Sau3AI and subcloned in the BamHI site of the exon trapping cosmid vector sCOGH6 (18). Cosmid clones containing human DNA inserts were identified by colony hybridization using the Alu consensus sequence (19). Alu-positive cosmid clones were subsequently hybridized with a radiolabeled (CTG)10 oligonucleotide. This resulted in selection of 41 Alu/CAG-containing cosmids clones, of which 33 were derived from YAC 882D9 and eight from YAC 830A12. Cosmid DNA was prepared, digested with Sau3AI, separated on agarose gel, blotted and hybridized with the radiolabeled (CTG)10 oligonucleotide. Eight cosmids were selected for further study since they contained different sized CAG-positive Sau3AI fragments. The selected cosmids were Sau3AI digested, plasmid subcloned and CAG-positive plasmids were selected by colony hybridization with the radiolabeled (CTG)10 oligonucleotide. Sequencing showed that in four plasmids the CAG-containing sequence was identical to a yeast sequence. The four remaining plasmids contained human DNA sequences. The sequence of the 600 bp Sau3AI insert fragment of one of these plasmids (cos14-1) showed homology with the (CAG)5CTGCAG-containing mRNA coding for the human FAN protein (GenBank accession no. X96586). All three remaining plasmids (cos5-10, cos18-3 and cos21-4) contained a 1.8 kb Sau3AI fragment; however, one plasmid contained an additional Sau3AI fragment of 1.4 kb, probably due to incomplete digestion of the cosmid DNA during plasmid subcloning (cos18-3). Sequencing showed that both ends of the 1.8 kb fragment are identical in cos5-10 and cos21-4 while only one end is identical in cos18-3. The complete sequence of the 1.8 kb Sau3AI fragment of cos21-4 was determined by transposon-based sequencing. Together with an internal 350 bp sequence of the partial 1.4 kb Sau3AI fragment in cos18-3, obtained by transposon-based sequencing, a genomic sequence of 2.18 kb was obtained containing an uninterrupted CAG repeat with 10 CAG units surrounded by a highly CG-rich sequence (Fig. 3A) (GenBank accession no. AF032102). Hybridization of the gridded 41 Alu/CAG-positive cosmids with the cos14-1 and cos21-4 plasmid inserts identified additional positive cosmids. In total 14 cosmid clones were positive for cos21-4 (all subclones from YAC 882D9), while cos14-1 recognized seven positive clones (five subclones from YAC 882D9 and two from 830A12).
To test whether one of the two CAG repeats identified was expanded in ADCAII patients we designed flanking primer pairs and PCR amplified genomic DNA of patients from family CA-1. PCR amplification of the cos14-1 CAG repeat produced only the expected PCR fragment (data not shown). PCR amplification of the cos21-4 CAG repeat from genomic DNA using primers H1 and H2 followed by agarose gel electrophoresis and hybridization with the radiolabeled (CTG)10 oligonucleotide showed not only the expected PCR fragment of 279 bp but also a longer PCR fragment that differed in size between patients (Fig. 4A). These data suggested that the CAG repeat in cos21-4 is expanded in SCA7 patients. To test this hypothesis we developed a fluorescence-based PCR test allowing analysis of CAG repeat fragments on an ABI automated sequencer equipped with GENESCAN software for size determination of the PCR fragments (Fig. 4B). First, we determined the polymorphic character of the CAG repeat in 50 control individuals. The size differences between polymorphic fragments were interpreted in terms of number of triplet repeats and were shown to vary from 4 to 18 CAG repeats, with 10 CAG repeats in the most common allele (80%). Next, we analyzed segregation of the CAG repeat in the complete CA-1 family (Fig. 2). In patients and carriers of the at- risk haplotype the number of CAG repeats varied from 38 to 55. In one patient, VI-85, with juvenile onset of ADCAII the exact size could not be determined, but was minimally 55 repeats. Furthermore, we observed a high degree of mosaicism in all expanded alleles from the analyzed patients (Fig. 4B). The location of the CAG repeat in the SCA7 candidate region was determined by PCR amplification of YACs from the YAC contig (Fig. 1). The CAG repeat mapped between markers D3S1600 and D3S1287, a location that is in agreement with the genetic mapping data obtained in family CA-1, mapping SCA7 centromeric of D3S1600.
The genomic sequence of the 2.18 kb fragment containing the SCA7 CAG repeat was subjected to a BLASTN analysis to identify homologous clones present in databases. Strong homology was obtained between the SCA7 CAG repeat and surrounding sequences (positions 1348-1581) in the 2.18 kb fragment (Fig. 3) with the NotI jumping clone J32Z180D, previously mapped to chromosome 3 (GenBank accession no. X95831). Also, two other regions within the 2.18 kb fragment showed significant homologies with ESTs: region 1 upstream of the CAG repeat with ESTs H41756, H40290 and H40285; region 2 downstream of the CAG repeat with EST AA099235 (Fig. 3A). Analysis of the 2.18 kb genomic sequence using the GRAIL program (20) predicted two putative exons of 324 and 69 bp respectively containing respectively the SCA7 CAG repeat and EST AA099235. Subsequent RT-PCR amplification of lymphoblast cDNA using primers H2 and 1220R (Fig. 3) produced a fragment of 404 bp. The cDNA clone 510718, corresponding to EST AA099235, was obtained from the UK HGMP resource centre. The cDNA had been isolated from a colon cDNA library (Stratagene z178a12r1). Restriction digestion identified an insert of 2.5 kb that was sequenced by transposon-based sequencing (Fig. 3B). The total length of the 510718 cDNA was 2461 bp, containing 137 bp of 3'-UTR but no CAG repeat (GenBank accession no. AF032103).
The 14 cosmids containing the SCA7 CAG repeat cloned in the exon trapping cosmid vector sCOGH6 (18) were used in an exon trapping experiment. Trapped products were obtained for seven cosmids and sequencing analysis revealed that two of them contained an identical 98 bp fragment which upon BLASTN analysis identified one EST, N30892, corresponding to a placental cDNA clone 258172. The other five trapped products were unique and ranged from 28 to 476 bp but did not identify homologies with known expressed sequences. The cDNA clone 258172 was obtained from the UK HGMP resource center andthe 750 bp insert was sequenced (GenBank accession no. AF032104). Sequence comparison identified an 89 bp overlap between clone 258172 and the SCA7 CAG exon (Fig. 3). Since the placenta cDNA library was cloned after NotI digestion, clone 258172 ended at the NotI site located 10 bp upstream of the SCA7 CAG repeat (Fig. 3), the same NotI site that is present in the NotI jumping clone J32Z180D. The trapped exon is located in the 3'-region of clone 258172, immediately upstream of the SCA7 CAG exon (Fig. 3B).
The sequences of cDNA clones 258172 and 510718 and of the RT-PCR products containing the SCA7 CAG repeat (Fig. 3B) were used to put together a consensus sequence for the SCA7 cDNA of 3465 bp, comprising an open reading frame (ORF) of 2727 bp, a 5'-UTR of 652 bp and a 3'-UTR of 137 bp. The SCA7 cDNA sequence was verified by RT-PCR sequencing of lymphoblast cDNA using four overlapping primer sets (GenBank accession no. AF032105).
Several lines of evidence had indicated that ADCAII is caused by a CAG triplet repeat expansion (3,14,21-23). This was confirmed by recent cloning of the SCA7 gene, containing an expanded CAG repeat in the 5'-coding region (13). We independently cloned the SCA7 gene using a combination of genomic sequencing, cosmid based exon trapping, cDNA sequencing and RT-PCR analysis.
We subcloned two overlapping YACs from the SCA7 region in a cosmid-based exon trap vector, sCOGH6. Human CAG-positive cosmids were selected and cosmids containing the SCA7 CAG repeat were used in exon trapping experiments. Also, the genomic sequence of a 2.18 kb CAG-containing fragment was determined by transposon-based sequencing. BLASTN analysis of the genomic and trapped sequences identified two ESTs, one colon cDNA clone 510718 and one placental cDNA clone 258172. Sequencing of the two cDNA clones together with RT-PCR sequencing of the SCA7 CAG repeat defined a consensus SCA7 cDNA of 3465 bp, with the CAG repeat located in an ORF of 2727 bp. The exon containing the SCA7 repeat is the first coding exon of the SCA7 gene and the SCA7 CAG repeat is translated into a polyglutamine stretch ranging from 38 to at least 55 amino acids in ADCAII patients of the Belgian SCA7 family CA-1 (12,14).
Sequence comparison with the published lymphoblast SCA7 cDNA sequence (13) showed that the coding sequence in our SCA7 cDNA was identical with the exception of 15 nt at the 3'-end of the ORF, which result in five different amino acids (VGNGL instead of PKARP). Also, the published 3'-UTR sequence is 734 bp long and completely different from the 137 bp sequence we obtained from colon cDNA clone 510718. However, sequencing of a 3'-RACE product obtained from lymphoblast RNA and of a fetal liver/spleen EST, H78392, identified by BLASTN analysis of our SCA7 cDNA consensus sequence, provided the same 137 bp 3'-UTR. Further, the 5'-UTR sequence contained within placental cDNA clone 258172 is 98 bp longer than in the published lymphoblast SCA7 cDNA (13). RT-PCR sequencing of lymphoblast cDNA confirmed the published 5'-UTR, showing the absence of this 98 bp exon and suggesting alternative splicing of 5'-UTR exons. From the position of the trapped 5'-UTR exon and the putative alternative spliced 5'-UTR exon we predict that the 5'-UTR of the SCA7 gene contains at least three exons. However, alternative splicing in the 5'-UTR of SCA7 will have to be demonstrated by analyzing 5'-UTR sequences by RT-PCR analysis of RNA obtained from different tissue or cell sources or by sequencing cDNA clones isolated from tissue-specific cDNA libraries.
The rationale behind the use of the sCOGH6 cosmid vector in our cloning experiments was that we could use the CAG-positive cosmids immediately in exon trapping experiments. Trapped exons would help us in the identification of the SCA7 gene since they can be used in screening cDNA libraries or EST databases. Although we were successful in trapping the 5'-UTR exon immediately upstream of the SCA7 CAG exon from two CAG-positive cosmids, our experiences suggest that there are a number of limitations to the use of the sCOGH6 trapping vector. First, although all selected cosmids were shown to contain the 1.8 kb fragment and thus had to contain the first exon of the SCA7 gene with the CAG repeat, this exon was never trapped. Also, the SCA7 CAG exon was not trapped together with the adjacent 5'-UTR exon from the two cosmids that were successful in the trapping experiment. However, the sequences of the intron/exon boundaries of the SCA7 CAG exon confirm the consensus splice site sequences. This observation is of particular importance if a similar strategy is followed in cloning CAG repeat-associated genes in other diseases, such as bipolar affective disorder (24) or spastic paraplegia (25). Also, the availability of the sCOGH6 cosmid sublibrary of YAC 882D9 was expected to be helpful in determining the exon/intron boundaries of the SCA7 gene by exon trapping of SCA7 cDNA-positive cosmids. We screened filters of the cosmid library with cDNA 510718 containing the ORF of the SCA7 cDNA and identified 10 positive cosmid clones, all different from our initial set of CAG-positive cosmids. However, analysis of these SCA7 cosmids showed that several of them had partially or completely lost their insert DNA, indicating that recombinant sCOGH6 clones are unstable. Instability had not been observed in previous experiments using an earlier version of the sCOGH cosmid vector, sCOGH2 (18,26). sCOGH6 is a derivative of sCOGH2 and was obtained by removal of the SVNeo gene and mutagenesis of the EcoRI site, upstream of the metallothionein-1 promoter. What exactly is causing the instability of recombinant clones in sCOGH6 is unclear.
The SCA7 gene was genetically localized in the 5 cM interval between D3S1600 and D3S3635 (13). We were able to refine the location of the SCA7 gene to the 2.5 cM interval between D3S1600 and D3S1287 (Fig. 1). This location is compatible with our previous mapping data (12), as well as with the observation in this study of an informative recombinant in one unaffected individual with D3S1600, mapping SCA7 centromeric of this marker (Fig. 2). When the SCA7 repeat was identified the recombinant individual was shown not to carry an expanded CAG repeat, mapping the recombinant event between D3S1600 and the SCA7 gene.
We developed a fluorescence-based PCR test for the SCA7 repeat allowing its analysis on an automated sequencer, in our case an ABI 373A system (Applied Biosystems). In the normal population the SCA7 CAG repeat ranged from 4 to 18 repeats, with 10 repeats as the most common allele (80%) (n = 50). In family CA-1 the SCA7 CAG repeat ranged from 38 to 55 repeats, with onset ages ranging from 20 to 65 years and inversely correlated with repeat length (Fig. 2). In one patient (VI-85) with an onset age of 3 years the SCA7 repeat was 55 repeats or longer, but its exact length could not be determined because of the low quality of the DNA sample. The range of SCA7 CAG repeat lengths and of onset ages is similar to that described by David et al. (13), who also found the majority of their patients having repeat lengths between 35 and 55, with only a few juvenile onset cases having longer repeats. Also, we observed somatic mosaicism for each expanded allele analyzed from leukocyte DNA. In seven affected parent/child transmissions in family CA-1 the mean increase in repeat length was 4.25 repeats (range 0-15 repeats). In four affected parent/child transmissions onset age decreased from a mean onset age of 60 years (range 56-65 years) in the parents to 31.2 years (range 21-36 years) in the affected children (VI-27, VI-29, VI-33 and V-21). The large decrease in onset age between two successive generations is hard to reconcile with the rather small increase in repeat length. However, in three of the four affected parent/child transmissions the onset age had been arbitrarily set at age at examination (VI-27, VI-29 and VI-30). These individuals received a complete clinical work-up within the framework of genetic counseling and were diagnosed with very mild to mild symptoms of cerebellar ataxia and retinal lesions in the absence of opthalmoplegia, decreased vibration sense and pyramidal signs. This active clinical evaluation at a young age may have biased onset age estimation in these children. On the other hand, all four affected parent/child transmissions were of paternal origin and it was previously shown that anticipation in SCA7 families is more pronounced in paternal transmission of ADCAII (3,14,27). Further, the large repeat in the juvenile case (VI-85) is also of paternal origin; however, we have not yet been able to analyse her father's DNA. Interestingly, the increment in repeat length (+15) is larger in this branch of the pedigree, suggesting that here other genetic factors contribute to gonadal mosaicism or that certain repeat lengths are more prone to expansion than others.
The SCA7 gene is a novel gene coding for a protein, ataxin-7, of unknown function that is ubiquitously expressed as a 7.5 kb transcript (13). Also, we detected a 7.5 kb transcript in various tissues of the brain upon Northern blot hybridization with colon cDNA clone 510718 containing the SCA7 cDNA ORF (data not shown). It was suggested, based on its amino acid composition and its nuclear localization (21,22), that ataxin-7 might be a potential transcription factor (13). However, more work, including functional studies in cellular and animal models, are needed to unravel the normal and pathological function of this new protein. Meanwhile, the availability of an easy test for SCA7 repeat expansion will be very helpful in differential diagnosis of ADCA patients and in delineating the ADCAII phenotype.
Family CA-1 is an extended SCA7 family of which the clinical and pathological characteristics have been described in detail (12,14). Following our more recent refinement of the SCA7 locus, DNA diagnosis was offered by the genetic counselor to affected and at-risk family members of family CA-1. Risk haplotypes were reconstructed using 12 STR markers located between D3S1300 and D3S1285. A counseling program similar to that used for Huntington's disease was followed. In addition, each individual who requested a DNA diagnosis received an extensive neurological examination followed by laboratory work-up, including brain-evoked potentials, electroretinography, visually evoked potentials, ophthalmological examination and magnetic resonance imaging (MRI). The clinical examination was done blind from the DNA analysis.
After informed consent, genomic DNA was isolated from whole blood and the STR markers were analyzed using published primers in PCR amplification. The PCR reaction was carried out in a total volume of 25 µl containing ~100 ng genomic DNA, 25 pmol each primer, 1.25 mM dNTPs, 20 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 and 1 U Taq DNA polymerase. One primer was chemically labeled with the fluorescent dye FAM or JOE (Applied Biosystems) and alleles were separated on a 6% polyacrylamide sequencing gel. The polymorphic alleles were analyzed on a model ABI 373A sequencer using GENESCAN 672 software (Applied Biosystems, Foster City, CA).
A total of 22 CEPH YACs were selected for the SCA7 candidate region, contained between D3S1300 and D3S1285, from three different databases (Baylor College of Medicine Genome center, http://kiwi.imgen.bcm.tmc.edu:8088/; Whitehead Institute for Biomedical Research, http://www.genome.wi.mit.edu/; the Généthon server, http://www.genethon.fr/genethon_en.html/) based on their marker content. YACs were obtained from CEPH (Paris, France) and the YAC Screening Center Leiden (Leiden, The Netherlands). The YAC clones were plated on selective agar plates and a single colony from each plate was grown in selective liquid culture medium for YAC DNA extraction using standard procedures.
YAC DNA was PCR amplified with the 12 STR markers used for the genetic analysis, D3S1239 and the trinucleotide repeat marker D3S4010. Further screening was performed with five STSs (D3S2984, D3S3424, D3S4003, D3S3395, D3S3068 and GATA-61E08) and with three ESTs (D3S3934, D3S3119 and D3S3913). Published primers were used in a standard PCR reaction. PCR amplifications were done in the presence of positive (human genomic DNA) and negative (water) controls and each marker was tested at least twice for each YAC/marker combination. YAC sizes were determined by pulsed field gel electrophoresis from embedded total yeast DNA. Pulsed field gel electrophoresis analysis was carried out using the CHEF Mapper XA apparatus (BioRad). Conditions for optimal separations were as determined by the embedded algorithm. YACs were visualized by ethidium bromide staining and their size was estimated using the chromosomes of Saccharomyces cerevisae YP148 as a size marker. YACs were also visualized after Southern blot hybridization with a radiolabeled Alu consensus probe or the SCA7 CAG repeat fragment of cos21-4.
High molecular weight yeast DNA embedded in 1% low melting point agarose was digested with the restriction enzyme Sau3AI using optimal conditions for partial digestion (28) after overnight incubation of the enzyme/DNA mixture on ice, generating fragments in the size range 20-100 kb. Yeast plugs were subsequently treated with agarase (Epicentre Technologies) according to the manufacturer's protocol. The partially digested yeast DNA was dephosphorylated with 1.5 U HK phosphatase (Epicentre Technologies)/µg DNA for 2 h followed by heat inactivation of the phosphatase for 20 min at 65°C. Next the DNA was ligated in a 1:10 molar ratio to sCOGH6 vector, digested with XbaI, dephosphorylated and digested with BamHI to generate the vector arms and the cloning site (17). After ligation the cosmid DNA was packaged using Gigapack gold II packaging extract (Stratagene) and recombinant cosmids were transfected into XL1-BlueMR (Stratagene).
For each YAC ~10 000 cosmids were plated, representing a 15-20 times coverage of the yeast genome. Cosmid clones containing human inserts were identified by colony hybridization with a radiolabeled Alu consensus probe and arrayed in 96-well plates. Gridded replica filters were prepared on Hybond-N+ membranes (Amersham) and were hybridized with [[gamma]-32P]ATP end-labeled (CTG)10 oligonucleotide. Cosmid DNA was isolated using the alkaline extraction method. Cosmid DNA was digested with NotI to determine insert size by agarose gel electrophoresis. The pGem3zf(-) vector (Promega) was digested with BamHI to completion and subsequently dephosphorylated with HK phosphatase (Epicentre Technologies) and purified. Cosmid DNA was digested with 2 U Sau3A I/µg DNA for 4 h and purified by phenol/chloroform extraction and ethanol precipitation. Sau3AI-digested cosmid DNA was ligated overnight to the BamHI-digested vector with 1 U ligase (Boehringer). The ligated DNA was dialyzed against 1× TE on VS 0.025 µm filters (catalogue no. VSWP 02500, Millipore) for at least 2 h. Aliquots of 2 µl dialyzed DNA were used to electroporate electrocompetent XL1-Blue cells, prepared according to Sheng et al. (29). Transformants were plated on ampicilin agar plates, prepared for colony hybridization and hybridized with [[gamma]-32P]ATP end-labeled (CAG)10 oligonucleotide. Hybridizations were done overnight in 7% SDS, 0.5 M Na2HPO4/NaH2PO4, pH 7.2, 1 mM EDTA. Hybridization temperatures were 58°C for the [gamma]-32P-labeled (CAG)10 oligonucleotide and 65°C for the [[alpha]-32P]-labeled probes. Filters were washed in 5× SSC (1× SSC is 0.15 M sodium chloride, 0.015 M sodium citrate) at 58°C for 15 min, followed by a 15 min wash at 58°C in 2× SSC and exposed to Kodak X-ray film for 4 h at room temperature for the oligonucleotide probes. [[alpha]-32P]-Probed filters were washed in 2× SSC, 1% SDS for 15 min, 1× SSC, 1% SDS for 15 min and 0.5× SSC, 1% SDS for 30 min, followed by overnight exposure to Kodak X-ray film. Plasmid DNA was prepared from an overnight 3 ml liquid bacterial culture with the High Pure Plasmid Isolation Kit according to the manufacturer's protocol (Boehringer Mannheim). The DNA was digested with Sau3AI to determine insert size by agarose gel electrophoresis.
Purified plasmid DNA was sequenced by PCR cycle sequencing using the fluorescent T7 dye termination system (Applied Biosystems) and the pUC/M13 forward and reverse primers.For transposon-based sequencing plasmid inserts were recloned in the pOCUS-2 vector supplied with the Locus-Pocus subcloning system (Novagen). Transformation, mating and analysis of the clones were performed according to the manufacturer's protocol.
RNA was isolated from ~107 EBV-transformed lymphoblast cells as described (30) and treated with RNase-free DNase (Promega). 3'-RACE products were obtained by nested PCR on first strand cDNA synthesized with the 3'-RACE system (Gibco BRL). A first PCR was done with the primers SCA7.3 (5'-TCCATCAAGAGGATGAGTGT-3') and the 3'-RACE adapter primer supplied with the kit. A nested PCR was done with primer SCA7.2 (5'-CATGAACAATGTCCACATGA-3') and UAP (Gibco BRL). The PCR product was gel purified and PCR cycle sequenced with primer SCA7.2. For the RT-PCR reactions first strand cDNA was synthesized by random priming using the Superscript II system (Gibco BRL) and PCR amplified using the primer set H1 and H2 or H2 and 1220R (Fig. 3). The SCA7 CAG fragments were size selected on 2% agarose gel, subcloned in pGem3zf(-) and sequenced. Verification of the lymphoblast cDNA sequence was done by RT-PCR using four overlapping primer sets. First strand cDNA was amplified with a standard PCR of 30 cycles using primer pairs: 562F and SCA7.H (5'-TGTGACTGTCCCTATCTTAAGG-3'); SCA7.8 and ctg3R (5'-GCATCCCATTCAGCAAAGTA-3' and 5'-CGTAATAGCCTCTTCCTATCTG- 3'); SCA7.6 and SCA7.E (5'-TAAACCTAAACCTCACACCC-3' and 5'-GCCAGATAGCTGACTCCACA-3'); SCA7.5 and SCA7.A (5'-TCTCCACACGTATTCCTCAC-3' and 5'-CCAAACACATTTCTGAGCTG-3'). The sequences of the amplification products were determined with cDNA primers 562F, H2, H1, SCA7.2, SCA7.8, ctg3R, SCA7.6, SCA7.5, H1IC (5'-CCTCTGCCCAGTCCTGAA-3'), SCA7.I (5'-ACAGAAACCAAATATTGGCA-3'), SCA7.7 (5'-TATTAGCCGAGCACAAAAAC-3'), SCA7.G (5'-CTACAAAAGGCTTTGATTCG-3'), SCA7.E (5'-GCCAGATAGCTGACTCCACA-3'), SCA7.4 (5'-AATCCAGCAAATCTTTGAGG-3'), SCA7.3 (5'-TCCATCAAGAGGATGAGTGT-3'), SCA7.2 (5'-CATGAACAATGTCCACATGA-3'), 562E (5'-ATTGCAGCAGATGAAGATCC-3') and 562A (5'-CTCGTTCGTCTTAATTCTGTTC-3'). Sequences were analyzed on an automated DNA sequencer model ABI 373A (Applied Biosystems) with the fluorescent T7 dye termination system (Applied Biosystem). The obtained cDNA sequences were aligned with DNAStar to generate the cDNA consensus sequence.
Exon trapping was essentially as published (18), with minor modifications. Cosmid DNA was purified by standard phenol/chlorofrom extractions. After electroporation (750 V, 900 µF) the V79 cells were transferred immediately to a 100 mm tissue culture dish containing 10 ml prewarmed DMEM + 10% FCS, to minimize cell death. To avoid DNA contamination of the exon trap products total RNA was first pretreated with RNase-free DNase (Promega). First strand cDNA synthesis was performed with primer hGHex4/5 (5'-CTTCCAGCCTCCCCATCAGC-3'), spanning two exon/intron boundaries. Aliquots of 5 µl first strand cDNA were used as template in a standard PCR reaction with primer hGHex1/2 (5'-ATGGCTACAGGCTCCCGGAC-3').
Aliquots of 100 ng genomic DNA was mixed with 10 pmol primers H1 and H2 (Fig. 3) in a total volume of 25 µl, containing 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 0.25 mM dNTPs, 0.05% W1 (Gibco BRL), 1 mM MgCl2, 10% DMSO and 1.5 U Taq DNA polymerase (Gibco BRL). Samples were denatured at 94°C for 2 min followed by 30 cycles of denaturation (94°C, 1 min), annealing (54°C, 1 min) and extension (72°C, 2 min) and a final extension at 72°C for 10 min. PCR product were analyzed on a 2% agarose gel, blotted onto Hybond-N+, hybridized with a radiolabeled (CTG)10 oligonucleotide, washed and exposed to Kodak X-ray film for 4 h at room temperature. In the case of fluorescent detection of the SCA7 CAG repeat the H2 primer was chemically labeled with the fluorescence dye JOE and PCR fragments were separated on a 6% polyacrylamide sequencing gel. The SCA7 repeat length was determined on a model ABI 373A sequencer using GENESCAN 672 software (Applied Biosystems) with the GENESCAN 2500ROX marker as internal size standard.
The authors are grateful to the patients and family members of family CA-1 for their cooperation in the research project, to Nicole Datson and Johan den Dunnen for providing the sCOGH6 vector and to Marleen Van den Broeck for help with the sequencing. The work described in this paper was in part funded by the Fund for Scientific Research-Flanders (Belgium), a research project of the University of Antwerp, Belgium and an EU grant BIOMED2 CT97-2466. L.K. and J.T. are grant holders of the Institute for Science and Technology.
Human Molecular Genetics Pages
Introduction
Results
DNA diagnosis in family CA-1
Physical mapping of the SCA7 region
Isolation of CAG-positive cosmids
Identification of the SCA7 CAG repeat
Isolation of SCA7 cDNA
Exon trapping of SCA7 cosmids
Discussion
Materials And Methods
Genetic and clinical studies
YAC contig mapping
Subcloning in sCOGH6
Identification of CAG-positive clones
Sequencing
Exon trapping
PCR amplification of the SCA7 repeat
Acknowledgements
References
REFERENCES
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 6 Jan 1998
Copyright© Oxford University Press, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Latouche, C. Lasbleiz, E. Martin, V. Monnier, T. Debeir, A. Mouatt-Prigent, M.-P. Muriel, L. Morel, M. Ruberg, A. Brice, et al. A Conditional Pan-Neuronal Drosophila Model of Spinocerebellar Ataxia 7 with a Reversible Adult Phenotype Suitable for Identifying Modifier Genes J. Neurosci., March 7, 2007; 27(10): 2483 - 2492. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Shukla, P. Bajwa, and S. R. Bhaumik SAGA-associated Sgf73p facilitates formation of the preinitiation complex assembly at the promoters either in a HAT-dependent or independent manner in vivo Nucleic Acids Res., December 4, 2006; 34(21): 6225 - 6232. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. McMahon, M. G. Pray-Grant, D. Schieltz, J. R. Yates III, and P. A. Grant Polyglutamine-expanded spinocerebellar ataxia-7 protein disrupts normal SAGA and SLIK histone acetyltransferase activity PNAS, June 14, 2005; 102(24): 8478 - 8482. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. D. Einum, A. M. Clark, J. J. Townsend, L. J. Ptacek, and Y.-H. Fu A Novel Central Nervous System-Enriched Spinocerebellar Ataxia Type 7 Gene Product Arch Neurol, January 1, 2003; 60(1): 97 - 103. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Cancel, C. Duyckaerts, M. Holmberg, C. Zander, G. Yvert, A.-S. Lebre, M. Ruberg, B. Faucheux, Y. Agid, E. Hirsch, et al. Distribution of ataxin-7 in normal human brain and retina Brain, December 1, 2000; 123(12): 2519 - 2530. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Yvert, K. S. Lindenberg, S. Picaud, G. B. Landwehrmeyer, J.-A. Sahel, and J.-L. Mandel Expanded polyglutamines induce neurodegeneration and trans-neuronal alterations in cerebellum and retina of SCA7 transgenic mice Hum. Mol. Genet., October 1, 2000; 9(17): 2491 - 2506. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Gu, Y. Wang, X. Liu, B. Zhou, Y. Zhou, and G. Wang Molecular and Clinical Study of Spinocerebellar Ataxia Type 7 in Chinese Kindreds Arch Neurol, October 1, 2000; 57(10): 1513 - 1518. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Yanagisawa, M. Bundo, T. Miyashita, Y. Okamura-Oho, K. Tadokoro, K. Tokunaga, and M. Yamada Protein binding of a DRPLA family through arginine-glutamic acid dipeptide repeats is enhanced by extended polyglutamine Hum. Mol. Genet., May 22, 2000; 9(9): 1433 - 1442. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. B. Martin Molecular Basis of the Neurodegenerative Disorders N. Engl. J. Med., June 24, 1999; 340(25): 1970 - 1980. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||