Human Molecular Genetics, 2002, Vol. 11, No. 13 1487-1496
© 2002 Oxford University Press
Strong aggregation and increased toxicity of polyleucine over polyglutamine stretches in mammalian cells
1Center of Human and Clinical Genetics, 2MCB1 and 3Neurology, LUMC, Leiden, The Netherlands
Received August 24, 2001; Revised April 9, 2002; Accepted April 22, 2002
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ABSTRACT |
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Expansion of a Glutamine (Gln) repeat above a specific critical size in certain proteins gives rise to aggregation-prone proteins that cause neurodegenerative disorders, such as Huntington's disease. However, proteins with long hydrophilic polyglutamine repeats are more frequently found in nature than proteins with long homogeneous repeats of other amino acids, such as hydrophobic (Ala)n and (Leu)n. To explore this finding, the effects of expression in mammalian cells of polyglutamine and polyleucine encoded by mixed DNA repeats were compared. It was found that polyleucine is significantly more toxic than polyglutamine. In addition, we show that polyleucine stretches display a high propensity for aggregation utilizing two complementary biochemical assays and that polyleucine stretches can also be detected by the monoclonal antibody 1C2, which specifically recognizes expanded pathogenic and aggregation-prone glutamine repeats. Together, these results suggest that polyglutamine stretches are in fact relatively well tolerated and that nature may select more strongly against DNA stretches that encode long hydrophobic homopolymeric amino acid stretches, such as polyleucine – possibly owing to their strong propensity for aggregation. In keeping with this notion, an increasing number of diseases are found to be associated with expansion of stretches of hydrophobic amino acids, including oculopharyngeal muscular dystrophy (OPMD), which is associated with expansion of a hydrophobic polyalanine stretch.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Expansions of glutamine (Gln) repeats above a specific critical size in certain proteins cause neurodegenerative disorders. The largest group of these diseases, including Huntington's disease (HD) and the spinocerebellar ataxias of types 1, 2, 3, 6 and 7, are associated with an expansion of a (CAG)n/(CTG)n repeat in the coding region of the gene (reviewed in 1). Proteins with an expanded Gln repeat have a high propensity for aggregation and can be found in inclusions both in model systems and in patient-derived tissues (1,2). The contribution of inclusions to polyglutamine-induced toxicity is not yet completely resolved. It is, however, generally believed that an altered, aggregation-prone, conformation of the mutant protein confers a dominant toxic phenotype on susceptible cells (1–3).
Studies of the occurrence of single amino acid repeats in proteins, nevertheless, revealed that repeats of hydrophilic amino acids, especially of Gln, are not rare in nature, in contrast to stretches of hydrophobic amino acids (see e.g. 4–6). The intriguing question thus remains of why amino acid repeats consisting of Gln, despite their toxicity upon expansion, are found relatively frequently, whereas several other homopolymeric repeats are quite rare. Since the instability of homogeneous [e.g. (CAG)n/(CTG)n] repeat sequences lies at the DNA level, there is no reason at this level why only proteins with long hydrophilic glutamine and, to a lesser extent, serine (Ser) repeats, are found. In principle, (CAG)n/(CTG)n could encode not only glutamine and serine, but also hydrophobic alanine (Ala) and leucine (Leu), and also cysteine (Cys) (which allows the formation of disulfide bonds), depending on the reading frame and the translated strand of the involved gene. In particular, the occurrence of proteins with homopolymeric stretches consisting of polyleucine and of polycysteine is infrequent.
In principle, various, not mutually exclusive, explanations can be given for these findings. Firstly, Gln and Ser stretches may encompass functional units and are therefore positively selected in nature. In agreement with this, various transcription factors contain polyglutamine stretches (see e.g. 7). A second possibility is that polyglutamine, despite being notorious for its toxicity in various neurodegenerative disorders, is in fact relatively well tolerated compared with other (long) homopolymeric amino acid repeats and that an even stronger selection occurs against other (long) homopolymeric stretches, such as hydrophobic polyleucine and polyalanine.
To address this question, we compared the properties of polyglutamine and polyleucine expressed in human cells. To overcome the pronounced instability of long homogeneous DNA repeats (see e.g. 8,9), mixed DNA-repeat constructs were designed, capable of encoding either (Gln)n or (Leu)n, for comparison. These were embedded in mammalian expression vectors. The proteins encompassing the homopolymeric amino acid stretches were subsequently analysed for their toxic and aggregation properties. The results suggest that nature may well select against genes with long (CAG)n/(CTG)n repeats encoding amino acids other than glutamine, especially hydrophobic stretches. In agreement with this proposal, extended computer searches facilitated by the various genome-wide sequencing projects corroborate the previous findings of a relatively preponderant occurrence of polyglutamine stretches, and the paucity of hydrophobic homopolymeric amino acid stretches, such as polyleucine.
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RESULTS |
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Localization and toxicity of polyglutamine and polyleucine in transfected human cells
To compare the properties of different homopolymeric amino acid stretches in mammalian cells, long mixed repeats were cloned in the mammalian expression vector pcDNA3.1. The two plasmids pcDNA3.1–(Gln)291 and pcDNA3.1–(Leu)291 express polyglutamine [(CAG-CAA-CAG-CAG-CAG-CAA-CAG)n reading frame, (Gln)291] or polyleucine [(CTG-CTG-TTG-CTG-CTG-CTG-TTG)n reading frame, (Leu)291] with C-terminal myc tags via the strong and ubiquitously expressed cytomegalovinus (CMV) promoter. In addition, green fluorescent Protein (GFP) derivatives of these vectors were constructed. The expression vectors were used for transfection of a variety of mammalian cells, including human osteosarcoma U-2 OS cells (10) and adenovirus-transformed human embryonic retinoblast 911 cells (11).
As a first approach to assess the consequences of expressing polyglutamine and polyleucine in mammalian cells immunofluorescence experiments were performed on U-2 OS cells transfected with plasmids encoding the myc-tagged constructs using a mouse monoclonal antibody 9E10 against the myc tag for detection of the polyglutamine and polyleucine proteins (Fig. 1) (12). A DAPI counterstain was performed to visualize the cell nucleus. Polyglutamine-expressing cells can contain one to five large aggregates 16–18 hours after transfection (Fig. 1A, on the left; see also 13), whereas in the case of polyleucine-expressing cells, the general pattern is that of a large number of speckles resembling small aggregates (Fig. 1A, on the right). No signals were observed when cells were analyzed transfected with vector alone or when incubations were performed in which the first antibody was omitted, and no small or larger aggregates were observed when cells were analysed transfected with vectors expressing unrelated proteins with a myc tag (results not shown).
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When the cells were analysed with the same technique at later time-points after transfection (24–72 hours), an increasing number of polyleucine-expressing cells showed a conspicuously different pattern, while polyglutamine-expressing cells did not change dramatically. Figure 1B shows the results of a representative experiment 24 hours after transfection. The vast majority of the polyleucine-expressing cells now displayed a rounded appearance (Fig. 1B, right panel) and highly aberrant DNA staining, i.e. a significant loss of DNA staining and/or shrunken appearance of the nucleus (Fig. 1B right panel; DAPI). In contrast, the majority of the polyglutamine-expressing cells at the same time-point appeared normal (Fig. 1B, left panel). Based on the intensities of the fluorescent signals per cell, there are no significant differences in the expression levels of the polyglutamine and the polyleucine proteins (see also Fig. 3).
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These results were quantified by counting the number of transfected cells that displayed highly aberrant DNA staining. For comparison, two different human cell lines, U-2 OS cells and 911, and two different transfection methods, the calcium phosphate and Fugene methods, were used. Three representative experiments, in which cells were counted 48 hours after transfection, are shown in Figure 1C. Analysis of U-2 OS cells after calcium phosphate transfection shows that the frequency of aberrantly DAPI-staining cells is significantly higher for polyleucine than for polyglutamine (Fig. 1C, on the left). Similar results were obtained when the same cells were transfected with the Fugene method (Fig. 1C, in the middle), or when 911 cells were transfected with the Fugene method (Fig. 1C, on the right).
To analyze the effects of expanded polyglutamine and polyleucine in another protein context, GFP fusion proteins encompassing long homopolymeric amino acid repeats were analysed in parallel with the GFP control protein. Figure 2 shows the results of a time-course experiment. At 6 hours after transfection, the majority of cells still displayed normal DNA staining and normal morphology, irrespective of the type of protein, GFP, (Gln)n–GFP or (Leu)n–GFP expressed (Fig. 2A). After 18 hours, the majority of cells expressing (Leu)n–GFP began to display aberrant DNA staining and a rounded morphology, whereas many cells expressing (Gln)n–GFP, while appearing normal, showed full-blown aggregates (Fig. 2B). In agreement with the myc-tag studies 24 hours after transfection, also in the case of polyleucine fused to GFP the majority of the cells displayed a rounded morphology and highly aberrant DNA staining (Fig. 2C). Together, these data show that cells expressing polyleucine quickly change from a healthy appearance to a rounded morphology with highly aberrant DNA staining, compared with the cells expressing expanded polyglutamine, and thus clearly argue for a stronger toxicity of polyleucine than polyglutamine.
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Analysis of protein aggregates containing polyglutamine or polyleucine proteins
Similar to polyglutamine accumulating in protein aggregates, the speckles observed with polyleucine most plausibly contain aggregated polyleucine proteins. Protein aggregation was further analysed using two additional and independent approaches: western analysis (see e.g. 14) and a membrane filter assay (15). For these approaches, cells were lysed under denaturing conditions in Laemmli sample buffer. This buffer solubilizes most cellular proteins, while leaving protein aggregates generally intact. For western analysis, estimated equal amounts of protein were analysed on a 10% PAGE gel. In this gel system, soluble proteins migrate according to their molecular weight, whereas proteins entrapped in large protein aggregates usually remain at the bottom of the slots or migrate only just into the stacking gel (see also 14). To analyse possible aggregate formation, both the stacking and the separating gel were blotted. As a first approach, we analysed extracts prepared from U-2 OS cells 24 hours after transfection with the polyglutamine- or polyleucine-expressing constructs. Electrophoresis was performed until the bromphenol blue marker reached the end of the gel, and the whole gel, including the stacking gel, was blotted. The myc-tagged polyglutamine and polyleucine proteins were detected with the antibody 9E10 against the myc tag. Neither the polyglutamine nor the polyleucine protein enters the separating gel (Fig. 3A, left panel), which suggests that these proteins accumulate efficiently in aggregates. As a control, the same blot was stripped and subsequently incubated with antibodies against an unrelated protein not known to aggregate. The putative tumour suppressor protein QM (Fig. 3A, middle panel) can be detected as a discrete band at the expected position (around 25 kDa). No signals could be detected in the slot region of the gel (see also Fig. 3A middle panel). Similar results were obtained when another protein, tropomyosin, was analysed (not shown). These results thus show the specific presence in aggregates of the myc-tagged polyglutamine and polyleucine proteins.
Extracts prepared from U-2 OS cells expressing either myc-tagged polyglutamine or myc-tagged polyleucine were also used in a membrane filter assay (15). This assay is based on the well-described difference in protein binding between cellulose acetate and nitrocellulose membranes. After dot-blotting on cellulose acetate, only proteins present in (large) aggregates are retained on the filter, whereas after dot-blotting on nitrocellulose, both soluble proteins and aggregates are retained. Two different amounts of the extracts were used for this assay. Both polyglutamine and polyleucine are retained on the cellulose acetate filter, independent of the quantity, underscoring the formation of both polyglutamine and polyleucine aggregates (Fig. 3A, on the right).
In addition, extracts were prepared 24 hours after transfection from U-2 OS cells expressing (Gln)n–GFP or (Leu)n–GFP and analysed in a western experiment using an antibody against GFP. A representative experiment is shown in Figure 3B. As expected, GFP alone migrates as a single band at the expected position (
27 kDa) in the gel, whereas (Gln)n–GFP shows a band in the separating gel and a signal in the slot region (Fig. 3B). The size of the polyglutamine protein is larger than the calculated size of around 56 kDa, which is in agreement with the anomalous migration of proteins with large expanded polyglutamine stretches (see e.g. 16). In the case of (Leu)n–GFP, all signals are around the border of the separating and the stacking gel, or in the slots, which indicates aggregated proteins. Therefore, also in the context of a larger protein (including the GFP moiety), polyglutamine and polyleucine are still capable of formation of protein aggregates, although apparently with somewhat lower efficiency [compare (Gln)n–GFP, which is partly present in the separating gel in Fig. 3B, with myc-tagged polyglutamine, which is exclusively present in the stacking gel/slot region in Fig. 3A]. In addition, the results underscore the results shown in Figures 1 and 2 that polyleucine is possibly more aggregation-prone than polyglutamine, since no soluble (Leu)n–GFP was detected in extracts prepared 24 hours after transfection with this technique, whereas at the same time point soluble (Gln)n–GFP could be detected (Fig. 3B).
Immunofluorescence studies with a monoclonal antibody 1C2 recognizing expanded polyglutamine repeats
Expanded polyglutamine stretches are known to display an altered protein conformation (see e.g. 1,2). The mouse monoclonal antibody 1C2 has the intriguing feature that it only recognizes the expanded, aggregation-prone polyglutamine stretches (17–19). As expected, this antibody efficiently recognizes expanded polyglutamine in U-2 OS cells 24 hours after transfection (Fig. 4A). A parallel analysis of the same transfection experiment with the antibody 9E10 against the myc tag, nevertheless, suggests that the antibody 1C2 does interact less efficiently with polyglutamine when it has converted to full-blown aggregates than the antibody 9E10 (compare the left panels of Fig. 4A and B). This result is in agreement with previous reports that, with some experimental protocols, 1C2 does not react, or reacts less efficiently, with expanded polyglutamine once it is present in full-blown (nuclear) aggregates (see e.g. 20). No signals above background were observed when the empty pcDNA3.1 vector was transfected (see Fig. 4A for control with 1C2 and Fig. 4B for control with 9E10). Since polyleucine stretches are also prone to aggregation, we tested the possibility whether these stretches could also be recognized by 1C2. As can be seen in Figure 4A, 1C2 is also capable of recognizing polyleucine-expressing cells. In contrast to the observations with polyglutamine, however, 1C2 seems capable of reacting only a subset of polyleucine-expressing cells. The majority of the 1C2-positive polyleucine-expressing cells 24 hours after transfection display only a slightly aberrant DNA staining and no rounded morphology, thus indicating early signs of toxicity (Fig. 4A), whereas in the parallel experiment with the anti-myc antibody 9E10, both of the rounded cells (the majority of cells; Fig. 4B, right panel on the left) and cells with a more normal shape can be detected (Fig. 4B; right panel on the right). These data suggest that there are structural similarities between polyglutamine and polyleucine, which can be recognized by the 1C2 antibody, but that 1C2 detects less efficiently more advanced forms of the aggregates.
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Occurrence of homopolymeric amino acid stretches in nature
Although targeted searches for the occurrence of homopolymeric amino acid stretches have already been reported, the rapid progress of the genome-wide sequencing projects now allows us to analyse the ocurrence of amino acid repeats on a unbiased genome-wide level in an increasing number of organisms. Searching the Saccharomyces cerevisiae YPD database for (Gln)n repeats with 20 or more amino acids results in eight hits (http://www.proteome.com/). Performing the same search for the other amino acid repeats that can be potentially encoded by (CAG)n/(CTG)n stretches depending on reading frame and translated strand in the involved gene, two hits are found for Ser and none for Leu, Ala or Cys (Table 1). A similar trend for the occurrence of long homopolymeric amino acid repeats is also found in higher eukaryotes (Table 1). In this context, it should be noted that the amino acid composition in percentage for the complete SWISS–PROT database indicates that the amino acids Ser, Leu and also Ala are more frequently present in proteins than Gln (http://www.expasy.ch/sprot/relnotes/relstat.html; Table 1). Furthermore, the longest consecutive stretch detected for Gln on searching the YPD database is 37, whereas the longest consecutive stretches found for Ser, Leu, Ala and Cys are 25, 19, 9 and 8, respectively. When the same search is performed for 15 or more or for 10 or more amino acids, similar results are obtained. These extended searches thus corroborate the previous findings of the preponderant occurrence of polyglutamine stretches, followed at some distance only by polyserine. These extended searches underscore the scarcity of hydrophobic homopolymeric amino acid stretches.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The documented deleterious effects of expansion of unstable homogeneous (CAG)n/(CTG)n repeats resulting in proteins with expanded glutamine stretches in various neurodegenerative disorders are at first sight inconsistent with the relatively preponderant occurrence of polyglutamine stretches in nature. In the same way, also searches for the occurrence of (CAG)n [or (CTG)n] stretches in, for example, human mRNAs yield a number of proteins with long Gln stretches (www.ncbi.nlm. nih.gov/genome/seq/HsBlast.html). Proteins encompassing long Ala, Leu or Cys stretches, which can also be potentially encoded by such DNA stretches, depending on reading frame and translated strand, are conspicuously lacking. Possible explanations for these findings include positive selection for polyglutamine and also (but to a lesser extent) polyserine, and/or negative selection against the other homopolymeric amino acid stretches. In agreement with the latter possibility, this study shows that polyleucine-expressing constructs are significantly more toxic than polyglutamine-expressing constructs when transfected into mammalian cells.
Since the actual expression levels of exogenously introduced proteins may influence toxicity, it is pertinent to note that the intensities of the signals in the immunofluorescence and the western experiments show no major differences in the expression levels of the polyglutamine and the polyleucine proteins. We note also that the use of C-terminally tagged (myc or GFP) fusion proteins by necessity implies that the detected proteins are in frame with the tags, i.e. that the polyglutamine and the polyleucine frames are indeed detected. This is important in light of the recent observation that some sequences (e.g. GAGAG or perfect CAG repeats) show +2 frameshifts, albeit at very low frequencies (21,22). Thus far, we have not obtained any evidence that translational frameshifting contributes to the observed toxicity of the polyleucine-expressing constructs (H.G. Kerkdijk, unpublished results). Together, these data are thus in agreement with the notion that more-hydrophilic amino acid stretches such as polyglutamine and polyserine are better tolerated than hydrophobic stretches such as polyleucine and polyalanine.
An important hallmark of expanded polyglutamine-induced toxicity is an alteration in conformation of the mutant protein, resulting in a significantly increased propensity for aggregation and subsequent accumulation in inclusions. In this study, we show that the introduction of polyleucine-encoding constructs in human cells, just as holds for the polyglutamine-expressing constructs also gives rise to the formation of protein aggregates as assayed by two biochemical methods, western and dot-blot assay, and in immunofluorescence experiments. The latter results suggest that there may be commonalities in the mechanisms of toxicity. In this respect, it is interesting that the monoclonal antibody 1C2, which interacts specifically with expanded, pathogenic and aggregation-prone polyglutamine stretches but not with short repeats (17–19), can also detect long polyleucine proteins. In the case of polyglutamine, 1C2 detects full-blown aggregates, but interacts more strongly with polyglutamine proteins that are aggregation-prone but have not yet converted to full-blown aggregates. For polyleucine, only cells seem to be recognized that display very early signs of toxicity, i.e. display only a slightly aberrant DNA staining. Given our total results, it is quite plausible that polyleucine can shift more efficiently or more rapidly to densely aggregated structures.
There are indications that other expanded, homopolymeric amino acid sequences may also confer new and toxic functions via similar mechanisms. For example, the disease oculopharyngeal muscular dystrophy (OPMD) has been found to be associated with expansion of a hydrophobic alanine (Ala) repeat (23,24). Unique intranuclear filament inclusions in skeletal muscle fibers are the morphological hallmark of OPMD, implying again that stretches of hydrophobic amino acids with a non-polar side-chain such as polyalanine, just as shown for polyleucine in this study, can also efficiently aggregate. In addition, expansion of a polyalanine repeat in the human and mouse homeobox protein Hoxd13 has been associated with synpolydactyly, characterized by abnormality of hands and feet (25,26). Interestingly, a Huntington's-disease-like CAG-repeat disorder has recently been described, which may be caused by either an expanded polyalanine or polyleucine stretch (27). Homopolymeric stretches other than polyglutamine may well have toxic properties already at shorter repeat length, since for polyglutamine the longest non-pathogenic consecutive stretches can be detected. In agreement with this view, OPMD is associated with alanine repeat expansions already when these exceed 10 copies, whereas most of the polyglutamine diseases are associated with glutamine expansions greater than 32.
In summary, it is likely that the expanded polyglutamine repeats associated with the neurodegenerative disorders are in fact relatively well tolerated compared with other homopolymeric amino acid repeat sequences, such as hydrophobic polyleucine and polyalanine. In addition, the observations that expression of several distinct (expanded) homopolymeric amino acid stretches induces protein aggregation and toxicity makes it likely that in the future more diseases that are caused by such expansions will be uncovered.
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MATERIALS AND METHODS |
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Construction of mixed-repeat sequences
Mixed CAG-CAA repeats constructed by annealing and ligating two complementary synthetic DNA oligonucleotides, 5'-G-CAG-CAA-CAG-CAG-CAG-CAA-CA-3' and 5'-TG-CTG-TTG-CTG-CTG-CTG-TTG-C-3', followed by subcloning in the pGEM-T Easy vector (Promega, Netherlands) of purified ligated fragments (28). One pGEM-T Easy vector clone, designated p98, contained a mixed repeat encoding 98 amino acid residues. This clone was used to generate derivatives of the mammalian expression vector pcDNA3.1 (InVitrogen, Netherlands) expressing (Leu)291 and (Gln)291 with C-terminal myc tags. In addition, pcDNA3.1 derivatives were generated that express (Leu)291 fused to the green fluorescent protein GFP-S65T (phGFP-S65T; Clontech, USA). Details of the construction procedure are available on request. All mixed repeats are stable upon propagation in Escherichia coli and can be efficiently and accurately replicated by Taq polymerase (data not shown), in contrast to the homogeneous repeats (see e.g. 8,9,29).
Culturing and transfection of mammalian cells
Human U-2 OS cells (a human osteosarcome cell line) (10) and adenovirus-transformed human embryonic retina 911 cells (11) were cultured on Dulbecco modified Eagle's medium (DMEM; Gibco) supplemented with fetal calf serum (Life Technologies BV, Netherlands) and antibiotics on cell culture dishes (Greiner, Germany). The cells were transfected at a density of approximately 30% with the calcium phosphate method essentially as described by van der Eb and Graham (30) or with the Fugene method according to the instructions of the manufacturer (Roche Diagnostics, Netherlands). For the immunofluorescence experiments, cells were grown on glass coverslips.
Immunofluorescence experiments
Immunofluorescence experiments were performed essentially as described in (31). As first antibodies, the mouse monoclonal 9E10 against the myc tag (12) or 1C2 (17) (Euromedex, France) were used at a dilution of 1 : 100. In the case of GFP, cells were fixed as described in (31). After the final rinsing step, the coverslips were mounted on glass slides in mounting medium containing DABCO (1,4-diazabicyclo(2,2,2)octane) and DAPI (4'-6-diamidino-2-phenylindole). Immunofluorescence was observed using an Olympus AX70 microscope (Olympus, NL). For counting experiments, at least 100 of the transfected cells as judged by a positive signal with the 9E10 antibody against the myc tag were analysed for aberrant nuclear DNA staining.
Preparation of extracts
Transfected cells were washed twice with PBS, subsequently Laemmli sample buffer was added, and the cells and debris were scraped from the dishes using a rubber policeman. Laemmli sample buffer was prepared as described in (32). The lysis buffer and cell debris were subsequently transferred to a 1.5 ml tube. DNA in the cell lysate was sheared by passing it 10 times through a needle using a l ml disposable syringe. Subsequently the lysate was boiled for 5 minutes. Extracts were centrifuged for 20 seconds at 10 000 r.p.m in an Eppendorf centrifuge. Extracts were stored in a -80°C freezer. The concentration of the protein extracts was estimated by analysing samples of the extracts by SDS–PAGE followed by staining with Coomassie–Brilliant Blue (32).
Western analysis
Estimated equal amounts of protein were loaded onto a 10% SDS–PAGE gel. The transfer of proteins to nitrocellulose filters (Schleicher and Schuell, Netherlands) was performed according to the procedure for wet electrophoretic transfer using transfer buffer 1 as described in (32). As a first antibody, the mouse monoclonal 9E10 against the myc tag (12) was used at a dilution of 1 : 1000. For the control Western, the rabbit polyclonal antibody QM (N-15) against the QM protein was used (Santa Cruz, USA) at a dilution of 1 : 1000. For the detection of GFP, the rabbit polyclonal antibody GFP–(FL) (Santa Cruz, USA) was used at a dilution of 1 : 1000. Proteins were detected with ECL according to the instructions of the manufacturer (Roche Diagnostics, Netherlands). The Cruz marker was used as molecular size marker (Santa Cruz, USA).
Membrane filter assay
The membrane filter assays were performed essentially as described in (15). Cellulose acetate was purchased from Schleicher and Schuell (Netherlands). Usually, an estimated amount of 10–30 µg protein in Laemmli sample buffer was used for the cellulose acetate filter assay. The polyglutamine and polyleucine proteins were subsequently detected via ECL as described under western analysis.
Computer searches
Proteins from different species were searched for the occurrence of homopolymeric amino acid stretches. Proteins in S. cerevisiae, S. pombe and C. elegans and humans encompassing long homopolymeric amino acid stretches were identified via searches of the YPD, PombePD, WormPD and HumanPSD databases, respectively (www.proteome.com/ databases/). Proteins in D. melanogaster containing such amino acid sequences were identified via a Berkeley Drosophila Genome Project (BDGP) pattern search using the curated database (www.fruitfly.org/seq_tools/patscan.html).
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FOOTNOTES |
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* To whom correspondence should be addressed at: MCB1/HCG, Sylvius Laboratory, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. Tel: +31 71 5276283; Fax: +31 71 5276075; Email: j.c.dorsman{at}lumc.nl
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