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Human Molecular Genetics Pages 1417-1424  

Organization, expression and polymorphism of the human persyn gene
Introduction
Results
   Cloning and analysis of the human persyn genomic locus
   Persyn mRNA expression in tumour cell lines
   Persyn protein expression in human tissues
   Sequence polymorphisms in the human persyn coding region
   Assignment of the human persyn gene to chromosome 10q23.2-q23.3
Discussion
Materials And Methods
   Molecular cloning
   Nucleotide sequence analysis
   Localization of persyn genes on human metaphase chromosomes
   RT-PCR analysis of the human persyn mRNA
   PCR analysis of the human persyn gene exons
   Western blotting
   Immunocytochemistry
Acknowledgements
Abbreviations
References

Organization, expression and polymorphism of the human persyn gene

Organization, expression and polymorphism of the human persyn gene

Natalia N. Ninkina1,2, Maria V. Alimova-Kost3, James W. E. Paterson1, Liz Delaney1, Brian B. Cohen1, Stefan Imreh3, Nikolai V. Gnuchev2, Alun M. Davies1,4 and Vladimir L. Buchman1,*

1School of Biomedical Sciences, University of St Andrews, Bute Medical Buildings, St Andrews, Fife KY16 9TS, UK, 2Institute of Gene Biology, Russian Academy of Sciences, 34/6 Vavilov Street, Moscow B-334, Russia, 3Microbiology and Tumor Biology Center (MTC), Karolinska Institute, Box 280, S-17177 Stockholm, Sweden and 4Neuropa Ltd, Robertson Building, Dumbarton Road, Glasgow G11 6NU, UK

Received April 23, 1998; Revised and Accepted June 18, 1998

DDBJ/EMBL/GenBank accession nos AF017256, AF037207

Persyn is a recently identified member of the synuclein family with a distinct pattern of expression during pre- and postnatal development of the mouse peripheral and central nervous systems. As with other synucleins, persyn is believed to be involved in the pathogenesis of human neurodegenerative diseases. However, in contrast to other synucleins, high levels of persyn mRNA expression were also found in advanced breast carcinomas, suggesting an involvement of the encoded protein in breast tumour progression. Here we have used an antibody specific to human persyn to demonstrate that the level of this protein is increased in ageing cerebral cortex and in breast tumours. We cloned, characterized and sequenced the human persyn genomic locus and localized it to the long arm of chromosome 10 in the q23.2-q23.3 region. Sequence information was used to search for specific mutations in the protein coding regions of persyn mRNA and the persyn gene in breast tumours and tumour cell lines. No tumour-specific mutations were found, but two linked polymorphisms in the coding region were detected, both in mRNA and exons III and IV of the gene. These results suggest that development of breast tumours correlates with overexpression of the wild-type persyn protein. Detailed characterization of the human persyn locus is important for further studies of the involvement of persyn in neurodegeneration and malignancy.

INTRODUCTION

Three genes of the synuclein family have been recently identified in the human genome. The first two code for [alpha]- and [beta]-synucleins, which are closely related presynaptic proteins that have similar patterns of expression and are most abundant in evolutionarily recent regions of the central nervous system (1-5). Both proteins are believed to be `natively unfolded' or random coiled (6,7) and tend to aggregate in solution, forming amyloid-like fibrils (8). Another important feature of synucleins is their ability to bind and trigger aggregation of amyloid A[beta]-peptides (9-11). These results, together with the detection of a peptide (NAC) derived from [alpha]-synuclein in the senile plaques of Alzheimer's brains (2,8), raise the possibility that synucleins are involved in the pathogenesis of Alzheimer's disease (12). Although mutations or polymorphisms in synuclein genes have yet to be correlated with Alzheimer's disease (13,14), two point mutations of the human [alpha]-synuclein gene were found in several families with early onset, autosomal dominant Parkinson's disease (15,16). These findings, together with the immunohistochemical detection of a high level of [alpha]-synuclein in Lewy bodies of Parkinson's disease patient brains (17,18), implicates [alpha]-synuclein in the pathogenesis of certain forms of this neurodegenerative disorder.

The most recently identified member of the synuclein family, persyn, shares all of the main structural features of [alpha]- and [beta]-synucleins, but has a distinctive pattern of expression (19). During mouse embryonic development, persyn is expressed only in primary sensory neurons and motoneurons. Postnatally, persyn expression remains high in these neurons and becomes detectable in sympathetic neurons and a subset of neurons of the cerebral cortex. In contrast to the decrease in [alpha]-synuclein expression in ageing cerebral cortex (20), persyn mRNA expression increases and persyn protein accumulates in the mouse cerebral cortex with age (19).

Although persyn is not present in senile plaques, Lewy bodies or neurofibrillary tangles in any of the clinical cases studied so far, a high level of persyn immunoreactivity is detectable in dot-like structures which are characteristic axonal lesions in the brains of patients with neurodegenerative diseases (V.L. Buchman, H.J.A. Hunter, L.G.P. Piñon, J. Thompson, E.M. Privalova, N.N. Ninkina, J. Lowe and A.M. Davies, submitted for publication). These clinical findings are consistent with the developmental and regional patterns of persyn expression and with experimental evidence which suggests that persyn plays a role in regulating neurofilament network integrity (19).

Persyn is nearly identical to a protein coded by an expressed sequence tag (EST), BCSG1, isolated from a breast cancer cDNA library (21). Although BCSG1 mRNA cannot be detected in normal breast tissue and in benign breast tumours, its level dramatically increases in highly infiltrating malignant carcinomas (21). If the level of the encoded protein reflects the level of persyn/BCSG1 mRNA, it could be useful as a breast cancer progression marker.

The dual role of persyn in neurodegeneration and malignancy could involve common mechanisms. Changes in organization of the cell cytoskeleton are among the most prominent characteristics of both processes. The involvement of persyn in regulating neurofilament network integrity raises the possibility that it may also affect the intermediate filament network in malignant breast epithelial cells. An important question is whether breast tumour progression correlates with a high level of expression of wild-type persyn or the presence of mutations in the persyn coding region is an obligatory characteristic of this process. Multiple differences (including three that resulted in amino acid substitutions) between BCSG1 isolated from tumour tissue and persyn isolated from normal brain tissue are consistent with the latter possibility. Such mutations could be somatic or germline and might reflect advanced tumour progression or predisposition of an individual to malignant progression of a tumour.

Clarification of these and other questions requires further study of the human persyn gene and persyn protein. Here we report the organization of the human persyn locus, its chromosomal localization and studies of human persyn protein expression. We also show that there are no mutations in the persyn gene specific for breast tumours, all confirmed differences being the result of natural sequence polymorphisms.

RESULTS

Cloning and analysis of the human persyn genomic locus

Two human persyn cDNA clones have previously been isolated from a juvenile human cDNA library. One of these cDNA clones (clone H1) was used to screen a human genomic library in [lambda]EMBL4 (22). Two identical, independent clones were isolated from this genomic library and the positions of the exons were determined by restriction analysis, hybridization with cDNA and oligonucleotide probes, direct sequencing of the locus and genomic Southern hybridization. Figure 1 shows that the human persyn gene consists of five exons that span ~5 kbp. There were no differences between the sequence of cDNA clone H1 used to probe the genomic library and the exonic sequences of the genomic clones isolated. However, in another shorter cDNA clone (4C) isolated from the same juvenile human cDNA library, two substitutions in the coding region were found (G->C at position 243 and T->A at position 377 of cDNA sequence AF017256, boxed in Fig. 1), the latter resulting in the amino acid substitution Val110Glu. Because the cDNA library was constructed from an individual juvenile brainstem, it is likely that these substitutions are the result of natural polymorphism of the persyn gene and that both alleles of the gene were transcriptionally active in this tissue.


Figure 1. Organization of the human persyn gene. Physical map of the locus is displayed on the upper part of the figure. The restriction endonuclease sites for EcoRI (E), SacI (S), KpnI (K) and XbaI (X) are shown. Exons (vertical black boxes) are numbered with Roman numerals. The sequences of exons and adjacent parts of introns are shown in the lower part of the figure. The encoded amino acids (one letter code) are shown below the nucleotide sequence. The nucleotide substitutions in EST BCSG1 are shown above the nucleotide sequence and the deduced amino acid substitutions are shown below the amino acid sequence. The two substitutions that were also found in human persyn clone 4C and are the result of gene polymorphism are boxed. The polyadenylation site is marked by an arrowhead. The full sequence of the human persyn locus is deposited in GenBank under accession no. AF037207.

Persyn mRNA expression in tumour cell lines

persyn gene expression in various normal and tumour tissues and cell lines was studied by northern blot analysis using the human persyn cDNA clone H1 as a hybridization probe. A single 0.8 kb transcript was observed in some tumour cell lines; the highest level of this transcript was detected in a sub-line (MCF7[prime]) of the breast tumour cell line MCF7, which does not normally express persyn mRNA (Fig. 2; 21). Similar results were obtained by semi-quantitative RT-PCR (not shown). However, by increasing the number of amplification cycles we detected a low level of persyn mRNA in tumour cell lines that were persyn-negative on northern and western blots (for example line GI101, as shown in Figs 2, 3a and 4).


Figure 2. persyn transcripts in breast tumour cell lines. Northern hybridization of total RNA isolated from tumour cell lines with a human persyn cDNA probe. After stripping off the probe, the same filter was hybridized with a nick-translated cDNA fragment encoding mouse GAPDH to provide an indication of the amount of total RNA from each cell line present on the filter. All cell lines are derived from breast cancers, except SKOV3, which is derived from an ovarian carcinoma.


Figure 3. Persyn protein in human tissues and cell lines. Western blot showing expression of persyn protein in human tissues and cell lines. Aliquots of 20 µg total cellular protein were separated on a 15% polyacrylamide gel, transferred to Hybond PVDF membrane and probed with SK109 anti-persyn antibody followed by ECL detection. Proteins were extracted from the following sources: (a) tumour cell lines (designations as in Fig. 2); (b) normal breast tissues (N) and breast tumours (T) from four patients; (c) cerebral cortices of two patients who suffered from Alzheimer's disease (12/95 and 256/96), two normal individuals from the same age group (109/95 and 197/96), fetal cerebral cortex, mouse spinal cord, unstimulated (PBL) and PHA-stimulated (PBL-PHA) peripheral blood lymphocytes.


Figure 4. Detection of persyn protein in breast cancer cells. Confocal microscope section of human breast cancer cells stained with antibodies against persyn (green channel) and keratin 8 (red channel).

Persyn protein expression in human tissues

To study persyn protein expression in human tissues we used western blotting with a polyclonal antibody raised against a synthetic peptide of human persyn protein (see Materials and Methods). This antibody does not cross-react with either [alpha]- or [beta]-synuclein and on western blots detects a single band that disappears if the antibody is preincubated with an excess of the immunizing peptide (not shown). Persyn was not detected in human fetal cerebral cortex, but was abundant in the cortex of elderly individuals (Fig. 3c). No substantial differences in the level of persyn were noticed in cerebral cortices of Alzheimer's disease patients and normal individuals of the same age group (Fig. 3c). Persyn mRNA and persyn protein were also detected in unstimulated and phytohaemagglutinin (PHA)-stimulated cultured lymphocytes from peripheral blood of normal donors (Figs 3c and 4 and data not shown).

In breast cancer cell lines the pattern of persyn protein expression corresponded to that of mRNA; it was detected by western blotting in the same cell lines in which persyn mRNA was detected by northern hybridization (Figs 2 and 3a).

It has been shown previously by in situ hybridization that persyn/BCSG1 mRNA is not expressed in normal adult breast tissue, but high levels of this mRNA are present in advanced infiltrating breast tumours (21). We detected persyn protein in breast tumours but not in normal breast tissue (Fig. 3b).

We used immunocytochemistry with an anti-persyn antibody to localize persyn in cultured breast cancer cells. In paraformaldehyde (PFA) fixed cells, persyn displayed punctuate cytoplasmic staining (Fig. 4), a pattern that is usually associated with markers of the endoplasmic reticulum or vesicular structures. Persyn was not co-localized with cytokeratin, actin or tubulin arrays in these cells (Fig. 4 and data not shown).

Sequence polymorphisms in the human persyn coding region

The nucleotide substitutions in the reported sequence of the BCSG1 transcript (21) are shown in Figure 1a. Two of these (at positions 243 and 377 of the cDNA sequence, boxed in Fig. 1a) are identical to the substitutions found in cDNA clone 4C. The other substitutions were not present in any of our clones and may be correlated with the transformed phenotype of the cells from which the BCSG1 EST was isolated. To study the presence and frequency of these mutations of the persyn gene in breast cancer cells, we carried out RT-PCR of mRNAs isolated from breast tumours and tumour cell lines. As a control, we used mRNA isolated from post-mortem cerebral cortices and PHA-stimulated peripheral blood lymphocytes of normal donors. The PCR amplification products were digested with restriction endonucleases HphI, StyI and MnlI. Using these digestions, four mutations in the coding region reported for BCSG1, including two polymorphisms present in cDNA clone 4C, could be detected. In all studied samples, digestion with MnlI generated the same pattern of fragments (Fig. 5a), which corresponds to the pattern predicted from the sequence of the persyn clones. Identical, predicted patterns were also obtained with StyI (not shown). These results reflect the absence of two G->A (and consequently Glu->Lys) substitutions reported for the BCSG1 EST in all studied tumour cell lines, tumours and control tissues. In contrast, when RT-PCR products were digested with HphI, three types of fragment patterns were revealed (Fig. 5a). The pattern represented in Figure 5a by the GI101 cell line corresponds to persyn transcripts with a sequence similar to H1 (G243, presence of HphI site, 120 bp but not 135 bp HphI fragment; T377, absence of HphI site, 155 bp but not 79 and 76 bp fragments). The pattern of MCF7[prime] in Figure 5a corresponds to persyn transcripts with a sequence similar to 4C (C243, absence of HphI site, 135 bp but not 120 bp HphI fragment; A377, presence of HphI site, 79 and 76 bp but not 155 bp fragment). PBL-N in Figure 5a is an example of cells in which both transcripts are expressed. Direct sequencing of RT-PCR fragments amplified from four persyn-expressing breast tumours did not reveal any alterations apart from G243C and T377A.


Figure 5. Detection of polymorphisms in the human persyn gene and its transcripts. Representative examples of different cell types (designations as in Fig. 4) and different combinations of persyn alleles are shown. The sizes of fragments are given in base pairs. (a) Ethidium bromide stained 4% MetaPhor agarose gels of the fragments amplified by RT-PCR from human RNA and digested with restriction endonucleases MnlI (upper) and HphI (lower). RNAs were isolated from breast cancer cell lines MCF7[prime] and GI101 and peripheral blood lymphocytes of donor N (PBL-N). (b) Ethidium bromide stained 2% agarose gels of the fragments amplified by PCR from human DNA and digested with restriction endonuclease HphI. The products of digestion of the 286 bp fragment that includes human persyn exon III are shown in the upper panel and those of the 330 bp fragment that includes human persyn exon IV are shown in the lower panel.

These data were confirmed by PCR amplification of individual exons of the persyn gene followed by digestion of the products with the restriction endonucleases HphI, StyI and MnlI. No polymorphic StyI and MnlI sites were found, but as illustrated in Figure 5b, the expected polymorphic HphI sites were present in exons three (G243C) and four (T377A) of the human persyn gene. Both alleles of the gene were expressed in heterozygotic cells (for instance in peripheral blood lymphocytes of donor N as demonstrated in Fig. 5a and b). As expected for the two nearby polymorphic sites in the genome, they were found linked in all studied DNA. The frequencies of these two alleles were the same in genomes of breast cancer and normal cells (20% G243/T377 and 80% C243/A377).

Assignment of the human persyn gene to chromosome 10q23.2-q23.3

DNA of the [lambda]W6H genomic clone was labelled with biotin and used for fluorescence in situ hybridization (FISH) with metaphase chromosomes prepared from donor peripheral blood lymphocytes. The unique hybridization site was assigned to the long arm of chromosome 10 in the q23.2-q23.3 region (Fig. 6).


Figure 6. Localization of the persyn gene on human metaphase chromosomes using FISH. (a) Metaphase plate. The arrow shows specific hybridization signals with the [lambda]W6H probe on chromosome 10. (b) Magnified images of chromosome 10 from different metaphase plates. Two images are shown for each chromosome, the FISH signal (white arrow) and inverted DAPI banding [the black arrow shows the DAPI band (10q 23.2-23.3) that hybridized with the [lambda]W6H probe].

DISCUSSION

Members of the synuclein family have been implicated in the pathogenesis of Alzheimer's, Parkinson's and other neurodegenerative diseases (12,19,23-25). In some families with a hereditary form of Parkinson's disease, a point mutation in the gene coding for [alpha]-synuclein is believed to be responsible for development of the pathology (15,26,27). This finding suggests that mutations in the genes encoding members of the synuclein family could be linked to neurodegenerative conditions and underlies the need for further studies of these genes.

We cloned and defined the structure of the human gene encoding persyn, a recently identified member of the synuclein family (19). The general organization of this gene in terms of the positions of the exon-intron junctions and the size of introns is very similar to that of the mouse persyn gene (unpublished data). Although the complete structures of the other genes of the synuclein family have not been published, the available sequence data (15) and deduction of the positions of some of the exon-intron junctions from known splice variants of [alpha]- and [beta]-synucleins (1,4,19) suggest close similarities in organization of all synuclein genes.

In addition to its potential involvement in neurodegeneration, persyn is possibly involved in breast tumour progression. It has been shown that expression of an EST (BCSG1) that is nearly identical in nucleotide sequence to persyn mRNA is increased dramatically in infiltrating breast cancers compared with normal breast tissues and benign tumours (21). We have shown that expression of persyn protein also follows this pattern: no persyn could be detected by western blotting in normal breast tissues, whereas persyn protein was detectable in breast tumour tissue. In established breast cancer cell lines, the levels of persyn protein expression were found to be substantially different and correlated with levels of persyn mRNA. We did not find amplification of the persyn gene in the studied cell lines (data not shown). These observations suggest that persyn expression is regulated predominantly at the level of persyn gene transcription and/or persyn mRNA stability. Thus, breast cancer cell lines could be used for further studies of the mechanisms that regulate persyn expression and to investigate the physiological consequences of persyn overexpression. We have shown previously that experimental overexpression of persyn in cultured sensory neurons leads to dramatic changes in the neurofilament network (19). It will be interesting to see if acute overexpression of persyn has a similar effect on the intermediate filament network in epithelial cells. Our immunocytochemical data suggest that persyn could be associated with membranous structures in the cytoplasm of breast cancer cells. However, subcellular fractionation of neural tissues and breast cancer cells demonstrated the presence of persyn in cytosolic but not in particulate fractions (data not shown). These results are similar to results obtained in studies of [alpha]-synuclein, which is believed to be very loosely associated with synaptic vesicle membranes in presynaptic terminals (5,28-30). This is interesting in the light of the recent demonstration that [alpha]-synuclein is able to bind synthetic membranes in vitro. This association dramatically changes the protein secondary structure from natively unfolded to predominantly [alpha]-helical and perhaps alters the functional features of the protein (31). One of these features is the ability of both [alpha]- and [beta]-synuclein to inhibit phospholipase D2 (32). This inhibition is dependent on the bulk lipid concentration in the reaction, suggesting that interaction with the enzyme occurs on the membrane surface (32) and thus involves [alpha]-helical but not natively unfolded synucleins. As phospholipase D2 is implicated in various intracellular pathways, including regulation of cell growth and differentiation, it will be interesting to study the effect of persyn on the activity of this enzyme in neurons and tumour cells.

Substantial changes in persyn expression not only accompany breast tumour progression but also take place during nervous system development. In our current study we have shown that persyn is not expressed in human fetal cerebral cortex but is abundant in cerebral cortices of elderly individuals. This is consistent with our previous observation that persyn expression is increased in ageing mouse cerebral cortex (19). We did not observe substantial differences in the levels of persyn protein in cerebral cortices of patients that suffered from Alzheimer's disease and normal age-matched individuals. These results are in agreement with immunohistochemical data that implicate persyn in the axonal pathology of neurodegenerative conditions (dot-like structures in white matter) without significant changes in persyn immunoreactivity in cortical neurons (V.L. Buchman, H.J.A. Hunter, L.G.P. Piñon, J. Thompson, E.M. Privalova, N.N. Ninkina, J. Lowe and A.M. Davies, submitted for publication).

The important question is whether wild-type or mutated persyn protein is expressed in breast tumours. Sequence analysis suggests that the protein from tumour cells (BCSG1) is different from the protein expressed in the nervous system (cDNA clones H1 and 4C) and that coded by the genomic clone W6H. Substitution of two lysines by glutamates in BCSG1 changes the overall charge of the persyn molecule and disturbs EKTKEGV repeats that are highly conserved in the synuclein family. Such changes should have serious consequences for protein structure and function. However, the fact that BCSG1 is only an EST generated by RT-PCR amplification raises the question of how accurately the sequence of this clone reflects the sequence of the mRNA in tumour cells. We studied a number of breast tumours and tumour cell lines for the presence of mutations that underlie Lys->Glu amino acid substitutions in the human persyn sequence. Although both persyn mRNA and exons of the persyn gene were analysed by MluI and StyI digestion of PCR products, no such mutations were found in any of the studied tissues or cell lines. In contrast, using HphI digestion, we were able to detect two nucleotide polymorphisms that had been initially recognized as sequence differences between persyn cDNA clones H1 and 4C. These two linked polymorphic sites discriminate two alleles of the human persyn gene. Both alleles are transcriptionally active and are expressed with similar efficiency in heterozygotes. We did not find any obvious correlation between the presence of particular persyn alleles and pathological phenotype. Although a limited number of samples were analysed, the two alleles in breast tumours and tumour cell lines occur with the same frequencies as in normal cells. These data suggest that, at least in spontaneous breast tumours, the high level of wild-type persyn protein correlates with malignant phenotype and that the reported mutations in the BCSG1 EST are artefactual and may be the result of Taq polymerase errors.

We have mapped the human persyn gene to the long arm of chromosome 10 in the q23.2-q23.3 region. A few hereditary disorders for which affected genes are not yet known have been located in this locus. Taking into account the pattern of persyn expression in the sensory neurons of the peripheral nervous system and motoneurons of the central nervous system, it will be interesting to check the possible involvement of the persyn gene in infantile onset spinocerebellar ataxia with sensory neuropathy (IOSCA, SCA8) (33). Also, it is intriguing that the loss of heterozygosity (LOH) at 10q23 occurs at high frequency in different human tumours. Mutations in the PTEN/MMAC1 gene, which is located in the region, have been implicated in many breast carcinomas and germline mutations of this gene are believed to be associated with Cowden disease, a rare autosomal dominant familial cancer syndrome with a high risk of breast cancer (34). However, not all families and individuals with Cowden disease have mutations in the PTEN/MMAC1 gene; somatic mutations of this gene occur in only a small fraction of primary breast cancers and LOH in breast cancers does not necessarily involve the PTEN/MMAC1 locus (35-37). Because of the correlation between persyn expression and breast tumour progression it will be interesting to search for mutations in the coding and regulatory regions of this gene in hereditary and spontaneous forms of breast cancer. Defining the organization of the human persyn gene reported here is an important step towards genetic studies of the involvement of this gene in neurodegenerative and neoplastic disorders.

MATERIALS AND METHODS

Molecular cloning

Human persyn cDNA clones H1 and 4C were isolated from a juvenile brainstem cDNA library (Stratagene) by hybridization with a mouse persyn cDNA probe at low stringency as described previously (38). The H1 clone was labelled by nick-translation and used as a probe for screening a genomic library and for Southern and northern hybridizations as described previously (39). Construction of the human genomic library in [lambda]EMBL4 was described previously (22).

Nucleotide sequence analysis

EcoRI fragments of [lambda]W6H DNA were subcloned into the pGEM3 vector and both strands were sequenced using specific oligonucleotide primers. Cycle sequencing was carried out with ABI dRhodamine `Big Dye' terminators and AmpliTaq FS (ABI). Reaction products were analysed on the ABI Prism 377 DNA Sequencer.

Localization of persyn genes on human metaphase chromosomes

FISH of biotin-labelled [lambda] DNA with metaphase chromosomes obtained by standard techniques from PHA-stimulated human lymphocytes was accomplished as described elsewhere (38,40). The signals were analysed using a Zeiss Axiophot fluorescence microscope equipped with a cooled CCD camera (Hamamatsu) and the Adobe Photoshop software package.

RT-PCR analysis of the human persyn mRNA

Total cellular RNA was reverse transcribed and amplified as described earlier (41). The amounts of total RNA in samples were normalized by amplification of a 437 bp fragment of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. The primers for amplification of the human persyn coding region were as follows: 5[prime]-AGCAGCACAACCCTGCACAC-3[prime] and 5[prime]-TCTTCAGGTCATCCACGCTG-3[prime]. Amplification of a 441 bp fragment was carried out for 40 cycles: 95°C for 45 s, 58°C for 30 s and 72°C for 60 s. For cells with a low level of persyn expression, an amplified fragment was purified by agarose gel electrophoresis and re-amplified for another 20 cycles. Amplified fragments were digested with the appropriate restriction endonuclease and analysed in a 4% MetaPhor agarose gel (FMC).

PCR analysis of the human persyn gene exons

For analysis of the human persyn gene exons, human genomic DNA was amplified using primers located inside introns in proximity to exon-intron junctions. The conditions of amplification and restriction endonuclease analysis were similar to the conditions used for RT-PCR products, except that the annealing temperatures were 64°C for exon III and 60°C for exon IV. Primers 5[prime]-TGCGAGCCTGACTCCAGCAG-3[prime] and 5[prime]-GGTGTGGAGTGGAGTGATGC-3[prime] were used for amplification of exon III and 5[prime]-TTGAGGCCAGGGTAGACAAG-3[prime] and 5[prime]-CCACTCAGGTTCAGGGTTAG-3[prime] for exon IV. DNA fragments were analysed in 2% agarose-TBE gels.

Western blotting

Western blotting was used to detect persyn protein in cell lysates as described earlier (42). The rabbit polyclonal antibody SK109 against a synthetic peptide of human persyn (V.L. Buchman, H.J.A. Hunter, L.G.P. Piñon, J. Thompson, E.M. Privalova, N.N. Ninkina, J. Lowe and A.M. Davies, submitted for publication) was affinity purified on recombinant human persyn protein bound to NHS-activated columns (Supelco) and used at a dilution of 1:500. The secondary antibody was a horseradish peroxidase-linked donkey anti-rabbit IgG (diluted 1:2000; Amersham). ECL detection was carried out according to the manufacturer's protocol (Amersham).

Immunocytochemistry

Cells on culture dishes were washed with phosphate-buffered saline (PBS), fixed in 4% PFA, PBS at 4°C for 10 min, washed with TBT (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100), blocked with 5% goat serum in TBT followed by incubation with rabbit polyclonal SK109 anti-persyn antibody (1:50) and mouse monoclonal anti-keratin 8 antibody (MAB1600, 1:50; Chemicon) at 4°C for 16 h in 1% goat serum, TBT. For detection, TRITC-conjugated anti-rabbit and FITC-conjugated anti-mouse secondary antibodies were used in dilutions recommended by the suppliers (Jackson Laboratories).

ACKNOWLEDGEMENTS

Our thanks are due to Jim Lowe (Department of Histopathology, University of Nottingham) for tissue samples, Alex Houston (DNA Sequencing Unit, University of St Andrews) for automatic DNA sequencing and S. Earnshaw and J. Allan for photographic work. This work was supported by grants from The Wellcome Trust and The Royal Society.

ABBREVIATIONS

EST, expression sequence tag; FISH, fluorescence in situ hybridization; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LOH, loss of heterozygosity; PBS, phosphate-buffered saline; PFA, paraformaldehyde; PHA, phytohaemagglutinin.

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*To whom correspondence should be addressed. Tel: +44 1334 463282; Fax: +44 1334 463600; Email: vlb@st-andrews.ac.uk

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