Bloom's syndrome (BS), a human recessive disorder associated with an increased risk of malignancy, arises through mutations in both alleles of the BLM gene, which was recently identified as a member of the RecQ helicase family. BS cells are characterized by an increased rate of sister chromatid exchange (SCE). However, a subpopulation of lymphocytes exhibiting a normal level of SCE is observed in some patients. It has been proposed that reversion to a low-SCE phenotype involves an intragenic crossing over between the paternal and maternal BLM alleles, generating a wild-type allele. In this study we characterize a new BLM mutation in a BS patient leading to the replacement, in the C-terminal region of Blm, of a highly conserved cysteine by a phenylalanine in codon 1036. Moreover, our data show that this patient also inherited a BLM allele carrying a mutation affecting its expression and that a somatic intragenic crossing over was involved in reversion to the low-SCE phenotype. Further, we show that both topoisomerase II[alpha] mRNA and protein levels are decreased in the high-SCE cells derived from this patient, whereas they are normal in the corresponding low-SCE cells. Altogether, our data led us to propose that besides its putative helicase activity, Blm could be involved in transcription regulation.
Bloom's syndrome (BS) is a rare human autosomal recessive disorder associated with growth retardation, immunodeficiency and increased risk of malignancy at an early age (1 ,2 ). Besides these clinical manifestations, BS cells exhibit a spontaneous mutation rate 10 times higher than normal cells and a number of cytogenetic abnormalities, including increases in chromosome breakage, symmetric quadriradial chromatid interchanges between homologous chromosomes and sister chromatid exchange (SCE) (1 ,2 ).Although most cells exhibit an elevated SCE rate, in about one in five BS patients normal levels of SCE are observed in a subpopulation of B and T lymphocytes (3 ,4 ).These low-SCE `revertant' BS cells have normal levels of both spontaneous chromosome breakage (5 )and mutation rate (6 ). It has been reported by Ellis et al. (4 )that cells with low SCE levels are found almost exclusively in BS-affected offspring of non-consanguineous matings, whose parental alleles were not identical by descent and therefore most likely carry different mutations in BLM. The observation that in some BS patients exhibiting a high-SCE/low-SCE mosaicism the polymorphic loci distal to BLM that are heterozygous in their high-SCE cells became homozygous in a subpopulation of their low-SCE cells led to the interpretation that an intragenic crossing over between the paternal and maternal BLM alleles generated a wild-type allele (4 ). Somatic crossing over point mapping helped identification of the BLM gene, which encodes a protein sharing motifs with the Escherichia coli RecQ helicase subfamily (7 ). Several lines of evidence suggest that Blm may in fact be the human homolog of Sgs1, the yeast homolog of RecQ helicase: (i) their sequence similarity extends beyond the helicase domains; (ii) their sizes are nearly identical, more than twice the size of E.coli RecQ and human RECQL; (iii) the position of the helicase domain is conserved; (iv) both their N-terminal domains contain two acidic amino acid clusters (8 ).Furthermore, mutations in either BLM or SGS1 confer a hyper-recombination phenotype to cells, whereas other recQ mutants exhibit decreased levels of recombination (8 ). It has been shown that Sgs1 protein interacts in vivo with both topoisomerase III (9 )and topoisomerase II (10 ), suggesting that Sgs1 could modulate their activity. DNA topoisomerases are nuclear enzymes providing the topological interconversions necessary for transcription, replication and chromosome segregation during both meiosis and mitosis (11 ,12 ). Although Blm function(s) is not established, it is tempting to speculate, on the basis of its primary structure, that, similarly to Sgs1, Blm may interact with topoisomerases. In addition, it has been suggested, on the basis of results showing decreased topoisomerase II activity in BS cells treated with bromodeoxyuridine, that topoisomerase II or a protein complexed with topoisomerase II is defective in BS cells(13 ).
In order to determine the molecular mechanism underlying the reversion of some BS lymphocytes to the low-SCE phenotype, we analyzed 11 highly polymorphic markers on chromosome 15 in DNA isolated from high-SCE fibroblast cell lines and from the corresponding low-SCE lymphoblastoid cell lines derived from two unrelated BS patients, 87 (AlFra) and PuCh (Fig. 1 ). This analysis was also performed on DNA isolated from total blood cells from patient PuCh and her parents. Concerning patient 87 (AlFra), the four loci distal to the BLM gene that were heterozygous in his high-SCE cells (D15S127, D15S130, D15S107 and D15S87) became homozygous in his low-SCE cells, confirming the results of Ellis et al. (4 ). Conversely, concerning patient PuCh, the two loci that were heterozygous in her high-SCE cells (IP15M9 and D15S107) remained heterozygous in her low-SCE cells (data not shown and Fig. 1 ). Diparental diploidy of chromosome 15 in patient PuCh was confirmed by analysis of the DNA isolated from her parents (Fig. 1 ).
To determine whether PuCh-L reversion to the low-SCE phenotype results from a somatic intragenic crossing over or from an alternative mechanism, we performed single strand conformation polymorphism (SSCP) analysis to characterize the mutation(s) of the BLM gene in the PuCh-F and PuCh-L cell lines. The only novel SSCP conformer identified was in the high-SCE PuCh-F cell line and corresponded to the segment amplified between positions 3143 and 3361 (Fig. 2 A). Direct sequencing of the RT-PCR products corresponding to this segment revealed an apparently homozygous base change at nucleic acid position 3181, consisting of a single base substitution (TGC -> TTC; Fig. 3 A), changing a cysteine residue to phenylalanine in the C-terminal region of the peptide, at amino acid position 1036 (Fig. 2 B). Although this G3181T base change was not detected by SSCP analysis in the PuCh-L low-SCE cells, we performed direct sequencing of the RT-PCR products corresponding to this region and confirmed the absence of the G3181T base change in the PuCh-L cell line (Fig. 3 B).
In order to test our hypothesis, we amplified the 124 bp fragment of BLM containing nucleic acid position 3181 directly from DNA extracted from the high-SCE PuCh-F cell line, the low-SCE PuCh-L cell line, PuCh's blood and PuCh's father's and mother's blood. As shown in Figure 4 , no EcoRI digestion was observed on the PCR products amplified from DNA extracted from PuCh's father's blood. Part of the PCR products amplified from DNAs extracted from PuCh-F, PuCh-L, PuCh's blood and PuCh's mother's blood were digested by EcoRI, indicating that they are heterozygous for the G3181T mutation (Fig. 4 ). Direct sequencing of the corresponding PCR products obtained from PuCh-F and PuCh-L genomic DNA confirmed the presence of both a wild-type sequence and a mutated sequence at nt 3181 in the PuCh-F and PuCh-L cell lines (Fig. 3 C and D). These results show that in patient PuCh the BLM gene G3181T mutationis heterozygous and inherited from her mother. Thus the fact that this mutation is present in the low-SCE PuCh-L cell line demonstrates that a somatic intragenic crossing over event, rather that a gene conversion event, occurred in these cells. As shown in Figure 5 , this intragenic recombination led to type II low-SCE PuCh-L cells that have an untranscribed allele carrying the two parental BLM mutations and a wild-type allele which allowed reversion to the low-SCE phenotype. According to our microsatellite analysis (Fig. 1 ), polymorphic loci distal to BLM remain heterozygous in type II low-SCE PuCh-L cells (Fig. 5 ).
It has been suggested that Blm may be the human homolog of Sgs1 (8 )and shown that Sgs1 protein interacts in vivo with both topoisomerase III (9 )and topoisomerase II (10 ).The fact that the high-SCE PuCh-F cell line expresses only one BLM allele, carrying the G3181T mutationwhich leads to the replacement of a highly conserved cysteine by a phenylalanine, allowed us to investigate the possible consequences of this mutation. Topoisomerase II[alpha] expression in nuclear extracts of PuCh-F, other BS and normal primary fibroblasts was analyzed by Western blot in exponentially growing cells, using specific antibodies against topoisomerase II[alpha]. As seen in Figure 6 A, the cellular level of topoisomerase II[alpha] was lower in the PuCh-F cell line than in control cells, whereas probing the same membrane with specific antibodies against topoisomerase I showed that the cellular level of topoisomerase I was comparable in all cell lines tested. Northern blot analysis was carried out to determine whether the decreased levels of DNA topoisomerase II[alpha] protein expression in the PuCh-F cell line was due to decreased expression of the corresponding gene. Expression of the 6.2 kb topoisomerase II[alpha] mRNA was severely decreased in the PuCh-F cell line, in comparison with control cells (Fig. 6 B). Thus, cellular levels of both DNA topoisomerase II[alpha] protein and its corresponding mRNA are much lower in the high-SCE PuCh-F cell line than in either other BS or normal cells used as controls. In order to determine whether the topoisomerase II[alpha] expression defect detected in the PuCh-F cell line could be related to the BLM gene G3181T mutation, topoisomerase II[alpha] expression was analyzed by Western blot of nuclear protein extracts of exponentially growing low-SCE PuCh-L and normal lymphoblastoid cell lines (Fig. 7 ). This experiment showed that the cellular levels of DNA topoisomerase II[alpha] protein are similar in the PuCh-L cell line and in the normal lymphoblastoid cell lines used as controls (Fig. 7 ). Altogether our results show that the high-SCE PuCh-F cell line, expressing only one BLM allele carrying the G3181T mutation, exhibits a severe reduction in both topoisomerase II[alpha] protein level and its corresponding mRNA, whereas the low-SCE PuCh-L cell line, expressing only one apparently wild-type BLM allele, has topoisomerase II[alpha] expression similar to that of normal cells. It has recently been reported that topoisomerase II[alpha] expression is cell cycle regulated mainly through changes in mRNA stability, with maximal mRNA levels in S phase (20 ). Cell cycle analysis of the high-SCE PuCh-F cells showed that it was similar to that of control fibroblasts, ruling out the possibility that the topoisomerase II[alpha] expression defect in PuCh-F cells resulted from cell cycle dysregulation (Fig. 8 ).
In this study we investigated the molecular basis of BS in an Italian patient from whom both a low-SCE lymphoblastoid PuCh-L cell line and a high-SCE fibroblast PuCh-F cell line were established. We showed that patient PuCh inherited a BLM allele carrying a G3181T mutation from her mother and a BLM allele carrying a mutation which affects its expression from her father. The high-SCE PuCh-F cells express only the BLM allele carrying the G3181T mutation, whereas only a wild-type BLM allele is expressed in the low-SCE PuCh-L cells. Furthermore, since the G3181T mutation is present at the genomic level in PuCh-L cells, we conclude that reversion to the low-SCE phenotype results from an intragenic crossing over event which leads to a wild-type allele and an untranscribed allele carrying the two parental BLM mutations. Because the genomic structure of the BLM gene is still unknown, we could neither investigate the exact borders of the crossing over nor search for the presence of specific sequences in the introns that may facilitate recombination. Since reversion to the low-SCE phenotype seems to be limited to lymphocytes (3 ),it is possible that heptamer/nonamer sequences involved in V-(D)-J recombination are present in BLM gene introns and favor recombination in these cells (21 ).
Blm belongs to the RecQ helicase family comprising Escherichia coli RecQ (22 ), Saccharomyces cerevisiae Sgs1 (9 ,10 ) and human RECQL (23 ,24 ) and Wrn, recently shown to be involved in Werner's syndrome (25 ). Primary structure comparison of several members of this helicase family showed that, unlike that observed for RecQ and RECQL, the sequence similarity between Blm, Wrn and Sgs1 extends beyond the seven helicase domains (7 ,8 ,25 ). As shown in Figure 9 , Cys1036 is highly conserved in Wrn, Sgs1 and in the less homologous RecQ and RECQL. In addition, Cys1036 is also conserved in the RecQ homologs identified in Schizosaccharomyces pombe (GenBank accession no. Q09811) and Caenorhabditis elegans (GenBank accession no. U29097). The high conservation of Cys1036 within a region of limited similarity among the members of the RecQ family emphasizes the potential functional importance of this residue. This is supported by our results showing that the high-SCE PuCh-F cell line, expressing only one BLM allele carrying a mutation replacing the highly conserved Cys1036 by a phenylalanine residue, exhibits a severe defect in both topoisomerase II[alpha] protein level and its corresponding mRNA, whereas the low-SCE PuCh-L cell line, expressing only one wild-type BLM allele, has normal topoisomerase II[alpha] expression. Although the decrease in expression of topoisomerase II[alpha] observed in the PuCh-F cell line could theoretically result from an additional mutation in the topoisomerase II[alpha] gene, this event is very unlikely to occur, even in the context of the mutator phenotype of BS. The defect in topoisomerase II[alpha] expression is not a common feature of the high-SCE BS cells; in particular, a high cellular level of topoisomerase II[alpha] is detected in GM01492 cells, in which BLM mutations are not located in this region of the gene (manuscript in preparation). The defect in topoisomerase II[alpha] expression observed in PuCh-F cells is most likely related to the Cys -> Phe substitution at codon 1036. This hypothesis raises the question of how the BLM gene product can regulate the transcriptional activity of the topoisomerase II[alpha] gene. Little is known concerning transcriptional regulation of the topoisomerase II[alpha] gene, apart from recent reports showing cell cycle regulation (20 ,26 ,27 ).
Analysis of the predicted sequence of the Blm protein allowed us to define a region of special interest. Indeed, Blm exhibits a high density of cysteine and histidine residues around Cys1036, reminiscent of zinc finger DNA binding domains (Fig. 9 ). Sequence alignments of various members of the RecQ family allowed us to extend the sequence homologies in the C-terminal region previously published (7 ,25 ). It is noteworthy that five of the six cysteine residues located in this region, including Cys1036, are conserved in at least four members of the RecQ family. Among them, Cys1055 has also been shown to be changed into a serine in an Italian BS patient (7 ). Four of the Cys residues may participate in a zinc finger DNA binding domain, although they do not strictly fit the published zinc finger consensus sequence (28 ,29 ; Fig. 9 ). Alternatively, it may correspond to another motif of importance that is only similar to the well-characterized zinc finger motif.
Our study further supports very recent data demonstrating that a mutated Sgs1 protein, lacking helicase activity, can still complement some yeast sgs1 mutants (30 ). In the light of these results showing that Sgs1 has an important function not related to its helicase activity, the authors propose that the genomic instability and subsequent syndromes caused by both BLM and WRN mutations may be due to a loss of a function other than the helicase activity (30 ). We propose that Blm protein may act as a transcriptional regulator or may be part of a transcription complex, as are the XPB and XPD helicases in the TFIIH complex (31 ).
In conclusion, we believe that BLM mutations located downstream of the helicase domain, such as we identified in patient PuCh, are of great significance and may provide insights into the structure-function relationship of the BLM gene product.
The high-SCE fibroblast cell line GM3498 and the low-SCE lymphoblastoid cell line (LCL) GM4408 were derived from patient 87 (AlFra) of the Bloom's syndrome registry and obtained from the NIGMS Human Genetic Mutant Cell Repository (Camden, NJ). Two cell lines were derived from patient PuCh, an Italian BS female: the high-SCE PuCh-F was kindly provided by Dr Alain Aurias (Institut Curie, Paris, France) and the corresponding low-SCE PuCh-L LCL was established in our laboratory by exposing 106 peripheral blood mononuclear cells (PBMC) to Epstein-Barr virus (EBV) (the culture is therefore presumably polyclonal). Forty two PuCh-L cell clones were established by a limiting dilution technique. Briefly, PuCh-L cells were seeded at 0.5 or 3 cells/well in the presence of 105 mitomycin-treated PBMC obtained from a normal individual. The human primary fibroblast cells GM09503 and GM08398, derived from normal individuals, as well as the high-SCE GM01492 derived from an unrelated BS patient (patient 44, AbRu), were obtained from the NIGMS Human Genetic Mutant Cell Repository and used as controls. Fibroblast cell lines were cultured in Dulbecco's modified Eagle's medium containing 20% heat-inactivated fetal calf serum (FCS). The lymphoblastoid cell lines Kas and Priess, derived from two normal individuals, were used as controls. Lymphoblastoid cell lines were cultured in RPMI 1640 medium containing 10% heat-inactivated FCS, except for the cloning experiments, which were performed in the presence of 20% FCS. Blood samples, obtained from patient PuCh, her parents and 106 control individuals, were used as a source of genomic DNA, prepared by a standard procedure (14 ).
Eleven microsatellite loci located in the vicinity of the BLM gene (D15S87, D15S107, D15S112, D15S116,D15S127, D15S130, D15S158, D15S175, D15S202, D15S211 and IP15M9) (15 ,16 )were analyzed as previously described (17 ).
RNA was isolated by the acid guanidium thiocyanate/cesium chloride method, as described (18 ). Poly(A)+ RNA were purified from 30 [mu]g total RNA preparations using oligo(dT)25 Dynabeads (Dynal, Oslo, Norway) according to the manufacturer's instructions. Half of the poly(A)+ RNA was used to synthesize cDNA using SuperScriptT reverse transcriptase (Gibco-BRL), following the manufacturer's instructions, in the presence of [[alpha]-32P]dCTP to quantify cDNA synthesis and to evaluate cDNA length.
After first-strand cDNA synthesis, PCR was performed using ~10 ng cDNA, 3% DMSO, 0.2 mM dNTPs (Pharmacia), 1.5 mM MgCl2, 1* reaction buffer (Perkin Elmer), 0.25 U Taq polymerase (Perkin Elmer) and 5 pmol each of the 31 pairs of primers described in Ellis et al. (7 ), with the exception of one pair that we designed (5'-CCTCTTCAAAATGCCTTAGTACG-3' and 5'-GTTTCAGTTTATCATCAGGAATAG-3'), and 7.5 [mu]Ci [[alpha]-32P]dCTP, in a total volume of 10 [mu]l. After a 5 min incubation at 94oC, samples were subjected to 35 cycles consisting of 1 min at 94oC, 1 min at 58oC and 1 min at 72oC, followed by a final elongation step at 72oC for 5 min. SSCP analysis was carried out as described by Ellis et al. (7 ), with minor modifications.
Aliquots of 500 ng either cDNA or genomic PCR products were purified using QIAquick PCR purification kit columns (Qiagen) and sent to Euro Séquences Gènes Services for direct sequencing using the PCR primers (Montigny-le-Bretonneux, France).
After first-strand cDNA synthesis, PCR was performed to amplify a 219 bp fragment between nt 3143 and 3361, as described in the SSCP analysis section, except that PCR was done in a total volume of 20 [mu]l in the absence of radiolabeled nucleotide and using 10 pmol of the oligonucleotide primers 5'-GTATAGCATGGTACATTACTGTG-3' and 5'-CCTTGTGATGAACTATGTTCTTG-3'.
PCR was carried out to amplify a 124 bp fragment between nt 3143 and 3266, using 250 ng genomic DNA, 0.2 mM dNTPs (Pharmacia), 1.5 mM MgCl2, 1* reaction buffer (Perkin Elmer), 2.5 U Taq polymerase (Perkin Elmer), 50 pmol of the oligonucleotide primers 5'-GTATAGCATGGTACATTACTGTG-3' and 5'-TATCACAAGAAACATCTGGGTGTT-3' and 2.0 [mu]Ci [[alpha]-32P]dCTP in a total volume of 100 [mu]l. After a 5 min incubation at 94oC, samples were subjected to 35 cycles consisting of 1 min at 94oC, 1 min at 43oC and 1 min at 72oC, followed by a final elongation step at 72oC for 5 min.
EithercDNA or genomic PCR products were subjected to digestion using the restriction enzyme EcoRI (Gibco-BRL). The digested cDNA PCR products were analyzed by agarose gel electrophoresis, whereas the digested genomic PCR products were run on a denaturating 6% acrylamide gel containing 7 M urea and 32% formamide after a 3 min denaturation at 95oC. The gel was dried and autoradiographied at -80oC (XAR; Kodak) for 1 week.
Denatured total RNA (20 [mu]g/lane) was electrophoresed in a 1.2% agarose gel containing formaldehyde and blotted overnight onto nylon filters (Hybond N; Amersham) with 10* SSC as transfer buffer. The membranes were prehybridized for 2 h at 42oC, hybridized for 48 h at 42oC in a buffer containing 50% formamide, 6* SSC, 1% SDS, 1* Denhart's solution, 100 [mu]g/ml salmon sperm DNA and 2 * 106 c.p.m./ml labeled probe. Membranes were washed for 10 min, once at room temperature in 1* SSC and 0.5% SDS, once at 65oC in 1* SSC and 0.5% SDS and once at 65oC in 1* SSC and 0.1% SDS. Membranes were exposed with intensifying screens at -80oC on Kodak XAR films for 15-48 h. Probes were removed from the blot by boiling in 0.1% SDS.
The topoisomerase II[alpha] and topoisomerase I probes used were respectively a 1280 bp cDNA fragment and a 1123 bp cDNA fragment obtained by RT-PCR using the following oligonucleotides: 5'-CCAGCTCGAGCGAGGAGCCACAGCTGAGTCAAAG-3' and 5'-CCAGCTCGAGGATTCGGATCCTGTGAAGGCCTGG-3' for topoisomerase II[alpha]; 5'-CCAGGAATTCGGCAACCACCCCAAGATGGGCATG-3' and 5'-CCAGGAATTCTTGGGA- CACCCCACTTCTTGCACC-3' for topoisomerase I. The rat GAPDH probe, which cross-hybridizes with human GAPDH, used as a loading control, was a kind gift from Dr C.Nahmias (Institut Cochin de Génétique Moléculaire, Paris, France). All probes were radiolabeled with [[alpha]-32P]dCTP using a random priming technique (Amersham).
Nuclear extracts were prepared using between 5 and 15 * 106 exponentially growing cells, previously washed in 1* PBS, as described (19 ). Aliquots of 20 [mu]g nuclear proteins were diluted in Laemmli buffer, denatured by heating for 4 min at 95oC and electrophoresed in a 7.5% SDS-polyacrylamide gel. Protein fractions from the gel were electrophoretically transferred to nitrocellulose filters (Hybond-ECL; Amersham) in 25 mM Tris-HCl, pH 8.3, 192 mM glycine, 20% methanol and 0.1% SDS for 12 h at 30 V. Nitrocellulose membranes were saturated for 1 h in PBS containing 6% dry non-fat milk, washed for 15 min in PBS containing 0.1% Tween 20 (Sigma) and further incubated for 4 h in PBS containing 2% dry non-fat milk in the presence of a polyclonal antibody against topoisomerase II[alpha] (1:1000; NeoMarkers, Freemont, CA). Membranes were rinsed in PBS containing 0.1% Tween 20, incubated with a secondary antibody coupled to peroxidase (NA 934; Amersham) and developed according to the manufacturer's specifications (ECL kit; Amersham). Antibodies were removed by incubating the membranes for 30 min at 50oC in a buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% SDS and 0.7% 2-mercaptoethanol, washed and reincubated with an antibody against topoisomerase I (1:2000; TopoGEN, Colombus, OH) prior to treatment with a secondary antibody coupled to peroxidase (NA933; Amersham), as described for topoisomerase II[alpha].
Cells were washed twice in ice-cold PBS and then suspended in 0.3 ml PBS. Then cells were fixed for 30 min on ice by addition of 0.9 ml 95% ethanol (-20oC). Cells were washed and resuspended in 0.8 ml PBS. Then samples were incubated for 30 min at 37oC in the presence of 100 [mu]g/ml RNase A (Sigma) and 50 [mu]g/ml propidium iodide (Sigma), before the DNA content was determined by measuring fluorescence intensities on a FACScalibur (Becton Dickinson) instrument.
We are grateful to the PuCh family for their cooperation, without which this work would not have been possible. We are grateful to Prof. A.Vierucci (Ospedal A.Meyer, Firenze, Italy), Prof. Claude Griscelli and Prof. Alain Fisher (Hôpital Necker, Paris, France) who allowed us to obtain BS patients' samples. We thank Dr Alain Aurias (Institut Curie, Paris, France) for providing us with the PuCh-F cells, Dr Renzo Barrasso (Hôpital Bichat, Paris, France) for providing us with blood samples used as controls and Yann Lécluse for expert assistance in FACS analysis. We also thank Dr Malek Djabali (Centre d'Immunologie de Marseille-Luminy, Marseille, France) and Prof. Pierre Netter (Institut Jacques Monod, Paris, France) for helpful discussions. This work was supported by grants from Institut National de la Santé et de la Recherche Médicale (CRE no 930701), Ligue Nationale Française contre le Cancer and Institut d'Oncologie Cellulaire et Moléculaire Humaine, Bobigny. F.F. was the recipient of a fellowship from the Association pour la Recherche sur le Cancer.
BS, Bloom's syndrome; SCE, sister chromatid exchange; SSCP, single strand conformation polymorphism.
*To whom correspondence should be addressed. Tel: +33 1 42 11 40 50; Fax: +33 1 42 11 54 94; Email: amor@igr.fr
+Present address: Institut Gustave Roussy, Centre National de la Recherche Scientifique, Unit de Recherche Associe 1156, 39 Rue Camille Desmoulins, 94805 Villejuif Cedex, France
Human Molecular Genetics
Pages
Introduction
Results
The polymorphic markers distal to the BLM gene remain heterozygous in the low-SCE PuCh-L cells
At the cDNA level, an apparently homozygous BLM base change is detected in the high-SCE PuCh-F cells, whereas only the wild-type BLM gene is detectable in the low-SCE PuCh-L cells
At the genomic level, both PuCh-F and PuCh-L cells are heterozygous for the BLM gene mutation at nucleic acid position 3181
The high-SCE PuCh-F cells exhibit a severe defect of topoisomerase II[alpha] expression, whereas the low-SCE PuCh-L cells have normal topoisomerase II[alpha] expression
Discussion
Materials And Methods
Cell lines and DNA
Polymorphic marker analysis
RNA extraction and cDNA synthesis
Single strand conformation polymorphism (SSCP) analysis
DNA sequencing of SSCP conformers
RT-PCR
Genomic PCR
EcoRI digestion
Northern blot analysis
Probes
Western blot analysis
Cell cycle analysis
Acknowledgements
Abbreviations
References
REFERENCES
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