We have previously shown a high frequency of allele loss at D6S193 (62%) on chromosomal arm 6q27 in ovarian tumours and mapped the minimal region of allele loss between D6S297 and D6S264 (3 cM). We isolated and mapped a single non-chimaeric YAC (17IA12, 260–280 kb) containing D6S193 and D6S297. A further extended bacterial contig (between D6S264 and D6S149) has been established using PACs and BACs and a transcript map has been established. We have mapped six new markers to the YAC; three of them are ESTs (WI-15078, WI-8751, and TCP10). We have isolated three cDNA clones of EST WI-15078 and one clone contains a complete open reading frame. The sequence shows homology to a new member of the ribonuclease family. The other two clones are splice variants of this new gene. The gene is expressed ubiquitously in normal tissues. It is expressed in 4/8 ovarian cancer cell lines by Northern analysis. The gene encodes for a 40 kDa protein. Direct sequencing of the gene in all the eight ovarian cancer cell lines did not identify any mutations. Clonogenic assays were performed by transfecting the full-length gene in to ovarian cancer cell lines and no suppression of growth was observed.
Chromosome 6 has been implicated to contain a putative tumour suppressor gene important in the pathogenesis of ovarian cancer, both by karyotypic analysis and allele loss studies (Shelling et al., 1995). The analysis of 70 malignant ovarian tumours using cosmids mapping to chromosomal arm 6q initially defined the minimal region of allele loss between D6S149 and D6S193 (1.9 cM) in one tumour (Figure 1) (Saito et al., 1992, 1996). Subsequent studies have shown increased frequency of allele loss on 6q around the same region, although a minimal region was not defined (Orphanos et al., 1995; Wan et al., 1994). Based on the analysis of 56 malignant ovarian tumours we showed previously that the minimal region of allele loss on 6q27 is between D6S297 and D6S264 (3 cM) (Figure 1) (Chenevix-Trench et al., 1997; Cooke et al., 1996b). The allele loss was observed in all types of epithelial ovarian cancer. The maximal frequency of allele loss occurred at D6S193 (62%) and D6S297 (52%). Three tumours showed loss of D6S193 only, while retaining flanking markers thus suggesting that the putative tumour suppressor gene was close to D6S193. Thus the extended region of allele loss taking the two previous studies into account suggests that the putative gene lies within D6S264–D6S149 (approximately 7.4 cM).
Further evidence that there might be a putative tumour suppressor gene around D6S193 is provided by FISH studies using YACs on direct metaphase spreads from fresh ovarian tumours which suggested that this change might be important even in early ovarian tumours (Tibiletti et al., 1996, 1998). It has also been shown that the same region is implicated in a subset of lymphomas and breast cancer (Gaidano et al., 1992; Menasce et al., 1994; Rodriguez et al., 2000; Tibiletti et al., 2000).
To identify the gene on chromosomal band 6q27 implicated in the pathogenesis of ovarian cancer, we undertook a positional cloning approach involving isolation of yeast artificial chromosomes (YACs), P1 derived artificial chromosomes (PACs), bacterial artificial chromosomes (BACs) and construction of a physical and transcript map around the key polymorphic markers. We have identified seven genes in the interval between D6S264 and D6S149. We present the detailed analysis of a gene located telomeric to D6S193 and D6S297, a member of the ribonuclease family (RNASE6PL).
Genetic and physical mapping
We had previously constructed an integrated genetic map of chromosome 6q24–6q27 using the Genethon map as a backbone (Figure 1) (Cooke et al., 1996a). This map improved the number and order of markers within 6q24–6q27 compared to the Genethon map. We initially screened the ICRF, CEPH and ICI YAC libraries by hybridization and PCR with markers from this region. Several YACs were isolated and characterized for sequence tagged site (STS) content, size and integrity using FISH. Three YACs were identified which contained the relevant markers D6S193 and D6S297 from the ICI YAC library. One non-chimaeric YAC 17IA12 (ICI YAC library) was shown to contain D6S193, D6S297, D6S1585 and D6S133. A detailed long-range restriction map of this YAC (260–280 kb) was constructed and an overlapping contig of cosmids was made. Thirty ESTs/STS mapping in the region were evaluated by PCR and three ESTs (WI8751, WI15078 and TCP10) were identified to be located in the YAC, in addition to the STS markers D6S193 and D6S297.
Simultaneously, PACs and BACs were isolated from whole genome libraries (Ioannou et al., 1994; Shizuya et al., 1992) for all the markers in this region and were assembled into a contig by STS content and fingerprinting (Mungall et al., 1997). The orientation of the minimal overlapping set of bacterial clones in relation to the genetic map and YAC map is shown in Figure 1. The PAC RP1-167A14 contained STSs stSG11162, stSG17631 that were not present in the YAC 17IA12. This suggested that there was an internal deletion in the YAC corresponding to these markers. The bacterial contig is not yet complete as there is still a gap between RP11-178P20 and the PAC that contains the marker D6S149 and part of the gene AF6 (RP3-431P23).
In order to facilitate the identification of genes within this region between D6S264–D6S149, sequencing of the entire contig was undertaken. Except for the BAC RP11-178P20, which has not yet been sequenced to completion, linear overlapping sequence is available from PAC RP4-655C5 until the PAC RP3-366N23.
Several methods were used to identify coding sequences within the region. They included homology search of the GenBank/EMBL databases using BLAST algorithms and a suite of exon prediction programme to identify genes (Altschul et al., 1990). In addition, experiments were also performed to assess ESTs mapped within the interval between D6S264–D6S149 and so far none have yielded new bona fide genes. The order of genes identified so far is D6S264–p90Rsk-3–RNASE6PL–FOP–CCR6–GPR31–TCP10–HUnc93–D6S149–AF6.
Initially, we focused on the three ESTs that had been mapped to the YAC before the PAC contig and the sequence data were available. The cDNA clone of the first EST WI-8751 (IMAGE clone 68587, size 1.1 kb) was obtained, sequenced and used to screen cDNA libraries for obtaining full-length cDNA clones. After extensive screening of several cDNA libraries (12) we were unable to isolate any full-length clones. The full-length sequence of the cDNA clone of EST WI-8751 did not contain an open reading frame. Further, on Northern analysis of multiple tissues using this clone as probe we were unable to identify any transcript (data not shown). On comparison with the sequence contained in BAC RP11-514O12 that overlapped with PAC RP3-505P2, it was clear that the entire coding sequence of EST WI-8751 was contained as one linear sequence without any intron–exon boundaries. We also did not identify any promoter sequences in the genomic sequence 5′ of the cDNA. Although it is possible that the EST WI-8751 is expressed at low levels in a specific tissue, it is most likely to be a pseudogene.
The second EST which we mapped to the YAC was a previously identified gene TCP10 (T complex protein 10A) (Blanche et al., 1992). Interestingly, on the bacterial contig the gene mapped quite far away on BAC RP11-517H2. As the size of the YAC was 260–280 kb, and did not contain markers within PACs RP4-505P2 and RP1-167A14, this suggested that there was a large deletion within the YAC 17IA12 corresponding to these sequences. This gene (26 kb) is encoded by nine exons and they are present on BAC RP11-517H2. Unusually, the last two exons are also present in PAC RP3-366N23 which is nearly 150 kb away (Figure 2). Further, exon 8 is telomeric to exon 9 and is transcribed in the antisense orientation. The intron-exon structure is as shown in Table 1. As the expression of this gene was exclusively limited to the testis (data not shown) we did not characterize this gene further in ovarian cancer (Islam et al., 1993).
The third EST which was identified and mapped to the YAC (WI-15078, IMAGE clone 174311, size 1049 bp) was sequenced in its entirety. We used this clone as a probe and isolated two larger partial cDNA clones (IMAGE clones 199297 1332 bp, 246630 1195 bp) after screening of cDNA libraries. The three clones were completely sequenced in both strands. Comparison and analysis of the cDNA sequences of all the three clones suggested that there was an open reading frame in clone 246630 of 242 amino acids that encoded for a protein of 29 kDa. A search of the database showed that the sequence was homologous to the family of ribonucleases (Hime et al., 1995). This family is characterized by two conserved motifs ‘IHGWLP’ and ‘KHGTC’ that span the active site of the enzyme (Kurihara et al., 1996). This gene is the human equivalent and was the first reported member of this family (Trubia et al., 1997). A comparison of the amino-acid sequence of the human protein with that of other species is shown (Figure 3a).
We compared the full-length cDNA sequence of clone 246630 against the genomic sequence contained within PAC RP3-505P2 and BAC RP11-514012. This showed that the gene is coded by nine exons spread over 27 kb (Figure 3b, Table 2). Comparison of the other two cDNA clones (199297 and 174311) suggested that these two were splice variants of the same gene. Interestingly, both clones 199297 and 174311 contained unique exons (5a, 791 bp; exon 5b, 46 bp) which are not present in the cDNA clone 246630. In clone 199297, exon 5a has an in-frame stop codon. This exon has sequence which is identical to exon 5b in clone 174311, the intervening sequence and exon 5 of clone 246630. The size of the full-length cDNA for clone 199297 was 1954 bp. The predicted peptide of this splice variant contains 124 amino acids. In clone 174311, the novel exon 5b is followed by exon 6a and exon 5 is spliced out. Exon 6a contains sequence identical to exon 6 of clone 246630, followed by intervening sequence which has an in-frame stop codon. The size of the full-length cDNA for clone 174311 was 1671 bp. The predicted peptide from this splice variant contains 240 amino acids. Alignment of the amino-acid sequence from all of the three splice variants is shown in Figure 3c. Thus, the two splice variants 199297 and 174311 produce truncated proteins. The splice variant 199297 was confirmed by RT–PCR using primers specific to exon 5a (Figure 3d). We were unable to confirm the existence of the second splice variant 174311 by using primers specific for exon 5b and 6a by RT–PCR on mRNA from different tissues.
We examined the expression of the three splice variants in various tissues using the full-length probe for clone 246630 and the original partial cDNA clones for 199297 and 174311 which contain unique sequences specific to these cDNAs. All of the three splice variants are expressed in all tissues with maximal expression in pancreas and lymphocytes. The major message is 1.35 kb. In addition, in clone 174311, a larger transcript of 2.5 kb is expressed in heart and muscle. There were minor larger transcripts detected with clone 199297 which are probably due to cross reactivity with another species. There was variation in the level of the major transcript detected by all three cDNA's in the different tissues, particularly by cDNA clones 174311 and 246630. All of the three splice variants were expressed in the ovary (Figure 4a).
Expression of all of the three splice variants were examined in a panel of eight ovarian cancer cell lines by Northern analysis. There was variable expression of the major transcript of 1.35 kb in four (OAW28, OAW42, SKOV-3, UC101) of the eight cell lines and on prolonged exposure at a lower level in the remaining cell lines (Figure 4b). Although a higher band of approximately 4 kb was detected in the cell lines OAW42 and SKOV-3, by all three probes, this probably does not represent a true transcript but hybridization to the 28S RNA which is of the same size. Expression of the splice variant 246630 was subsequently confirmed by the more sensitive RT–PCR in all of these eight cell lines (Figure 4c).
In order to determine the size of the protein encoded by the full-length clone 246630, we transfected a HA-epitope tagged construct in Cos-7 cells. This showed a fusion protein of 40 kDa (Figure 4d). The molecular weight of the fusion protein was higher than that predicted (29 kDa) from the open reading frame. This suggests that post-translational modifications may account for the increase in the size of the mature protein.
As the RNASE6PL gene was close to the markers D6S193 and D6S297 and had variable expression in cell lines, we sequenced the coding region in its entirety in all of the eight cell lines. The sequence did not show any variation compared to the wild type sequence. To further assess whether the RNASE6PL gene was a potential candidate tumour suppressor gene, we transfected the cDNA (HA epitope tagged 246630 clone) into the four ovarian cancer cell lines (UC101, PEO1, OAW28 and 41M) and assessed colony formation in the presence of the selectable marker G418. In the two cell lines PEO1 and 41M, where there was relatively little expression of the gene on Northern analysis, there was no difference in the number of colonies compared to the vector transfected clones (Figure 5). In the other two cell lines where the RNASE6PL gene was expressed, there was no difference in the number of colonies when compared to vector transfected controls either.
Other candidate genes within the internal D6S264–D6S149
The gene FOP (FGFR1 Oncogene Partner) was identified to be wholly contained within the PAC RP1-167A14. It was isolated as a fusion partner of a rare translocation in leukaemia (Popovici et al., 1999). The gene is expressed in the ovary (data not shown) as previously published and was uniformly expressed in all the ovarian cell lines (Figure 6). In addition, transfection of the full-length cDNA into ovarian cancer cell lines and evaluation in a clonogenic assay showed there was no suppression of growth compared to vector control (data not shown). We have not analysed the gene further for mutations.
CCR-6 and GPR31
These two genes have been isolated previously (Baba et al., 1997; Greaves et al., 1997; Liao et al., 1997a; Zingoni et al., 1997). They encode for chemokine receptors and are expressed primarily in lymphoid cells by Northern analysis. Further, they are contained in the BAC RP11-517H2 as one linear sequence without any introns, a characteristic of other chemokine receptors. Although it was previously reported that CCR-6 is coded by two exons (Liao et al., 1997a), comparison of the cDNA sequence with the genomic sequence showed the absence of any introns. In the absence of their expression in the ovary (data not shown) we have not analysed these genes further in ovarian cancer.
The human homologue for Unc-93 which is contained within PAC RP3-366N23 was examined in detail (Liu et al., manuscript submitted) in ovarian cancer. Similarly, p90 Rsk-3 which is contained within PACs RP3-497J21 and RP1-168L15 is the basis of a separate report (Emilion et al., manuscript submitted). AF-6 which is partly contained within RP3-431P23 has been previously examined by FISH and analysed for mutations in ovarian cancer (Saha et al., 1995; Saito et al., 1996). It maps outside the interval of deletion at chromosome 6q27 and no mutations have been identified in ovarian tumours.
The clue to the location of a putative tumour suppressor gene at 6q27 was primarily provided by allele loss studies using polymorphic markers on chromosomal arm 6q (Cooke et al., 1996b). This was supported by FISH studies performed directly on metaphase spreads from ovarian tumours using YACs (Tibiletti et al., 1996, 1998). Precise delineation of the minimal interval of allele loss was shown to be between D6S264 and D6S297 by us previously, which overlapped with that between D6S193 and D6S149 (Saito et al., 1992). Thus, the extended interval was established to be between D6S264 and D6S149. The high frequency of allele loss at D6S193 was confirmed by analysis of a different set of samples from Australia (Chenevix-Trench et al., 1997). The predicted distance between D6S264 and D6S149 was 7.4 cM. To identify the putative tumour suppressor gene on 6q27, we undertook a positional cloning approach. Although our initial strategy was based on YACs, chimaerism proved a particular difficulty in assembling an overlapping contig within this interval. We identified one YAC 17IA12, which on FISH was non-chimaeric and contained D6S193 and D6S297. However, with the availability of bacterial genomic libraries (PAC and BAC), the establishment of a physical contig was pursued using these resources. To accelerate the identification of genes within this interval, we initially mapped all ESTs within 6q27 in the database to the YAC 17IA12 and subsequently to the PACs. We then undertook direct sequencing of the entire PAC/BAC contig as part of the genome project. The entire contig from D6S264 until D6S149 has one gap between RP11-178P20 and RP3-431P23. We estimate this to be of one-two PAC/BAC length. The overall linear sequence available within this interval is approximately 1.1 Mb. Comparison of the YAC map with that of the order of STS/genes on the bacterial contig suggested that there was an internal deletion of sequence within the YAC corresponding to the PAC RP1-167A14.
The primary method of identification of genes was by comparison with GenBank/EMBL databases. In addition, suites of programs were used to predict coding sequences. Overall, seven genes were identified within this interval. In addition, several EST matches have been identified but we have not been able to show that any of them represent bona fide genes experimentally.
As the RNASE6PL gene was located telomeric to D6S193, it was investigated in some detail. Members of the Rh/T2/S-Glycoprotein ribonuclease gene family are found predominantly in fungi, plants, and bacteria, where they have been implicated in functions such as the phosphate-starvation response and self-incompatibility. Self-incompatibility, the ability that stops a plant's pollen from fertilizing its own pistel, is controlled by a single multi-allelic locus in Nicotiana alata (Anderson et al., 1989; Ioerger et al., 1990; McClure et al., 1989). In Nicotiana alata, this locus encodes proteins termed the stylar glycoproteins (S-glycoprotein) which exhibit homology with two of the most studied families of extracellular fungal RNAses T2 and Rh, from Aspergillus oryzae and Rhizopus niveus (McClure et al., 1989). Therefore this gene family is termed as Rh/T2/S-Glycoprotein ribonuclease. These ‘stylar RNAses’, serve as a plant immune system, aiding in maintaining genetic diversity and strength of the species (Sassa et al., 1996). The first member of this family found in invertebrates was the DmRNAse-66B gene in the Drosophila melanogaster genome in 1995 (Hime et al., 1995). Ribonucleases (RNAses) are a group of enzymes of varying specifity to control the RNA population post-transcriptionally. Once regarded as ubiquitous ‘housekeeping enzymes’, they are now recognized to control processes ranging from splicing to organogenesis (Schein, 1997). It has been known that both plant and mammalian RNAses are produced in response to stress (Ye and Droste, 1996), and pathogenic agents, particularly viruses (Walter et al., 1996). RNAse active in greenhouse wheat increases during senescence, as does RNAse activity in human tissues (Francesconi et al., 1984). The role of RNAses in the mammalian immune system has not yet been investigated.
The gene encoding for a novel ribonuclease (RNASE6PL) was unusual in several aspects. Firstly, as previously reported, it is the first human homologue of an extra-cellular ribonuclease observed primarily in plants (Trubia et al., 1997). The predicted protein has two motifs, which are completely conserved in evolution suggesting that they are functionally important. Indeed, the motifs, IHGWLP and KGHTC have been shown to be the catalytic site by resolution of the tertiary structure (Kurihara et al., 1996). The two histidines in these motifs are completely conserved suggesting that there is functional conservation across species. Secondly, the two splice variants are unusual in that the translation proceeds into the intervening sequence in frame and terminates with an in-frame stop codon. The expression of the three splice variants is broadly similar in that a major transcript of 1.3 kb is present in all tissues although of varying levels. However, there was a larger transcript expressed in the heart and skeletal muscle (2.5 kb) by the splice variant 174311. The size of the cDNA clone 246630 correlates with the major message of 1.35 kb. However, the correlation of mRNA message size with clones 199297 and 174311 is less clear. Although only four of the cell lines had expression of all three transcripts, longer exposure revealed faint bands in the remaining cell lines. The expression of the full-length cDNA encoded by clone 246630 was confirmed by the more sensitive RT–PCR in all of the cell lines. The full-length transcript was expressed in an epitope tagged vector and encoded for a protein of approximately 40 kDa. The increase in size compared to that predicted is probably accounted by post-translational modification. To exclude the possibility that the gene might be mutated in ovarian cancer cell lines, we sequenced the coding region in its entirety and it was completely normal. The clonogenic assays provide further support that this gene is unlikely to be a putative tumour suppressor gene.
However, it was recently reported that RNASE6PL might be the putative gene for senescence and tumour suppressor gene in ovarian cancer (Acquati et al., 2001). Although the authors found loss of expression in 30% of ovarian tumours and 75% of cell lines, the expression was not completely absent but reduced which was similar to our results. Further they did not perform RT–PCR, a more sensitive method of analysing expression of RNASE6PL. Similar to our results, they did not identify mutations within the coding region of the gene either. Instead of clonogenic assays, they established stable cell lines by transfection of RNASE6PL (HEY4, an ovarian cancer cell line which shows 10% expression of this gene compared to that in normal ovarian tissue; SG10G, an ovarian cancer cell line has lost chromosome 6; XP12ROSV, a SV40-immortalized fibroblast cell line having no expression of the gene; pRPc, a mouse cell line derived from a tumour induced in nude mice by the human/mouse monochromosomal hybrid harbouring a deletion at 6q27). Examination of their data did not show statistically significant difference between propagated live colonies and control (in HEY4, 60.2%, vs 100%; in SG10G, 72.1% vs 100.0%; in XP12ROSV, 10.3% vs 66.7%; and in pRPc, 86.2% vs 100%), except possibly in XP12ROSV. Therefore, the results from this clonogenic assay was again similar to our results in that the colony formation was reduced, but not significantly, after the transfection of this gene when compared to the vector control in ovarian cancer cell lines. We did not perform nude mice tumorigenicity experiments as our clonogenic assays did not suggest that RNASE6PL inhibited cell growth. Further, even in their experiments there was no significant reduction in the number of tumours in mice. The principal results was that tumour xenografts showed loss of expression of the transfected RNASE6PL gene. However, other genes in the region between D6S264 and D6S149 were not examined to evaluate whether there was loss of their expression in these tumours.
The remaining ESTs identified on the YAC were WI-8751 and TCP10. WI-8751 was unlikely to represent a bona fide gene, as not only expression was absent on conventional Northern blots but also there was no defined intron–exon structure or an open reading frame. TCP10 was again not examined further because of its lack of expression in the ovary. It has an unusual intron–exon structure. Although all the nine exons of TCP10 were contained within the BAC RP11-517H2, the last two exons were also present in the PAC RP3-366N23. This might represent a duplication of this gene. This remains to be verified, as there is a gap in the contig after RP3-366N23. Of the remaining genes, CCR6 and GPR31 were not expressed in the ovary as previously published (data not shown) (Baba et al., 1997; Greaves et al., 1997; Liao et al., 1997b; Zingoni et al., 1997). FOP, which was isolated as a translocation partner in a rare type of leukaemia (Popovici et al., 1999), was expressed uniformly in all the ovarian cell lines. Further, in the clonogenic assay, there was no suppression in the growth of ovarian cancer cell lines upon transfection of full-length FOP cDNA. This suggests that it is unlikely to be a putative tumour suppressor gene. It is possible, however, that missense mutations may occur in ovarian tumours which do not interrupt translation.
In ovarian cancer, only P53 and PTEN have been shown to be mutated (Obata et al., 1998; Shelling et al., 1995). P53 mutations are observed in all subtypes of cancer and are more common in advanced stages of the disease. PTEN, in contrast is mutated in only 20% of endometrioid type of epithelial ovarian cancer. The majority of genes identified as potential tumour suppressor genes in ovarian cancer either by positional cloning or differential mRNA display have not been shown to harbour mutations in ovarian cancer although they do suppress growth in clonogenic assays (Liu and Ganesan, 2001). The mechanism of loss of expression of the putative gene has been either by methylation of the promoter or imprinting. Indeed, the genes important in hereditary forms of ovarian cancer such as BRCA1, BRCA2 or the mismatch repair genes have not been shown to be mutated frequently in sporadic ovarian cancer, thus deviating from the classical model (Katso et al., 1997). However, BRCA1 is not expressed in a proportion of tumours due to methylation of the promoter (Catteau et al., 1999; Mancini et al., 1998). Thus mechanisms other than mutations might be more common in the inactivation of tumour suppressor genes in ovarian tumours. This nearly completed integrated physical and transcript map in a 1 Mb region at chromosome 6q27 has been instrumental in evaluation of candidate tumour suppressor genes important in ovarian cancer.
Materials and methods
Ovarian cancer cell lines (Laval et al., 1994), and Hela, Cos-7 cell lines were supplied by Cell Service, Clare Hall, ICRF. The cells were cultured in the media with 10% FCS and grown at 37°C in the presence of 5% CO2.
Preparation of yeast, YAC DNA blocks and PFGE
YAC DNA in yeast cells was prepared in agarose blocks for FISH and Pulsed Field Gel Electrophoresis (PFGE). Intact chromosomal YAC DNA was generated by the Lithium method (Anand et al., 1990) in which whole yeast cells are embedded in agarose blocks before spheroplasting, lysis and deproteinization. Two per cent (w/v) low melting gel in 1 M sorbitol and 20 mM EDTA was dissolved by boiling, and cooled to 50°C in a water bath, and 14 mM of 2-ME was added. Block moulds (Pharmacia) were laid out on a glass plate placed on ice. Yeast cells from 50 ml culture were pelleted at 1000 r.p.m. for 10 min. The pellet was resuspended in 400 μl of solution I (1 M sorbitol, 20 mM EDTA, 14 mM 2-ME) and mixed with 500 μl of the molten agarose and dispensed into the slots of the block mould on ice. The set agarose inserts were released in a 50 ml Falcon tube containing 5 ml of 1 M sorbitol, 20 mM EDTA, 14 mM 2-ME, 10 mM Tris-HCL, pH 7.5, 2 U/ml Zymolyase and incubated at 37°C for 2 h. The agarose inserts were incubated in the lysis solution (1% lithium dodecyl sulphate, 100 mM EDTA, 10 mM Tris-HCL, pH 8.0) at 37°C for 1 h and stored in TE at 4°C until loading on a gel for sizing and purifying YAC DNA fragment from agarose gel using standard protocol. PFGE was performed according to standard protocol.
cDNA library screening
cDNA libraries of human brain, liver, lung (all foetal) and placenta were available as membranes with immobilized cDNA clones (Resource Centre/Primary Database of the German Human Genome Project). cDNA probes were labelled with 32P-dCTP using MegaprimeTM labelling systems (Amersham). The membranes were prehybridized in 20 ml Churchill & Gilbert buffer (0.22 M NaH2PO4, 0.35 M Na2HPO4, 7% SDS, 1 mM EDTA) 65°C for 2 h and then hybridized at 65°C with denatured probe in the same buffer overnight. The membranes were washed with 2×SSC/0.5% SDS for 15–20 min at 65°C twice, with 0.1×SSC/0.1% SDS for 30 min once at 65°C.
Total cellular RNA was isolated from 5–10×107 cells of each cell line with RNAeasy Midi/Maxi kit (QIAGEN) according to manufacturer's protocol. Ten μg of RNA from each cell line was separated in 1% denaturing formaldehyde/agarose gels and immobilized to Hybond-N+ (Amersham) membrane by standard capillary blotting in 20×SSC overnight and baking at 80°C for 2 h. Membranes were prehybridized in ExpressionHybTM Hybridisation Solution (Clontech) at 68°C for 1–2 h. 174311, 1992–97, 246630 cDNA probes were labelled with 32P-dCTP using MegaprimeTM labelling systems (Amersham) and purified through G-50 Sephadex columns. Membranes were hybridized in ExpressionHybTM Hybridisation Solution (Clontech) at 68°C with recommended amount of denatured probe for overnight and washed according to manufacturer's protocol. The probes used for labelling and hybridization were: full-length cDNA 246630, partial cDNA of 199297 containing exons 5a up to 9, partial cDNA of clone 174311 containing exons 5b and 6a, and for FOP full-length cDNA.
RNA isolated from each cell line as described above was used in a RT–PCR reaction using QIAGEN One Step RT–PCR Kit (QIAGEN) according to the manufacturer's protocol. All the reactions were carried out on a PTC-200 Thermocycler (GRI Lab Care Service) for the reverse transcription (50°C, 40 min), initial PCR activation step (95°C, 15 min), 40 cycles of denaturation (94°C, 40 s), annealing (Tm for each pair of primers, 1 min), extension (72°C, 1 min), and 1 cycle of final extension (72°C, 10 min). Primer sequences for expression of 246630 in cell lines were: Forward primer (244630F2) 5′-taatagatcttggcccttca-3′ and reverse primer 5′-caagggcatctttaaaatctgc-3′ (246630R2). Primer sequences for expression of 199297 were (199297F1) 5′-cgtcgttggaatcatacag,-3′ and (246630R2) 5′-caagggcatctttaaaatctgc-3′.
Plasmids and genomic DNA were sequenced using BigDye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer) on the Applied Biosystems model 373 DNA sequencing system (PE Applied Biosystems). One μg of plasmid DNA, 10 pmol of primer and 4 μl of Terminator mix were used for a 12 μl sequencing reaction. Primers from both 5′ and 3′ sides were used, while primers were also designed from cDNA sequence when necessary. A Thermal Cycler-480 (Perkin Elmer) was used to carry out the 25 cycles of denaturation (96°C, 30 s), annealing (50°C, 15 s), and extension (60°C, 4 min). The DNA was precipitated, spun down, washed, resuspended in loading buffer, denatured, and loaded onto ABI 373 Sequencher according to manufacturer's instruction.
Construction of epitope tagged RNASE6PL
Primers (with restriction sites) were designed to amplify the 246630 open reading frame (ORF). It was subcloned into the epitope tagged vector pMH (Boehringer Mannheim) with the cDNA in frame with the HA tag at the 3′ end. Primer sequences to amplify the full-length cDNA and clone it in frame were 5′-ctgaaagcttcaggtcggcaccatgcgc-3′ and 5′-tcacgaattctcgcaatgcttggtctttttaggtgg-3′.
Immunoprecipitation and immunoblotting
Cos-7 cells on 100-mm plate were transfected by the 246630-ORF-pMH construct with Effectene (QIAGEN) according to manufacturer's instructions. After incubation for 48 h at 37°C, transfected cells were lysed by adding 1.0 ml lysis buffer (1% Triton, 0.5% NP40, 1 mM EDTA in phosphate-buffered saline PBSA, which contains 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4,) containing freshly added protease inhibitors (1 μg each of Leupeptin and Pepstin per ml, 10 μg each of Aprotinin and Trypsin per ml, and 0.5 mM of Phenylmethylsulfonyl Floride per ml) at 0°C for 10 min. Insoluble material was removed by centrifugation for 30 min at 11 000 r.p.m. at 4°C. The cell lysate was incubated with 1 μg anti-HA antibody (Roche) at 4°C for 3 h and then absorbed with 25 μl of protein G-agarose beads at 4°C overnight. The immune complexes were washed twice in ice-cold cell lysis buffer supplemented with protease inhibitors and then in 0.5 M NaCl, supplemented with protease inhibitors. The immunoprecipitates were eluted and denatured by boiling for 5 min in sodium dodecyl sulphate (SDS) sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.25% bromophenol blue). The immunoprecipitates were resolved by 12% SDS polyacrylamide gel electrophoresis (PAGE) and then transferred to PVDF membrane (Amersham) according to standard protocol. The membrane was blocked for 2 h in PBSA (5% nonfat milk, 0.1% Tween 20), rinsed, with PBSA (0.1% Tween 20), and then incubated with Anti-HA-peroxidase (1 : 1000 dilution after reconstitution as 25 u/μl) (Boehringer Mannheim) for 1 h in PBSA (0.5% nonfat milk, 0.1% Tween 20). The membrane was washed in PBSA (0.5% nonfat milk, 0.1% Tween 20) for 50 min with changing solution every 10 min. The proteins were detected by the Western blotting detection reagents ECLTM (Amersham).
The 246630-ORF-pMH construct was transfected into several ovarian cancer cell lines by Effectene (QIAGEN) according to manufacturer's instructions. After incubation for 48 h at 37°C, 105–106 transfected cells were trypsinized and seeded to 100-mm dishes. Medium was added with G418 (400 μg/ml to 1200 μg/ml) for surviving selection. Two to four weeks later, colonies on plates were fixed with Methanol/Glacial Acid (3 : 1 in volume) and stained with Crystal Violet in H2O (1 mg/ml). Colonies were counted manually and the results were analysed by paired t-test for means for statistical significance.
The accession numbers for PAC/BAC sequences RP4-655C5:AL121956; RP3-427A4:Z98049, RP3-497J21:AL023775, RP1-168L15:AL022069, RP11-514012:AL159163, RP4,505P2:AL133458, RP1-167A14:Z94721, RP11-517H2:AL121935, RP11-366H19:AL353591, RP11-568A1:AL353747, RP3-366N23:AL021331, RP3-431P23:AL009178, RP11-178P20: AL592444. The accession numbers for cDNA sequences are, TCP10:U03399; CCR6: U45984, GPR31:U65402, FOP:Y18046. The accession numbers for clones 246630: AJ419865; 199297: AJ419866; 174311: AJ419867.
Acquati F, Morelli C, Cinquetti R, Bianchi MG, Porrini D, Varesco L, Gismondi V, Rochetti R, Talevi S, Possati L, Magnanini C, Tibiletti MG, Bernasconi B, Daidone MG, Shridhar V, Smith DI, Negrini M, Barbanti-Brodano G, Taramelli R . 2001 Oncogene 20: 980–988
Altschul SF, Gish W, Miller W, Myers EW, Lipman D . 1990 J. Mol. Biol. 215: 403–410
Anand R, Riley JH, Butler R, Smith JC, Markham AF . 1990 Nucleic Acids Res. 18: 1951–1956
Anderson MA, McFadden GI, Bernatzky R, Atkinson A, Orpin T, Dedman H, Tregear G, Fernley R, Clarke AE . 1989 Plant Cell. 1: 483–491
Baba M, Imai T, Nishimura M, Kakizaki M, Takagi S, Hieshima K, Nomiyama H, Yoshie O . 1997 J. Biol. Chem. 272: 14893–14898
Blanche H, Wright LG, Vergnaud G, de Gouyon B, Lauthier V, Silver LM, Dausset J, Cann HM, Spielman RS . 1992 Genomics 12: 826–828
Catteau A, Harris WH, Xu CF, Solomon E . 1999 Oncogene 18: 1957–1965
Chenevix-Trench G, Kerr J, Hurst T, Shih YC, Purdie D, Bergman L, Friedlander M, Sanderson B, Zournazi A, Coombs T, Leary JA, Crawford E, Shelling AN, Cooke I, Ganesan TS, Searle J, Choi C, Barrett JC, Khoo SK, Ward B . 1997 Genes Chromosomes Cancer 18: 75–83
Cooke IE, Cox SA, Shelling AN, Le Meuth VG, Spurr NK, Ganesan TS . 1996a Mamm. Genome 7: 157–159
Cooke IE, Shelling AN, Le Meuth VG, Charnock ML, Ganesan TS . 1996b Genes Chromosomes Cancer 15: 223–233
Francesconi M, Meryn S, Rogan AM, Szalay S, Graninger W, Schmidbauer CP . 1984 Cancer 53: 1927–1930
Gaidano G, Hauptschein RS, Parsa NZ, Offit K, Rao PH, Lenoir G, Knowles DM, Chagnati RS, Dalla-Favera R . 1992 Blood 80: 1781–1787
Greaves DR, Wang W, Dairaghi DJ, Dieu MC, Saint-Vis B, Franz-Bacon K, Rossi D, Caux C, McClanahan T, Gordon S, Zlotnik A, Schall TJ . 1997 J. Exper. Med. 186: 837–844
Hime G, Prior L, Saint R . 1995 Gene 158: 203–207
Ioannou PA, Amemiya CT, Garnes J, Kroisel PM, Shizuya H, Chen C, Batzer MA, de Jong PJ . 1994 Nat. Genet. 6: 84–89
Ioerger TR, Clark AG, Kao TH . 1990 Proc. Natl. Acad. Sci. USA 87: 9732–9735
Islam SD, Pilder SH, Decker CL, Cebra-Thomas JA, Silver LM . 1993 Human Mol. Genet. 2: 2075–2079
Katso RMT, Manek S, O'Byrne K, Playford MP, Le Meuth V, Ganesan TS . 1997 Cancer Metas. Rev. 16: 81–107
Kurihara H, Nonaka T, Mitsui Y, Ohgi K, Irie M, Nakamura KT . 1996 J. Mol. Biol. 255: 310–320
Laval S, Butler R, Shelling AN, Hanby AM, Poulsom R, Ganesan TS . 1994 Cell Growth Differ. 5: 1173–1183
Liao F, Alderson R, Su J, Ullrich SJ, Kreider BL, Farber JM . 1997a Biochem. Biophys. Res. Comm. 236: 212–217
Liao F, Lee HH, Farber JM . 1997b Genomics 40: 175–180
Liu Y, Ganesan TS . 2001 Re. Reprod in press
Mancini DN, Rodenhiser DI, Ainsworth PJ, O'Malley FP, Singh SM, Xing W, Archer TK . 1998 Oncogene 16: 1161–1169
McClure BA, Haring V, Ebert PR, Anderson MA, Simpson RJ, Sakiyama F, Clarke AE . 1989 Nature 342: 955–957
Menasce LP, Orphanos V, Santibanez-Koref M, Boyle JM, Harrison CJ . 1994 Genes Chromosomes Cancer 10: 286–288
Mungall AJ, Humphray SJ, Ranby SA, Edwards CA, Heathcott RW, Clee CM, Holloway E, Peck AI, Harrison P, Green LD, Butler AP, Langford CF, William RG, Huckle EJ, Baron L, Smith A, Leversha MA, Ramsey YH, Clegg SM, Rice CM, Maslen GL, Hunt SE, Scott CE, Soderlund CA, Theaker AJ, Carter NP, Ross MT, Deloukas P, Bentley DR, Dunham I . 1997 DNA Sequence 8: 151–154
Obata K, Morland SJ, Watson RH, Hitchcock A, Chenevix-Trench G, Thomas EJ, Campbell IG . 1998 Cancer Res. 58: 2095–2097
Orphanos V, McGown G, Hey Y, Thorncroft M, Santibanez-Koref M, Russell SE, Hickey I, Atkinson RJ, Boyle JM . 1995 Br. J. Cancer 71: 666–669
Popovici C, Zhang B, Gregoire MJ, Jonveaux P, Lafage-Pochitaloff M, Birnbaum D, Pebusque MJ . 1999 Blood 93: 1381–1389
Rodriguez C, Causse A, Ursule E, Theillet C . 2000 Genes Chromosomes Cancer 27: 76–84
Saha V, Lillington DM, Shelling AN, Chaplin T, Yaspo ML, Ganesan TS, Young BD . 1995 Genes Chromosomes Cancer 14: 220–222
Saito S, Saito H, Koi S, Sagae S, Kudo R, Saito J, Noda K, Nakamura Y . 1992 Cancer Res. 52: 5815–5817
Saito S, Sirahama S, Matsushima M, Suzuki M, Sagae S, Kudo R, Saito J, Noda K, Nakamura Y . 1996 Cancer Res. 56: 5586–5589
Sassa H, Nishio T, Kowyama Y, Hirano H, Koba T, Ikehashi H . 1996 Mol. Gen. Genet. 252: 222–
Schein CH . 1997 Nature Biotech. 15: 529–536
Shelling AN, Cooke IE, Ganesan TS . 1995 Br. J. Cancer 71: 521–527
Shizuya H, Birren B, Kim UJ, Mancino V, Slepak T, Tachiiri Y, Simon M . 1992 Proc. Natl. Acad. Sci. USA 89: 8794–8797
Tibiletti MG, Bernasconi B, Furlan D, Riva C, Trubia M, Buraggi G, Franchi M, Bolis P, Mariani A, Frigerio L, Capella C, Taramelli R . 1996 Cancer Res. 56: 4493–4498
Tibiletti MG, Sessa F, Bernasconi B, Cerutti R, Broggi B, Furlan D, Acquiti F, Bianchi M, Russo A, Capella C, Taramelli R . 2000 Clin. Cancer Res. 6: 1422–1431
Tibiletti MG, Trubia M, Ponti E, Sessa L, Acquati F, Furlan D, Bernasconi B, Fichera M, Mihalich A, Ziegler A, Volz A, Facco C, Riva C, Cremonesi L, Ferrari M, Taramelli R . 1998 Oncogene 16: 1639–1642
Trubia M, Sessa L, Taramelli R . 1997 Genomics 42: 342–344
Walter MH, Liu JW, Wunn J, Hess D . 1996 Eur. J. Biochem. 239: 281–293
Wan M, Zweizig S, D'Ablaing G, Zheng J, Velicescu M, Dubeau L . 1994 Int. J. Oncol. 5: 1043–1048
Ye ZH, Droste D . 1996 Plant Mol. Biol. 30: 697–709
Zingoni A, Rocchi M, Storlazzi CT, Bernardini G, Santoni A, Napolitano M . 1997 Genomics 42: 519–523
Dr Iain Goldsmith is acknowledged for synthesis of oligonucleotides, Mr Reginald Boone and Dr Anna Richardson for assistance with sequencing. The FOP clone was obtained from Dr MJ Pebusque. YAC 17IA12 was obtained from Dr J Boyle. Gridded YAC and cDNA libraries were obtained from the MRC HGMP centre. We would like to thank all the members of the chromosome 6 project group at Sanger Centre. The work described in this paper is supported by the Imperial Cancer Research Fund, Association of International Cancer Research, Wellbeing and Human mapping/sequencing at the Sanger Centre is funded by the Wellcome Trust.
About this article
PLoS ONE (2014)
A Role for the Mitochondrial Pyruvate Carrier as a Repressor of the Warburg Effect and Colon Cancer Cell Growth
Molecular Cell (2014)
Journal of Proteomics (2012)
Proceedings of the National Academy of Sciences (2011)