The MLL gene is reciprocally translocated with one of a number of different partner genes in a proportion of human acute leukaemias. The precise mechanism of oncogenic transformation is unclear since most of the partner genes encode unrelated proteins. However, two partner genes, AF10 and AF17 are related through the presence of a cysteine rich region and a leucine zipper. The identification of other proteins with these structures will aid our understanding of their role in normal and leukaemic cells. We report the cloning of a novel human gene (BRL) which encodes a protein containing a cysteine rich region related to that of AF10 and AF17 and is overall most closely related to the previously known protein BR140. BRL maps to chromosome 22q13 and shows high levels of expression in testis and several cell lines. The deduced protein sequence also contains a bromodomain, four potential LXXLL motifs and four predicted nuclear localization signals. A monoclonal antibody raised to a BRL peptide sequence confirmed its widespread expression as a 120 Kd protein and demonstrated localization to the nucleus within spermatocytes.
The analysis of chromosomal translocations affecting chromosome 11 at band q23 in leukaemias has shown that these events involve fusions between the human trithorax (MLL) gene (Djabali et al., 1992; Gu et al., 1992; Tkachuk et al., 1992; Ziemin-van der Poel et al., 1991) and a variety of partner genes (Bernard and Berger, 1995) each providing a COOH region to the resultant fusion protein. These translocations result in in-frame fusions suggesting an important role for the different COOH regions in the oncogenic transformation. This is supported by the demonstration that a germline fusion between MLL and AF9, created by homologous recombination, results in leukaemias in chimeric mice (Corral et al., 1996).
The t(10;11)(p12;q23) translocation which occurs in acute myeloid leukaemia (AML) has been shown to result in fusion of the 3′ portion of the AF10 gene to the 5′ portion of the MLL gene (Chaplin et al., 1995a). The AF10 gene encodes a protein with two identifiable features, a cysteine rich region (CRR) at its amino terminus and a putative leucine zipper sequence towards the carboxy terminus. It is now clear that the MLL-AF10 fusion is rather unusual in that the MLL and AF10 genes lie in opposite orientations with respect to the chromosomal telomeres (Beverloo et al., 1995). Inversion of one of the genes has to occur in order to achieve a stable fusion, thus accounting for the inversion and insertion of parts of the long arm of chromosome 11 into the short arm of chromosome 10 noted in previous cytogenetic studies (Le Beau et al., 1985). Despite the complexity of the AF10 translocations a consistent feature seems to be the fusion of the leucine zipper domain to MLL (Chaplin et al., 1995b). Another important feature of AF10, so far unusual for MLL partner genes, is that it has been found rearranged with genes other than MLL, namely CALM, in the U937 cell line (Dreyling et al., 1996) and HEAB in an invins(10;11)(p12;q23q12) in a 2 year old child with acute monoblastic leukaemia (Tanabe et al., 1996). AF17, the gene involved in the t(11;17) translocation (Prasad et al., 1994) encodes a protein containing a cysteine rich region and a potential leucine zipper domain, both highly homologous with the same features in AF10. The breakpoints within AF17 also occur between the CRR and the leucine zipper, and similarly result in the fusion of the 3′ end of the AF17 gene with the 5′ end of the MLL gene.
The first part of the CRR in AF10 and AF17 consists of a C4HC3 conserved putative zinc finger motif known as the LAP domain (Saha et al., 1995), PHD finger (Aasland et al., 1995) or TTC domain (Koken et al., 1995). The remainder of the CRR in AF10 and AF17 (116 amino acids) contains a cluster of 12 conserved cysteines and histidines. A similar CRR structure has been identified in a protein of unknown function encoded by the gene BR140 (Thompson et al., 1994). Interestingly, the MLL protein also contains the elements found in this CRR, i.e. LAP/PHD with a similar cluster of 12 cysteines and histidines located 250 residues downstream. An important observation is that in the translocations so far described, the CRR regions of MLL, AF10 and AF17 are either lost or disrupted following translocation, suggesting an important oncogenic role for this region. We describe the cloning, chromosomal mapping and expression of a new human gene designated BRL (BR140 Like gene) which encodes a protein with a CRR related to similar structures in MLL, AF10, AF17 and BR140.
Cloning and sequence analysis of human BRL
The homology between the cysteine rich regions of AF10, AF17 and BR140 suggested that this may be a functionally important domain which might be found in other proteins. Comparison of the cDNA sequences for these genes indicted that certain regions could be sufficiently conserved to allow a degenerate PCR approach to search for new members of this family. A clone with 200 bp of novel sequence (corresponding to nt 1352 – 1551) was isolated (pb1) which had greatest homology to BR140. cDNA clones were identified on screening a Jurkat library with pb1 and all but two of the clones were overlapping. The contiguous sequence contained that in pb1 and appeared to lack only small 5′ and 3′ portions of the gene. A RACE technique was used to obtain the 5′ sequence and database searches identified overlapping EST entries (Accession nos H07921, H11244 and H11599) which provided 3′ sequence up to the poly(A)+ tail. To confirm the derived sequence, the full length coding sequence of BRL was amplified from adaptor ligated placental cDNA (Clontech) using primers BRL for and BRL Rev (Table 1). The product (4.2 kb) was cloned into the TA Topo vector (Invitrogen) and its analysis confirmed the derived sequence and did not identify any extra exons.
The full nucleic acid sequence obtained (4617 nucleotides) is shown in Figure 1. The largest open reading frame translates to a protein of 1058 amino acids in length. Database searches have established that the deduced amino acid and nucleic acid sequences have the highest homology to those encoded by BR140, a human gene of unknown function which has been mapped to chromosome 3p25 (Gregorini et al., 1996). There is overall 56% identity at the amino acid level (Figure 2) and the new gene has therefore been named BRL (BR140 Like). The region of greatest homology (84% identity) between BRL and BR140 corresponds to a cysteine rich domain from C217 to H388. This region also has significant homology to the equivalent cysteine rich domains in AF10, AF17 and CEZF. The CRR can be considered in two parts: the first 46 amino acids can be configured as a LAP or PHD motif, a feature which has been identified in a wide range of proteins from different species where it is found as a single or repeated domain (Saha et al., 1995). The remaining carboxy part of the CRR in BRL is homologous to similar regions in AF10, AF17 and BR140. Two further regions of BRL are also highly homologous to BR140: residues 501 – 688 contain a bromodomain and show 61% identity; residues 925 – 1058 show 63% identity but contain no recognized motif. The bromodomain lies between residues 560 and 667 and is a motif which is found regularly in transcriptional coactivators (Jeanmougin et al., 1997). At least four potential nuclear localization signals, distributed throughout the protein sequence, were identified using PSORT, a program designed to search for protein localization motifs (Nakai and Kanehisa, 1992). Interestingly we have also identified four motifs which could potentially bind nuclear receptors (Heery et al., 1997). These have the consensus sequence LXXLL where L represents leucine and X represents any amino acid and they are found at residues 538 – 542; 567 – 571; 718 – 722 and 791 – 795.
After the cloning of the cDNA sequence of BRL, database searches identified segments within a genomic entry (Accession No Z98885) with 100% identity to BRL. It was evident that this represented the genomic sequence of BRL and analysing the start and finish sequences of the matches showed that these were consistent with donor/acceptor splice sites and that the matched sequences corresponded to exons. Based on this analysis, 12 exons can be determined for the sequence we have cloned and these are listed in Table 2. Exon 1 is the largest exon and comprises the 5′ UTR as well as the coding region for the entire cysteine rich region and two potential nuclear localization signals.
Figure 3a shows that BRL is present as a single transcript of approximately 4.6 kb and that there is a variable level of expression with the highest levels observed in testis. Probing of the cell lines (Figure 3b) again demonstrates a single band of the same size with a high level of expression seen in each lane, except for the Raji cell line where the β-actin control suggests inadequate loading of this lane. No evidence for alternatively spliced transcripts was found.
In situ hybridization
The high level of BRL mRNA expression in testis prompted us to investigate which cells were responsible for this. Using a radioactive in-situ hybridization technique there was clear positive staining with a BRL antisense probe mainly within spermatocytes (Figure 4). Spermatogonia at the basement membrane of the seminiferous tubules and the more mature spermatids and spermatozoa towards the tubular lumina tended to be negative. Under the same conditions a BRL control sense probe was negative and the antisense β-actin probe showed staining within all cell types within the tubules and also gave a signal within the smooth muscle of vessels.
Examination of the database of murine EST sequences suggested that murine and human BRL sequences would be highly conserved, as are murine and human AF10. Using appropriate murine EST sequences, sense and antisense BRL oligonucleotide probes were created and used for non-radioactive in situ hybridization experiments on mouse tissues (Figure 5). A pattern of staining similar to that in human tissues was obtained. It was then possible to extend this technique to other mouse tissues. Within the thymus, a proportion of lymphocytes within the cortex and medulla stained positively with the BRL antisense oligonucleotide probe. Neurons in the cerebral cortex and hippocampus gave a positive signal whereas all three cell layers of the cerebellar cortex showed no signal. In contrast, the β-actin probe did not give a strong signal in the neurons of the cerebellar cortex. In all cases the BRL sense probe gave no detectable signal.
The predicted size of the BRL protein is approximately 119.5 kD. Figure 6 shows a blot of whole cell lysates of the HeLa (S3) cell line and various human tissues probed with a monoclonal anti-BRL antibody against peptide 1 (see Materials and methods). The HeLa (S3) cell line shows a strong band of the expected size. In each of the tissues examined there is, in addition to a band of the expected size, a second smaller band: in testis, thymus, ovary and kidney the smaller band is in the region of 74 kD whereas in the brain the lower band is approximately 83 kD.
Subcellular distribution of AF10
The subcellular localization of BRL protein (Figure 7) was determined using immunohistochemistry with the monoclonal anti-BRL antibody. In testis it can be seen that the protein localizes to the nucleus and that, as predicted from the in-situ hybridization studies, the spermatocyte population is clearly positive whereas spermatogonia and spermatozoa are negative. In the thymus, the staining is also nuclear but is weaker in intensity and, as predicted from the in-situ hybridization, is present in only a proportion of the lymphocytes. In the cerebral cortex and hippocampus, the neurons stain positively within the cytoplasm, with little if any staining in the nuclei. In the cerebellar cortex, the neurons of the molecular layer showed positive nuclear staining while the Purkinje cells show both nuclear and cytoplasmic staining. About 50% of the neurons of the granular cell layer gave a clear positive nuclear signal while the remainder were completely negative. A range of other tissues was analysed (results not shown). Strong positive nuclear signals were present in myeloid and erythroid precursor cells in bone marrow, within duodenal and colonic mucosal epithelium, in tonsillar epithelium and in breast acinar epithelium. Weak staining was seen within tonsillar lymphocytes and ovarian stromal cells.
Since the AF10 and AF17 genes are involved in leukaemic translocations the chromosomal location of BRL might indicate whether it also could be involved in such events. FISH analysis using a P1 clone containing BRL demonstrated a clear signal on chromosome 22 band q13 (Figure 8). This finding was confirmed by examination of 15 ESTs that matched BRL with high homology. Among these, a particular sequence (Accession No H55108) had been derived by exon amplification from a cosmid sequence (Trofatter et al., 1995) previously mapped to human chromosome 22. This EST corresponds to bases 1492 – 1648 of the BRL sequence. Additionally, bases 3137 – 3209 of the BRL sequence were found in an STS (Accession No L04558) which mapped to chromosome 22 (D22S55) (MacCollin et al., 1993). Additionally, the genomic clone match for BRL was also described as originating from chromosome 22 band q13 (Accession No Z98885).
The deduced BRL protein sequence reveals a number of interesting motifs. The first 46 amino acids of the CRR contain a LAP/PHD motif, a feature which has been found in a wide variety of proteins and has been described in detail elsewhere (Saha et al., 1995). There are currently little experimental data on the functional role of the LAP/PHD domain. Fusion constructs of Drosophila TRX containing the LAP/PHD domain were used to demonstrate that this region could bind zinc more strongly than other cysteine rich regions within this protein (Mazo et al., 1990). The N terminus of the plant protein HAT3.1 contains a LAP/PHD motif and it can bind non-specifically in vitro to any fragment of DNA greater than 100 bp (Schindler et al., 1993). Regulatory proteins such as those in the trithorax and polycomb group have the capacity to modify chromatin structure and it has been suggested that the LAP/PHD domain may be instrumental in this interaction (Aasland et al., 1995). Although the exact role of the LAP/PHD domain in AF10 and related family members is not yet known, the PHD/LAP fingers of a protein known as AIRE have been shown to be necessary for correct nuclear localization (Rinderle et al., 1999). It is interesting that the translocation breakpoints in MLL, AF10 and AF17 usually occur outside the cysteine rich regions such that these elements are lost from the critical fusion proteins (Bernard and Berger, 1995). The exceptions to this include one adult case of acute myeloid leukaemia demonstrating a t(10;11) translocation (Chaplin et al., 1995a,b) and also the t(10;11) translocation of U937 cell line (Dreyling et al., 1996), in both of which the breaks in AF10 occur within the CRR. It is possible that the cysteine rich regions are important to the transcriptional regulation of myeloid cells (Saha et al., 1995) and that their loss or disruption is critical to malignant transformation in haematopoietic cells.
In addition to the cysteine rich region, BRL also has a bromodomain. This motif was first described following the cloning of the brahma (brm) gene in drosophila (Haynes et al., 1992; Tamkun et al., 1992) and has since been identified in other transcriptional co-activators including the human brahma protein, BRM (Muchardt and Yaniv, 1993) and the yeast protein SNF2/SWI2 (Laurent et al., 1993). The cell cycle gene 1 (CCG1) (Sekiguchi et al., 1991) which was later identified as hTAFII 250 (Hisatake et al., 1993; Ruppert et al., 1993) and shown to be necessary for G1 progression in mammalian cells also contains a bromodomain. More than one bromodomain can be present within a protein: polybromo, a gene of unknown function isolated from chicken monoblasts, encodes a putative protein with five bromodomains (Nicolas and Goodwin, 1996). The bromodomain was originally described as a sequence of about 60 amino acids, potentially forming two α-helices (Haynes et al., 1992). However this motif has recently been redefined (Jeanmougin et al., 1997) extending to approximately 110 residues with two additional α-helices, each on either side of the two existing helices. The bromodomain in BRL exhibits these same conserved features.
The bromodomain appears to be highly conserved through evolution and is associated with many transcription factors having a role in histone acetylation including yeast and human GCN5 (Brownell et al., 1996; Candau et al., 1996; Georgakopoulos and Thireos, 1992; Wang et al., 1997), p300/CBP (Arany et al., 1994; Arias et al., 1994; Bannister and Kouzarides, 1996; Chrivia et al., 1993; Kwok et al., 1994; Ogryzko et al., 1996), pCAF (Yang et al., 1996) and hTAFII 250 (Mizzen et al., 1996). It has, however, been shown by several groups that the bromodomain is dispensable for HAT activity (Candau et al., 1997; Mizzen, 1996 #77; Ogryzko, 1996 #76). It may provide a surface for protein-protein contacts (Haynes et al., 1992) and it has been suggested that it has a role in directing HAT activity to chromatin `receptors' (Brownell and Allis, 1996). Studies of the binding of p300 to the activating transcription factor-2 (ATF-2) have highlighted the importance of the bromodomain of p300 (Kawasaki et al., 1998). Specifically, the binding of p300 to ATF-2 requires both the bromodomain and the second cysteine/histidine rich region of p300. ATF-2 is the first protein shown to interact with the bromodomain of p300. The helix – turn – helix – turn region of the bromodomain of human GCN5 has recently been shown to bind to the p70 subunit of the human Ku heterodimer which is the DNA binding component of the DNA dependent protein kinase (DNA-PK) holoenzyme (Barlev et al., 1998). The downstream effect of this interaction is the phosphorylation of GCN5 and inhibition of its HAT activity. Evidence is therefore growing to support the idea that the bromodomain has the potential to interact with different types of protein and, at least in certain situations may regulate HAT activity.
It is interesting that there are some genes involved in leukaemic translocations in which one or other of the patterns may encode a LAP motif or a bromodomain. CBP contains a bromodomain and has been implicated in the t(11;16) translocation with MLL and in the t(8;16) translocation with MOZ (Borrow et al., 1996). CBP has been shown to mediate the transcription of cyclic AMP response element (CRE) or mitogen responsive genes (Arias et al., 1994; Chrivia et al., 1993; Kwok et al., 1994) and interact with the basal transcription apparatus through TFIIB (Kwok et al., 1994). CBP or its close homologue, p300, can also interact with p300/CBP associated factor (P/CAF) which, as part of a protein complex is capable of histone acetylation (Brownell et al., 1996). p300 also contains a bromodomain and has been implicated in a t(11;22) translocation with MLL (Ida et al., 1997). In each of these cases, the partner gene to CBP or p300 contains one or more LAP domains. MOZ is an interesting partner in that the fusion transcript resulting from the t(8;16) translocation retains the coding sequences for most of the key elements from both proteins including the LAP domains from MOZ. The MOZ LAP domains were also retained in a recently analysed case of acute mixed lineage leukaemia with an inv(8)(p11q13) where MOZ was fused in frame with TIF2 (Liang et al., 1998). In the fusion proteins which result from these cases it is possible that the normal functioning of the LAP domains is modified or redirected in some way, possibly through aberrant histone acetylation since it is predicted that MOZ has type A acetyltransferase activity. The presence of a predicted LAP containing cysteine rich region and bromodomain in BRL together with the recurring presence of these motifs in leukaemic translocations strongly suggest that this is a potential transcription factor and one which, at the very least, can be considered as a potential target for malignant transformation. (Rinderle et al., 1999).
In view of the putative leucine zipper region which has been described for AF10 (aa 766 – 794) and BR140 (aa 605 – 651), we searched but could not find a similar motif in BRL. We subsequently employed a database resource (Lupas et al., 1991) to search more generally for coiled coil motifs. This programme accurately predicted the coiled coil configuration of the putative leucine zipper region of AF10. It failed, however to reliably predict coiled coil structures in either BRL or BR140: neither contained a sequence of greater than 35 residues with probabilities of >80 – 90% that were predictive of a coiled coil structure.
Northern analysis shows that the strongest signals were obtained in testis and in cell lines where a rapid and regular cell turnover is expected. The in-situ hybridization analysis and immunohistochemistry on human and mouse testis and also in thymus confirms this impression. However, similar levels of mRNA and similar strengths of nuclear protein signal were also seen in cerebral and hippocampal neurons, cell populations which are considered permanent and in which there is no cell turnover. These findings suggest that BRL is not simply associated with cell cycle and must relate to other cellular functions common to a range of cell types.
The Western blot data on the human tissues shows a second smaller band which is approximately 74 kD in all tissues except brain where it is approximately 83 kD. This is unlikely to be due to alternative splicing in BRL mRNA since a single species is consistently observed on Northern blotting. Other possibilities include alternative start sites for translation or variations in post-translational modifications. Clearly, in the brain at least, where the lower band differed in size from the other tissues, there is a differential pattern of staining on immunohistochemistry. The cerebral cortical neurons show only cytoplasmic staining, Purkinje cells show both nuclear and cytoplasmic staining and only a proportion of the granular layer neurons are positively stained. It is possible that the 83 kD band represents a truncated BRL protein lacking key nuclear localization signals resulting in cytoplasmic staining.
The homology between BRL and AF10 and its mapping to chromosome 22q13 suggests that BRL could be involved in translocations affecting this region. However reports of translocations in leukaemia involving chromosome 22q13 are rare. The first reported case was described in 1992 (Lai et al., 1992) in a 73 year old man who had an acute monocytic leukaemia with a t(8;22)(p11;q13) translocation. A similar translocation in a second patient was reported by the same group in 1996 (Soenen et al., 1996) in a patient who had AML-M5, which transformed from a chronic myelomonocytic leukaemia. The p300 gene which has also been located on 22q13 was cloned as a partner gene to MLL in an AML (Ida et al., 1997). It remains to be established whether BRL will also be implicated in haematopoietic neoplasia.
Materials and methods
The cDNA sequences of AF10, AF17 and BR140 were compared and several conserved regions were noted within the encoded cysteine rich region. Degenerate oligonucleotide primers (DP1 and DP2) were synthesized to two such regions (see Table 1) and used to amplify products from randomly primed cDNA synthesized from peripheral blood cells as previously described (Chaplin et al., 1995a,b). Fresh PCR products were cloned using the Original TA cloning kit (Invitrogen, San Diego, CA, USA). One clone, pb1, was identified as containing novel sequence related to BR140, AF10 and AF17.
A cDNA library prepared from the Jurkat cell line was screened with pb1 and subsequently with a second probe (pb2) which was constructed by amplifying a 385 bp fragment from within the non-conserved region of the sequence provided by pb1 screening. Probes were radiolabelled with α-32P-CTP using the Multiprime labelling kit (Amersham, UK). The library filters were prehybridized for 1 h at 65°C and hybridized overnight at 65°C in buffer containing 7% sodium dodecyl sulphate (SDS), 0.5 M sodium phosphate (pH 7.2) and 1 mM ethylenediaminetetra-acetic acid (EDTA). Washing was performed at room temperature and then stringently at 65°C for 1 h in 0.1% SDS, 40 mM sodium phosphate (pH 7.2). Filters were exposed to X-ray film (Genetic Research Instrumentation Ltd., UK) overnight at −70°C with intensifying screens. Three rounds of hybridization were performed to isolate the clones of interest.
5′ rapid amplification of cDNA ends (RACE)
This was performed using the Marathon cDNA amplification kit (Clontech Laboratories, CA, USA). Adaptor ligated oligo-dT primed cDNA was synthesized from placental mRNA supplied with the kit. Primary amplifications were performed using a gene specific primer (Mac 69) and a kit adaptor primer (AP1). A second internal gene specific primer (Mac 16) and a kit adaptor primer (AP2) were used in the nested amplifications (see Table 1). Mac 69 and Mac 16 were each located close to the 5′ end of the gene and separated by 115 nt. Fifty μl reactions were set up as follows: 5 μl adaptor ligated cDNA, 5 μl Klentaq buffer, 1 μl Advantage Klentaq polymerase mix (50×), 1 μl gene specific primer (10 μM), 1 μl kit primer (10 μM), 1 μl dNTP (10 mM), 36 μl water. A touchdown PCR protocol was used in primary amplifications: 94°C for 1 min; five cycles at 94°C (30 s), 72°C (4 min); five cycles at 94°C (30 s), 70°C (4 min); 25 cycles at 94°C (20 s), 68°C (4 min). In nested amplifications, the cycling parameters were: 94°C for 1 min; 20 cycles at 94°C (30 s), 55°C (30 s), 68°C (2 min). The PCR reactions were performed in a Perkin-Elmer TC-1 thermocycler and the nested products were cloned (Original TA cloning kit) and sequenced.
Amplifiction of full length BRL
Marathon ready cDNA (Clontech) was used as template for amplification of full length BRL in order to confirm part of the sequence obtained from library screening. Only a single round of amplification was required. The reaction was set up as follows: 5 μl of Marathon ready cDNA, 1 μl each of gene specific primers at 10 μM (BRL For and BRL Rev, Table 1), 5 μl Klentaq buffer, 1 μl Advantage Klentaq polymerase mix (50×), 1 μl dNTP (10 mM) and 36 μl water. The PCR was performed as follows: 94°C (30 s) followed by 30 cycles of 94°C (5 s), 68°C (4 min).
Standard miniprep or caesium chloride maxiprep protocols were followed to prepare plasmid DNA from positive clones. Sequencing along both strands was performed using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer). The protocols were sequenced on an automated ABI 373 sequencer and analysed using DNASTAR software.
The Human Multiple Tissue Northern Blot II (Clontech Laboratories, CA, USA) was probed with α-32P-CTP-labelled pb2 following the manufacturer's protocol. The blot was prehybridized for 30 min and then hybridized for 1 h at 68°C in Expresshyb buffer (Clontech). Several washes were performed at room temperature in 2×SSC, 0.05% SDS for 40 min and then two washes in 0.1% SSC, 0.1% SDS each for 20 min at 50°C. The blot was then exposed to X-ray film for up to 3 weeks. The level of RNA loading in each lane on the blot was determined by probing with the supplied β-actin probe under the same hybridization conditions and then exposed to X-ray film with intensifying screens for 80 min. A Northern blot of cell lines (Clontech) was probed under similar conditions.
All glassware was wrapped in aluminium foil and baked at 180°C. Human tissue sections were permeabilized with proteinase K (20 mg/ml in PBS) at 37°C for 10 min when the reaction was stopped by two 5 min washes in a 0.2%(w/v) solution of glycine in PBS. The tissues were then post-fixed with freshly made 4% paraformaldehyde for 20 min and acetylated for 10 min in a solution of 0.1 M triethanolamine containing 1.25 ml acetic anhydride. Sections were then washed in PBS, dehydrated through ethanol and air-dried.
Sense and antisense RNA transcripts of BRL were prepared from the same 385 bp sequence as the Northern blot probe. SacI and XbaI restriction sites were added to the primer sequences to facilitate cloning of the amplified product in the PGEM-3Zf(+) vector. The plasmid was linearized with EcoRI to prepare BRL antisense transcript and AccI to prepare BRL sense transcript. The β-actin probe represents an EcoRI/RsaI fragment of an original clone pHFβA 3′ ut (Ponte et al., 1983) from the 3′ untranslated region of human β-actin. This was cloned to the pSP73 vector (Promega) and its use as a positive control has been documented previously (Zandvliet et al., 1996). The Promega Riboprobe in vitro Transcription system was used to label the probes with 35S-UTP (Amersham). Unincorporated label was removed using a Chromaspin-30 column (Clontech) and the integrity of the labelled probe was assessed on a 6% polyacrylamide gel. The counts per minute (c.p.m.) were determined on a scintillation counter and the probe concentration was adjusted such that 2×106 c.p.m. in hybridization buffer were applied to each section. The hybridization solution consisted of the following volumes: 10% of a 0.2% Denhardt's solution (Sigma) in a 10× salts solution (3 M NaCl, 100 mM Na2HPO4, 100 mM Tris-HCl, 50 mM EDTA); 50% formamide (Sigma); 3% rRNA (Sigma); 20% dextran sulphate (Sigma); 1% 1 M DTT (Sigma); 16% DEPC treated water and labelled riboprobe. The hybridization solution was boiled at 100°C for 1 min, spun briefly and 25 μl applied to each section. Sections were coverslipped and incubated at 55°C overnight in slide boxes with blotting paper soaked in 50% formamide and 1×SSC solution.
The post hybridization washes were as follows: four washes with 50% formamide at 55°C over 3 – 4 h followed by ten 5 min washes with TNE to remove all formamide. The slides were then incubated with 100 mg/ml RNase A in TNE buffer at 37°C for 1 h to remove any non-hybridized probe and washed twice in 2×SSC for 30 min at 65°C followed by a single wash in 0.5×SSC for 30 min at 65°C. The sections were then dehydrated through graded ethanols containing 0.3 M ammonium acetate and air dried.
The slides were coated in a solution of Ilford K5 emulsion, allowed to dry and then sealed in wooden slide rack holders and stored at 4°C for 10 days (β-actin control) or 15 days (BRL sense and antisense). The sections were subsequently developed in Kodak D-19 developer, fixed, counterstained in Giemsa and mounted in DPX.
Non radioactive in situ hybridization (NISH)
The cDNA sequence of BRL was used to search the non-redundant and mouse est databases for murine sequences which matched human BRL. Several overlapping sequences were identified within the 5′ end of the gene and also within the 3′UTR which matched the human sequence, confirming that these sequences did in fact represent the murine homologue of BRL. BRL sense and antisense oligonucleotide probes (Table 1) were synthesized to a 33 bp sequence within the 5′ region of the murine homolgue and labelled at both the 5′ and 3′ ends with digoxigenin (Boehringer-Mannheim, Germany). A 41-mer mouse β-actin antisense oligonucleotide probe was also synthesized and similarly labelled (MWG-Biotech, UK). Mouse tissue sections were dewaxed and pre-hybridized in 1× Denhardt's solution, 36.5% formamide, 2.5×SSC and 0.25 mg/ml t-RNA for 1 h at 42°C. Sections were then dehydrated and hybridized for 18 h at 42°C within a sealed wet chamber in a solution containing 100 ng of oligonucleotide probe, 1× Denhardt's solution, 36.5% formamide, 2.5× SSC and 0.17 mg/ml t-RNA. Subsequently the sections had three 15 min washes at 42°C in ×2 SSC followed by a 30 min wash in 1× SSC at room temperature and finally two 5 min washes in 0.1 M PBS. Sections were then blocked for 2 min in 0.1% Triton X-100/0.1 M PBS, washed in PBS and incubated in 0.2% alkaline phosphatase conjugated anti-digoxigenin antibody (Boehringer-Mannheim, Germany) and 5% goat serum in 0.1 M PBS for 1 h at room temperature. Subsequently the tissues had two 10 min washes in 10 mM KPO4 buffer, 0.5 M NaCl, 1 mM EDTA, 0.5% Triton X-100 and 0.1% BSA. The reaction product was detected using NBT/BCIP (Sigma) as substrate in buffer (100 mM Tris-HCl, 100 mM NaCl, 16 mM MgCl2) in an overnight incubation in the dark. Levamisole (Sigma) was added in the final detection step at a concentration of 10 – 15 mM to inhibit endogenous alkaline phosphatase activity.
Production of anti-BRL antibodies
The amino acid sequences of BRL and BR140 were compared and two peptide sequences were chosen for synthesis from regions which were dissimilar. Peptide 1 corresponded to amino acids 692 – 702 (RRPFSWEDVDR) and peptide 2 corresponded to amino acids 805 – 818 (CGDSEVEEESPGKR). The peptides were made as amides and conjugated to KLH (Affiniti Ltd). Two mice were immunized with each peptide as follows: the first innoculation consisted of 10 μg of peptide made up to 100 μl in PBS and admixed with 100 μl of Freund's complete adjuvant. Three subsequent booster doses were given at 3, 7 and 11 weeks each with 10 μg of peptide and Freund's incomplete adjuvant. Test bleeds were performed at 10 days following each booster dose and assayed by enzyme-linked immunosorbent assay (ELISA) or Western blotting. Following the last test bleed, one mouse was selected as showing a stronger response than the rest and this mouse was boosted intravenously at 15 weeks with 10 μg of peptide containing no adjuvant. The mouse was sacrificed 4 days later and fusion performed on splenic cells. The hybridomas were then plated out on 96 well plates and the supernatants screened by ELISA. Those wells which screened positive were expanded; single cell cloning was then performed by doubling dilution and the wells screened by ELISA. Positive clones were analysed by Western blotting and immunohistochemistry. A monoclonal antibody to peptide 1 was derived by this approach and used for all subsequent studies.
One hundred microlitres of a 1 mg/mL solution of unconjugated peptide was added to the wells of a Falcon 3912 microassay plate and incubated overnight at 4°C and washed in PBS and tap water. Free protein binding sites on the plate were blocked overnight with 10% Marvel in PBS. The plates were washed with tap water and incubated with 90 μl of hybridoma supernatent for 2 h at room temperature. The plate was washed with 0.05% Tween 20 in PBS, left for 3 min and then washed a further 5 – 6 times with water. Eight microlitres of peroxidase conjugated goat anti-mouse Ig (BioRad), diluted 1/2500 in PBS was added to each well and incubated for 1 h at room temperature. After a further wash with 0.05% Tween 20 in PBS, blocking was performed with 10% Marvel in PBS for 10 min. The wells were then rinsed in tap water and the reaction developed using o-phenylenediamine (Sigma) as substrate. Those supernatants which gave a positive reaction were subsequently analysed by Western blotting and immunohistochemistry.
Tissue specimens and HeLa (S3) cell line
Normal human tissues, formalin fixed and paraffin embedded, were obtained from the archives of the Histopathology Unit at the Imperial Cancer Research Fund (ICRF). Normal mouse tissues were taken from 3 month old C57Black males, fixed immediately in 4% freshly made paraformaldehyde solution and kept at 4°C overnight prior to paraffin embedding. Five micrometre sections were taken of tissues for immunohistochemistry or in situ hybridization. Those taken for in situ analysis were cut using a new disposable DEPC washed microtome blade and the sections floated out onto DEPC treated water. The HeLa (S3) cell line was obtained from the Cell Production Laboratory at the ICRF and the cells were maintained in RPMI 1640 medium containing 10% foetal calf serum with penicillin and streptomycin and incubated at 37°C in 5% CO2.
Human tissue sections were dewaxed and endogenous peroxidase blocked by incubating with 0.5% methanol in H2O2 for 30 min and then washing in water for 5 – 10 min. Microwave antigen retrieval was performed by immersing the sections in a pressure cooker (Nordic Ware, USA) containing pre-boiled 0.01 M sodium citrate buffer, pH 6, and subsequently heated further on high setting for 8 – 10 min. The sections were washed three times in PBS and then blocked with 200 μl of 1/25 rabbit serum (Dako). Two hundred microlitres of 1/250 BRL antibody was incubated with each tissue section for 1 h at room temperature. After a further three washes in PBS, the sections were incubated with 1/300 biotin conjugated rabbit anti-mouse Ig (Dako) for 1 h at room temperature and then washed again before applying a 1/500 dilution of horseradish peroxidase conjugated streptavidin (Dako) for 30 min. The reaction was detected using diaminobenzidine tetrahydrochloride (Sigma) and hydrogen peroxide. The slides were counterstained with haematoxylin and mounted in Pertex mounting medium (Cellpath Ltd, UK).
Protein from HeLa (S3) cells was extracted using standard techniques (Harlow and Lane, 1988). Human protein samples of testis, thymus, brain, ovary and kidney were obtained commercially (Clontech). Fifty micrograms of each protein sample was electrophoresed under denaturing conditions on a 7.5% acrylamide gel, blotted to Hybond-C extra (Amersham) using a semi-dry technique and blocked overnight at 4°C in 5% Marvel and 0.02% Tween-20 in PBS. Blots were probed with anti-BRL antibody for 1 h at room temperature in 3% Marvel, 0.02% Tween-20, washed three times in 0.5% Tween-20 in PBS followed by a 20 min incubation with secondary antibody (1 : 1000 goat anti-mouse horseradish peroxidase linked F(ab)2 fragments. After three further washes, positive bands were detected using the ECL system (Amersham).
Chromosomal localization of BRL
A P1 library (supplied by the Reference Library Database at the Max Planck Institut fur Molekulare Genetik, Berlin) was screened to obtain a probe large enough to perform fluorescent in situ hybridization (FISH): P1 clones have an average insertsize of 70 kb. The gridded filters were probed with radiolabelled pb2 as before. Two positive clones were identified and DNA was prepared from one. The P1 probe was fluorescein isothiocyanate (FITC) labelled by nick-translation using biotin d-ATP and the Gibco Bio-nick kit (Gibco – BRL Life Technologies, UK) and competitively hybridized with metaphase spreads from a trisomy 21 patient. The results were analysed on a Biorad-Lasersharp MCR 600 confocal microscope.
The sequence reported in this paper has been deposited in the GenBank database (Accession no: AF005067).
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This work was funded by the Camilla Samuel Research Fellowship held by P McCullagh at the ICRF. We gratefully acknowledge the support and advice of Professor TA Lister. We are grateful for the support of Jane Steele and Maggie Stubbs of the Hybridoma Laboratory, ICRF. The Jurkat cell line cDNA library was a gift from Dr J Dunne (ICRF). We thank Miss V Lewis (medical student) for technical assistance.
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