Transcriptional Mapping in a 700-Kb Region Around the DXS52 Locus in Xq28: Isolation of Six Novel Transcripts and a Novel Atpase Lsoform (Hpmcas) Nina S

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RESEARCH Transcriptional Mapping in a 700-kb Region Around the DXS52 Locus in Xq28: Isolation of Six Novel Transcripts and a Novel ATPase lsoform (hPMCAS) Nina S. Heiss, Ute C. Rogner, Petra Kioschis, Bernhard Korn, and Annemarie Poustkal

Deutsches Krebsforschungszentrum, Abteilung Molekulare Genomanalyse, 69120 Heidelberg, Germany

The chromosomal band Xq28 has been a focus of interest in human genetics because >20 hereditary diseases have been mapped to this region. However, about two-thirds of the disease genes remain uncloned. The region around the polymorphic DXS52 locus (ST14} within Xq28 lies in the candidate regions for several as-yet-uncloned disease genes. So far, only four melanoma antigen genes (MAGE) and the human biglycan (BGN} gene, have been mapped within the 700-kb stretch around DXS52, suggesting that more genes may reside in this region. By combining exon trapping and direct cDNA selection methods, we sought to identify novel transcripts around the DXS52 locus, in addition to recovering the MAGE and BGN genes, we isolated and mapped six putative novel genes (XAPI03-XAPI08), the caltractin gene, and a gene encoding a novel Ca2§ ATPase isoform (hPMCA5). The newly isolated sequences were considered as representing parts of putative genes if they contained at least one unique exon-trap product and/or at least one expressed sequence tag {EST) from sequence data bases and if, in addition, they showed evidence of expression by RT-PCR and/or Northern blot analysis. Our data facilitated the integration of the transcript map with the physical map around the DXS52 locus. Future analysis of the novel genes as candidates for Barth syndrome (BTHS) and chondrodysplasia punctata {CDPX2) is in progress.

The most distal region of the long arm of the The 7.5-Mb region that spans Xq28, exhibits human X chromosome, especially band Xq28, drastic variations in chromosomal substructure has been a focus of interest with respect to gene that is exemplified by diverging distributions of identification. The correlation of an exception- Alu and L1 repetitive elements (Rogner et al. ally high CpG island density and GC-content 1994), CpG islands (Pilia et al. 1993), and gene with a high gene density in Xq28 (Bernardi 1989; content (Palmieri et al. 1994). Because the GC- Pilia et al. 1993), has made it the most intensively content was found to peak between the color vi- studied region of the human genome. Accord- sion and G6PD loci (Pilia et al. 1993), the hunt ingly, biological and medical interest has esca- for genes in this particular region was most in- lated because of the many disease loci that are triguing. Although the region around more linked to this region. The construction and inte- proximal loci such as DXS52 has been of lesser gration of physical (Poustka et al. 1991; Dietrich interest with respect to gene identification, it has et al. 1992; Kioschis et al. 1994; Palmieri et al. nonetheless deserved continuous attention be- 1994; Rogner et al. 1994) and transcript maps cause of its interesting chromosomal structure. (Korn et al. 1992; Bione et al. 1993; Sedlacek et al. The DXS52 loci, also referred to as the ST14 se- 1993), as well as the cloning of disease genes has quence family, belong to the most polymorphic progressed rapidly and is ongoing. Of the 22 ge- loci in the human genome and consist of three netic disorders with Xq28 linkage, however, 14 reiterating copies within 60 kb (Oberl~ et al. remain uncloned. 1985). It is suggested that this polymorphism arose owing to a variation in a number of tandem 1Corresponding author. repeats (VNTRs). Alternatively, rearrangements E-MAIL [email protected]; FAX (40) 6221 423454. or point mutations may have contributed to such

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TRANSCRIPT MAP OF DXS52 REGION IN Xo28

heterogeneity (Oberl6 et al. 1985). Furthermore, a tion of the disadvantages. In this report, we de- separate polymorphic locus, DXS15, is linked to scribe the isolation and mapping of known and the ST14 sequence cluster (Oberl6 et al. 1985; Pat- novel genes in a 700-kb region around the DXS52 terson et al. 1987). Despite the close proximity of loci between the DXS1104 (Chatterjee et al. the DXS52 and DXS15 markers, recombinational 1994) and ST35.638 (Palmieri et al. 1994) events were observed between these loci, indica- markers. tive of a hot spot for recombination in this region (Brown et al. 1988; Bell et al. 1989). Although the recombinational activity between DXS52 and RESULTS DXS15 was refuted by Patterson et al. (1989) and Strategies Employed for the Isolation of Feil et al. (1990), the data of Brown et al. (1988), Transcripts together with the problems associated with the subcloning of this region into yeast artificial Two strategies were employed for the isolation of chromosomes (YACs) (Rogner at al. 1994) and coding sequences in the DXS52 region. One ap- cosmids (P. Kioschis, pers. comm.), suggests an proach involved using a combination of the com- inherent, regional instability. Conceivably, the plex exon-trap and enriched cDNA-selection instability may contribute to a higher mutation products as probes on the region-specific cDNA rate and consequently to an altered disease geno- and exon-trap sublibraries, respectively. The sec- type. Although the significance of such polymor- ond approach involved the hybridization of spe- phisms with respect to gene density and expres- cific cosmid fragments onto the same subli- sion is unknown, the DXS52 polymorphic locus braries. was found to be highly conserved among mouse The complexities of the exon-trap and cDNA- and hamster, suggestive of a small cluster of a selection products were initially compared by hy- related, expressed gene family (Oberl6 et al. 1985; bridization onto the cosmids from which they Feil et al. 1990). were derived (Fig. 1). Most of the cosmid frag- In light of the many uncloned disease-caus- ments hybridizing strongly with the exon-trap ing genes such as Barth syndrome (BTHS) (Adds probe were also positive for the cDNA-selection et al. 1993), Waisman syndrome (Gregg et al. probe (Fig. 1). Although the complexity of the 1991), Happle syndrome (Traupe et al. 1992), cDNA-selection probe was higher in that many oto-palatodigital syndrome (Biancalana et al. more cosmid fragments were positive, hybridiza- 1991), Goeminne syndrome (Zuffardi et al. tion with a CA-positive probe (poly[d(A-C)] Phar- 1982), X-linked mental retardation (MRX3; macia) showed that most of these extra hybridiz- Gedeon et al. 1991; Nordstr6m et al. 1992), and ing bands were CA-repeat containing fragments dyskeratosis congenita (Connor et al. 1986), the (Fig. 1). Despite the CA-richness of the region, candidate regions of which span the DXS52 loci, the cDNA-selection products (Fig. 1) also hybrid- a considerably small amount of genes have been ized to fragments that were unique for the cDNA- mapped to this area. To date, only the melanoma selection probe only. For completeness, we there- antigen genes (MAGE)-2, -3, -6, and -12 (Rogner fore isolated these extra hybridizing cosmid et al. 1995) and the human biglycan genes (BGN) bands and utilized them as probes on the grid- (Traupe et al. 1991; Kioschis et al. 1994) have ded, enriched cDNA and exon-trap sublibraries. been mapped around the DXS52 loci. By combining exon-trapping and cDNA-selection tech- Isolation of Known Genes in the DXS52 Region niques, we endeavoured to isolate novel genes in a 700-kb region around the DXS52 loci. By implementing the cosmid fragment hybrid- Although cDNA selection (Lovett et al. 1991; ization approach, two individual cDNA clones Parimoo et al. 1991; Korn et al. 1992) and exon and two unique exon-trap products that had trapping (Buckler et al. 1991; Church et al. 1993; high homologies to the MAGE-2, -3, -6, and -12 North et al. 1993) are sufficiently sensitive to iso- genes were isolated. Upon hybridizing a 3' end late rarely expressed transcripts from large chro- conserved MAGE probe (CH089; Rogner et al. mosomal regions, both methods have their draw- 1995) onto the cosmid digests, the MAGE genes backs when used in isolation (Brennan and were found to lie on cosmids LLNL K0238 [iso- Hochgeschwender 1995). A combination of the lated from the Lawrence Livermore National two methods, however, is synergistic, facilitating Laboratory (LLNL)-chromosome X cosmid li- complementation of the advantages and elimina- brary], Qc4G10, and QclD2, from which the cos-

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HEISS ET AL.

Figure 1 Cosmid digests showing the distal part of the contig extending from the most proximal of the three DXS52 markers to ST35.638. The top, middle and bottom panels show the positive bands on hybridizing with the complex exon-trap probe, the complex cDNA-selection probe, and the CA- repeat probe respectively. All of the strongly positive exon-trap bands (top) were in common with the strongly positive cDNA-selection bands (middle) and are marked by closed triangles. (bottom) Four of these exon-trap- and cDNA-selection-positive bands were CA-positive (T). Most of the extra hybridizing bands positive with the cDNA-selection probe were CA-containing fragments (V) (middle, bottom). The map positions of the novel genes and the BGN gene are indicated at the top of the panels. Cosmids labeled Qc were isolated by hybridization screening of gridded clone libraries of an Xq28-specific cosmid library constructed from the hamster/human cell hybrid QIZ. Cosmids isolated from the Lawrence Livermore National Laboratory chromosome X cosmid library are preceded by LLNL.

mid-fragment probes had been generated. This provided confirmation of the physical map posi- tion of the MAGE genes proximal to the DXS52 VNTR (Fig. 2; Rogner et al. 1995). The weak hybridizations of the complex cDNA selection and exon-trap probes with the MAGE-positive cosmid fragments (data not shown), were in agreement with the observation of a low abundance of MAGE clones in both sublibraries. The BGN gene was recovered from the enriched cDNA library as a 1040-bp cDNA contig, corresponding to exons 2-7, by hybridizing specific fragments from cosmids LLNL M1937 and Qc12H4 (Fig. 1). No BGN exons were recovered from the exon trap library. Upon hybridizing a BGN probe (PG1; Fisher et al. 1991) onto the cosmid digests, the BGN-positive bands correlated well with the same signals obtained for the cDNA-selection probe and their ab- sence in the exon-trap probe (Fig. 1). Thus, the mapping of BGN distal to the third DXS52 locus was also confirmed (Fig. 2). Recently, the caltractin gene was mapped to Xq28 (Tanaka et al. 1 1994) and its refined mapping to the DXS52 re- more, hybridization of a KpnI probe (Shafit- gion at the YAC level was shown (Chatterjee et al. Zagardo et al. 1982) onto the cosmid digests (data 1995). By isolating a 870-bp caltractin cDNA con- not shown), facilitated integration of the gene tig from the enriched cDNA sublibrary, and by mapping data with the distribution of LINE ele- remapping these clones onto cosmids Qc9C8 and ments in the DXS52 region. This 700-kb stretch Qc17C4, a more precise mapping of the caltractin of DNA was shown to contain three short gene at the cosmid level and more specifically stretches of LINE-containing and three short distal to the MAGE genes but proximal to the first stretches of LINE-free regions. The MAGE genes DXS52 locus was accomplished (Fig. 2). Further- mapped to the first LINE-free region, whereas cal-

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TRANSCRIPT MAP OF DXS52 REGION IN )(028

to contain at least one unique exon-trap product. In an attempt to extend the length of the cDNA contigs and to find additional exon- trap products, specific cDNA clones were hybridized back onto the exon- trap and cDNA sublibraries. Consequently, 138 additional cDNA and 310 additional exon-trap clones were identified. However, owing to a high degree of redundancy for both the cDNA and exon-trap libraries, only extensively overlapping sequences were found such that neither the length of the cDNA contigs nor the number of unique exon- trap products were in- creased substantially. Ulti- mately, the cDNA contigs were composed of 99 sequenced cDNA clones from Figure 2 Map showing the physical cosmid and YAC maps in conjunction the enriched cDNA library with the transcription map of the DXS52 region from DXS1104 to ST35.638. The and 43 sequenced exon-trap marker positions are shown as dotted, vertical lines, whereas the known genes products. and novel cDNAs are shown as solid, vertical lines. The right ends of the YACs We defined a cDNA are indicated by -91. Probes Y458-1 to Y458-3 correspond to IRS-PCR frag- contig as being part of a ments isolated from YAC 458 (Rogner et al. 1994). The scale at the top of the novel gene if it fulfilled at map refers to the distance of the DXS52 region in megabases from the telomere least two of four criteria: (1) (Poustka et al. 1991 ). The light- and dark-shaded horizontal bar at the bottom of the presence of at least one the map shows the LINE-free (light) and LINE-containing (dark) regions with the genes mapping to the LINE-free regions. Cosmids indicated by Qc are described exon-trap product; (2) ho- in Fig. 1. Cosmids isolated from the Lawrence Livermore National Laboratory mology to at least one ex- chromosome X cosmid library are preceded by L, which is an abbreviation for pressed sequence tag (EST) LLNL. from the data base (the sub- sequent sequencing of EST clones followed by primer tractin and BGN mapped to the second and third walking facilitated a consid- LINE-free regions, respectively (Fig. 2). erable increase in the length of those cDNA contigs that contained known ESTs); (3) the presence of an RT-PCR product that coincided with the Isolation of Novel cDNAs in the DX$52 Region length of the expected fragment as calculated Upon hybridizing the complex exon-trap and from the existing sequence; and (4) evidence of cDNA-selection probes onto the enriched cDNA expression from Northern blot hybridizations. By and exon-trap libraries, respectively, a total of 82 subjecting each of the cDNA contigs to these initial cDNA clones and 50 initial exon-trap analyses, evidence that they were parts of poten- clones were identified and sequenced. Compari- tially six novel genes was obtained. sons of the sequences of these clones in XGAP showed that the cDNAs, which had an average Analysis of the Novel cDNA Contigs length of 300 bp, frequently overlapped to form distinct cDNA contigs, most of which were found The sequenced cDNAs were assembled into over-

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HEISS ET AL. lapping contigs and were named XAPI03 to quence indicated that XAP103a represented the XAP108. Because of the strong redundancy of the 3' end of a novel gene (Table 1). An open reading clones it was possible to calculate an accurate frame (ORF) of 120 amino acids was also con- consensus sequence for each cDNA contig. The tained in the XAPIO3a sequence and additionally length of the consensus sequences ranged from revealed the presence of part of the coding re- 630 to 3310 bp (Table 1). BLASTN and BLASTX gion. (Table 1). XAP103b evidently represented alignments were carried out on all individual part of the S' coding region of the gene that con- cDNA clones as well as on the final consensus tained one unique sequenced exon-trap product sequences. (Table 1). At the DNA level, XAP103b demon- XAP103 consisted of two cDNA contigs, strated a 61% homology to two human ESTs as XAP103a and XAP103b, with lengths of 3310 bp well as a 62% and 66% homology, respectively, and 830 bp, respectively (Table 1). Both XAP103a to mouse KRAB and mouse mkrS mRNA se- and XAP103b exhibited a 7.S-kb transcript ex- quences encoding zinc finger proteins (Table 1). pressed mainly in heart, brain, skeletal muscle, XAPIO3b contained one unique sequenced exon- and pancreas but expressed weakly in placenta, trap product (Table 1). BLASTX analyses showed liver, and kidney (Fig. 3A). XAP103a and that the ORF of 219 amino acids exhibited ho- XAP103b also mapped to the same cosmids and mologies of 33% identical and 62% similar accordingly these two cDNA contigs were consid- amino acids to a human ZNF43 zinc finger pro- ered as belonging to the same gene. RT-PCR tein and an average of 37% identical and SS% analyses revealed that XAPIO3a and XAPIO3b ex- similar amino acids to zinc finger proteins from hibited the expected 1110- and 400-bp products, mouse, rat, and chicken (Table 1). respectively, in the three tissues tested which in- XAP104 consisted of a sequence of 1260 bp cluded brain, muscle, and liver (Table 1). and exhibited ubiquitous expression of a 2-kb XAP103a contained two unique, sequenced transcript in all tissues present on the multiple exon-trap products. BLASTN alignments revealed tissue Northern blot (Fig. 3A). Evidently, the that XAPIO3a exhibited homologies of between length of the consensus sequence comprised al- 93% and 100% to 19 largely redundant ESTs most a full-length cDNA. RT-PCR facilitated am- while BLASTX comparisons demonstrated that plification of an expected 1030-bp fragment in XAP103a exhibited no homologies to any known brain, muscle, and liver (Table 1). XAP104 con- proteins (Table 1). The presence of an AATAAA tained two unique, sequenced exon-trap prod- polyadenylation signal and a polyadenylation ucts. BLASTN analysis showed that XAPI04 ex- tail at the end of the XAPIO3a consensus se- hibited homologies of ~<98% to three human

Figure 3 (A) Multiple tissue Northern blots showing size and tissue distribution of the transcripts obtained for XAP103, XAP104, and XAP105. (B) Agarose gel showing size of RT-PCR products and tissue expression for XAP106, XAP107, and XAP108.

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TRANSCRIPT MAP OF DXS52 REGION IN X028

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HEISS ET AL.

ESTs (Table 1). BLASTX protein comparisons pro- primer combinations used in the RT-PCR experi- vided evidence for this gene representing a novel ments on genomic DNA. In this way, however, dehydrogenase because an average amino acid no exon-intron boundaries were detected. We identity of 40% and 65% similarity to the choles- considered that, owing to the strong conserva- terol NAD(P)-dependent dehydrogenase of Nocar- tion between human and mouse coding se- dia over the entire ORF of 413 amino acids was quences, hybridization of the novel cDNAs to detected (Table 1). This sequence also demon- mouse genomic DNA would provide further evi- strated homologies to the 3-~-hydroxysteroid de- dence that XAP106, XAP107, and XAP108 repre- hydrogenase of diverse species such as mouse, sented parts of genes. Strong cross-hybridization rat, human, rhesus macaque, cattle, and rainbow signals were obtained for all the novel cDNA con- trout (Table 1). tigs when hybridized onto YACs derived from the The XAP105 cDNA contig comprised 3100 bp homologous mouse DXS52 region (data not (Table 1) and hybridized to a 5-kb transcript on shown; Chatterjee et al. 1994). Northern blots showing obvious expression in placenta but weak expression in kidney and pancreas (Fig. 3A). The more sensitive RT-PCR analy- Mapping of the Novel cDNA Contigs ses showed that XAPI05 is also expressed in High-resolution mapping of the novel cDNAs brain, muscle, and liver because the expected was achieved by hybridizing specific cDNAs from 1190-bp fragment was amplified in all these tis- each contig onto the cosmid digests. In this way, sues (Table 1). BLASTN alignments showed that XAP103-XAP108 were shown to map distal to XAPI05 exhibited high homologies of between the three polymorphic DXS52 loci (Fig. 2). It also 93% and 100% to clusters of redundant ESTs became apparent that the novel genes mapped to (Table 1). BLASTX comparisons failed to find any the second and third LINE-free DNA stretches homologies to any known proteins or protein around the DXS52 polymorphism (Fig. 2). motifs for the ORF comprising 307 amino acids (Table 1). XAPI06, XAP107, and XAP108 exhibited Isolation, Analysis, and Mapping of the Novel lengths of 1500 bp, 680 bp, and 630 bp, respec- ATPase lsoform ~hPMCA5~ tively (Table 1). The expression of these cDNAs was beneath the detection level of a Northern Fragments from cosmids Qcl2H4, LLNL F0731, blot but was nonetheless evident by RT-PCR and LLNL C092 positive for the cDNA selection analysis. The presence of the expected 500-bp probe only (Fig. 1), led to the isolation of cDNA fragment in human fetal liver but not in fetal contigs of 450 bp and 899 bp. These consensus brain and adult skeletal muscle (Fig. 3B), showed sequences exhibited highest homologies to iso- a selective expression of XAP106 for the three form 3 of the rat calmodulin-sensitive plasma tissues tested. The expected 220-bp fragment was membrane Ca2+-transporting ATPase (rPMCA3; amplified from all three tissues for XAPI07 (Fig. J05087) cDNA sequence with values ranging 3B). The selective expression of XAPI08 in hu- from 81% to 92% identity for different segments man adult skeletal muscle and human fetal brain along the sequence. Homologies to the known became evident when the expected 250-bp frag- human isoforms were generally lower than to the ment was amplified only from these two tissues rPMCA3 isoform and were highest to the human (Fig. 3B). Although XAPI06 contained two ATPase isoform 2 (hPMCA2; J03754, L00620) unique, sequenced exon-trap products, and with 78%. Similarly, at the protein level, the XAP107 and XAP108 contained one sequenced novel hPMCA consensus sequences revealed exon-trap product each, none of these cDNA con- highest homologies to the rPMCA3 (pir2:A34308) tigs demonstrated homologies to any known protein with 80% identity and 82% similarity. DNA or protein sequences (Table 1). All three Homologies to the human isoforms 2 cDNA contigs did however contain ORFs of 112, (pir2:A38871) and 3 (hPMCA3; pir:A35547) were 102, and 51 amino acids for XAPI06, XAPi07, again lower with identities averaging 77% and and XAP108, respectively (Table 1). Despite the 73% and similar amino acids averaging 84% and presence of exon-trap products and positive ex- 81% for the hPMCA2 and hPMCA3 proteins, re- pression in the RT-PCR assay, we attempted to spectively. To demonstrate the extent of homol- verify further that XAP106, XAPI07, and XAPI08 ogy between the novel hPMCA cDNAs, the represented parts of novel genes by testing the rPMCA3, hPMCA2, and hPMCA3 isoforms, a

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CLUSTAL alignment of the consensus sequences of the novel hPMCA sequence with any of the was performed in HUSAR (Fig. 4). This shows known PMCA sequences in the databank, was that although the homologies of all PMCA se- further confirmed at the genomic level by shot- quences are very high, each protein had a distinct gun cloning the cosmids known to bear the novel sequence. Lower homologies were also observed hPMCA sequence and recovering the same cod- to the human Ca2+-ATPase isoform 4, the rat iso- ing sequence as before. We name this novel forms 1, 2, and 4, and the PMCA1 isoforms of pig hPMCA isoform hPMCA5. Localization of and rabbit (data not shown). The lack of identity hPMCA5 was verified by hybridizing the cosmid digests with a known hPMCA3 probe (Burk and Shull 1992; Fig. 2), which rPMCA3 SLGLSFYAPPGEESEACGNVSGGAEDEGEAL~GWIEGAAILLSVICVVLVTAFNDWSKEK hPMCA2 SLGLSFYHPPGEGNEGCATAQGGAEDEGEAEAGWIEGAAZLLSVICVVLVTAFNDWSKEK because of its high con- hPMCA3 SLVLSFYRPAGEENELCGQVATTPEDENEAQAGWZEGAAILFSVIIVVLVTAFNDWSKEK servation gave the same hPMCA5 ...... GWIEGAAILLSVICVVLVTAFNDWSKEK ********* ee, ************** signals as the hPMCA5

rPMCA3 QFRGLQSRIEQEQKFTVIRNGQLLQVPVAALWGDIAQVKYGDLLPADGVLIQGNDLKID probe. Hence, we have iden- hPMCA2 QFRGLQSRIEQEQKFTVVRAGQWQIPVAEIWGDIAQVKYGDLLPADGLFIQGNDLKID tified a novel hPMCA iso- hPMCA3 QFRGLQCRIEQEQKFSIIRNGQLIQLPVAEIVVGDIAQVKYGDLLPADGILIQGNDLKID hPMCA5 QFRGLQSRIEQEQKFTVIRNGQLLQVPVAALVVGDIAQVKYGDLLPAIXgVLIQANDLKID form, hPMCA5, that maps ******~ **.~ ~ ** ****** to the DXS52 region imme- rPMCA3 ESSLTGESDHVRKSADKDPMLLSGTHVMEGSGRMVVTAVGVNSQTGIIFTLLGAGGEEEE diately distal to BGN in hPMCA2 ESSLTGESDQVRKSVDKDPMLLSGTHVMEGSGRMLVTAVGVNSQTGIIFTLLGAGGEEEE hPMCA3 ESSLTGESDHVKKSLDKDPMLLSGTHVMEGSGVVTAVGVNSQTGIILTLLGVNEDDEG the third LINE-free region hPMCA5 ESSLTGESDHVCKSADKNPMLLSGTHVMEGSGRMVVTAVGVNSQTGIIFTLLGAGGEEEE ******,wt., ,, ** ****************************** **** ~ .., (Fig. 2).

rPMCA3 KKDKK ...... AKKQDGAVAM hPMCA2 KKDKKGVKKGDGLQLPAAIXgAAASNAADSANASLVNGIQ~QDGN S QSKAKQQDGAAAM hPMCA3 EKI~KKGKKQG ...... VPENRNKAKTQDGVAL hPMCA5 KKDKKGKQ ...... QDGAMESSQTKAKKQDGAVAM DISCUSSION , ** ,o

rPMCA3 EMQPLKSAEGGEMEEREKKKANVPKKEKSVLQGKLTKLAVQIGKAGLVMSAITVIILVLY To date only the MAGE-2, hPMCA2 EMQPLKSAEGGDADDR--KKASMHKKEKSVLQGKLTKLAVQIGKAGLVMSAITVIILVLY -3, -6, and -12 (Rogner et al. hPMCA3 EIQPLNSQEGIDNEEKDKKAVKVPKKEKSVLQGKLTRLAVQIGKAGLLMSALTVFILILY hPMCA5 EMQPLKSAEGGEMEEREKI~KANAPKKEESVLQGKLTKLAVQIGKAGLVMSAITVIZLVLY 1995) and BGN (Traupe et ..*** , ** . ~ * *** ************************** **.** al. 1991; Kioschis et al. rPMCA3 FVIETFVVIX~RVWLAECTPVYVQYFVKFFIIGVTVLVVAVPEGLPLAVTISLAY~ 1994) genes have been hPMCA2 FTVDTFVVNKKPWLPECTPVYVQYFVKFFIIGVTVLVVAVPEGLPLAVTISLAY~ hPMCA3 FVIDNFVINRRPWLPECTPIYIQYFVKFFIIGITVLWAVPEGLPLAVTISLAYSVIKIKMM mapped to the DXS52 re- hPMCA5 LVIETFVVEGRTWLAECT~QYFVKFFIIGVTVLWAVPEGLPLAVTISLAY~ .o.**. ** ********************************************* gion (see Fig. 2). In retro- spect of the high disease rPM~3 KDNNLVRHLDACETMGNATAICSDKTGTLTTNRMTVVQSYLGDTHYKEIPAPSALTPKIL hPM~2 KDNNLVRHLDACETMGNATAICSDKTGTLTTNRMTVVQAYVGDVHYKEIPDPSSINTKTM gene density in Xq28, of ~M~3 KDNNLVRHLDACETMGNATAICSDKTGTLTMNRMTVVQAYIGGIHYRQIPSPDVFLPKVL ~M~S ~D ...... --- which seven uncloned dis- ** ease genes lie within the DXS52 region, it was antici-

rPMCA3 DLLVHAISINSAYTTKILPPEKEGALPRQVGNKTECALLGFILDLKRDFQPVREQIPEDQ pated that potential novel hPMCA2 ELLINAIAZNSAYTTKILPPEKEGALPRQVGNKTECGLLGFVLDLKQDYEPVRSQMPEEK disease genes could be iso- hPMCA3 DLIVNGISINSAYTSKILPPEKEGGLPRQVGNKTECALLGFVTDLKQDYQAVRNEVPEE K hPMCAS ---VHAISINSAYTTKILPPEKEGALPRQVGNKTEYALLGFVLDLKRDFQPVREQIPEDK lated. To accomplish the iso- .. *~ ********** **** ***o*o. ** ~176 lation of new transcripts rPMCA3 LYKVYTFNSVRKSMSTVIRMPDGGFRLFSKGASEILLKKCTNILNSNGELRGFRPRDRDD from the 700-kb DXS52 re- hPMCA2 LYKVYTFNSVRKSMSTVIKLPDEHVRMYSKGASEIVLKKCCKILNGAGEPRVFRPRDRDE hPMCA3 LYKVYTFNSVRKSMSTVIRNPNGGFRMYSKGASEIILRKCNRILDRKGEAVPFKNKDRDD gion defined by markers hPMCA5 LYKVYTFNSVRKSMSTVIRMPDGGFRLFSF~GSEILLKKCTNILNSNGELRGFRPRDGAA DXS1104 (Chatterjee et al. 1994) and ST35.638 (Palm- rPMCA3 MVKKZ I EPMAC DGLRTI C IAYRDF SAI QEPDWDNENEVVGDLTC IAVVGI EDPVRPEVPE hPMOA2 MVKKVI EPMACDGLRT I CVAYRDFP S S pE PDWDNENDI LNELTC I CVVGI EDPVRPEVPE ieri et al. 1994), we em- hPMCA3 MVRTVI EPMACDGLRT I C IAYRDFDDT - Ep SWDNENE I LTELTC IAVVGI EDPVRPEVPD ployed a combination of hPMCA5 LLGRFLGPMACDGLRT I C IAYRDFSAGQEPDW ...... ***********, ***** ** * exon trapping, cDNA selec- Figure 4 Protein CLUSTAL alignment of the rat ATPase isoform 3 (rPMCA3), tion, and cosmid fragment human ATPase isoforms 2 (hPMCA2) and 3 (hPMCA3) with the novel human hybridizations. isoform hPMCA5. (*) Positions of identical amino acids; (.) positions of similar Comparisons of the but conserved amino acids; spaces refer to different amino acid positions that exon-trapping and cDNA- may alter the protein structure. A break in the hPMCA5 sequence is shown as selection products dem- dots on the left-hand side of the alignment. onstrated the specificity

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of the combined approach. Unwanted contami- ther indicative of the presence of coding regions. nating background products such as vector cryp- Based on this reasoning, we considered that the tic splice products and genomic repetitive se- presence of at least one EST and/or one exon-trap quences that are accumulated by the exon trap- product together with an indication of expres- ping and cDNA selection methods, respectively, sion detected by RT-PCR and/or Northern blot were automatically eliminated. The specificity analysis was sufficient as a verification that we was evident when using the exon-trap and were dealing with expressed sequences. cDNA-selection products as probes on cosmid di- XAPI03 consisted of two cDNA contigs, gests, as well as when using them as probes on XAPIO3a and XAP103b, that corresponded to the the gridded libraries. Considering that all cDNA 3' end and part of the 5' coding regions respec- isolation methods may lead to the extraction of tively of the same gene. Considering the large contaminating genomic sequences and/or un- size of the 7.5 kb XAPI03 transcript and the find- spliced cDNAs, the combined approach offers the ing that XAPI03, XAPI06, and XAP107 mapped opportunity of recovering genuine cDNAs with a to the same cosmids, it is conceivable that the reasonably high probability. This was also con- latter two cDNA contigs also belonged to the firmed by the observation that most of the extra same gene. A reason for the failure of XAPI06 products that were enriched by cDNA selection and XAP107 to hybridize on Northern blots only, were genomic, CA-containing fragments. could be explained in terms of alternative splic- The selectivity of the approach, however, also being mechanisms. Because such products may ei- came evident because the cDNA selection prod- ther be absent, present in selected tissues, or oc- ucts also exhibited unique signals on cosmid di- cur in concentrations lower than the detection gests. This phenomenon was reflected by the in- level of a Northern blot, it is possible that complete recovery of genes when using the XAPI06 and XAP107 arose via alternative splic- combined approach. Clearly, although the two ing. Moreover, the explanation correlates with methods synergize in that the isolation of arti- the ability to isolate differentially spliced tran- facts is almost completely eliminated, the com- scripts when utilizing the exon-trap method bination of the methods lacks completeness in (North et al. 1993). From the Northern data it was that cDNA selection can enrich for coding se- clear that XAP104 and XAP105 represented indi- quences not arnplifiable by exon trapping and vidual transcripts each representing a single tran- vice versa (Brennan and Hochgeschwender script of 2 kb and 5 kb, respectively. XAPI08 did 1995). The selectivity of the combined approach not exhibit a transcript at the Northern blot level prompted a more thorough search for transcripts but expression was observed with the RT-PCR that entailed further screening of the cDNA and analysis. Although this cDNA contig mapped on exon trap libraries with specific cosmid frag- one of the cosmids also positive for XAP104, ments. XAP108 is likely to represent a transcription unit A combination of these methods resulted in of its own because XAP104 contained a full cod- the recovery of the MAGE-2, -3, -6, -12 (Rogner et ing region. XAP106, XAP107, and XAP108 each ah 1995) and BGN (Traupe et al. 1991; Kioschis et contained exon-trap products and were positive ah 1994) genes, which confirmed their map po- in RT-PCR analysis but no transcripts were ob- sitions at the transcript level. Isolation of the cal- served and no exon-intron boundaries were de- tractin gene, which to date was mapped to the tected at the genomic level. Considering the con- DXS52 region at the YAC level (Chatterjee et ah served nature of coding regions between species 1995), was significant because it permitted a fine- and because cross-hybridisation signals were ob- mapping of this gene at the cosmid level (Fig. 2). tained for XAPI06, XAPI07, and XAP108 on Besides isolating and mapping the known genes, mouse YAC DNA (Chatterjee et al. 1994), these the additional isolation of six putative novel data provided further evidence that all cDNA genes was noteworthy insofar as the diseases that contigs represented true coding sequences. have been linked to the DXS52 region remain We identified a novel hPMCA isoform that, unidentified. The relevance of the abundance of because of its lack of identity with any of the ESTs in four of the contigs lay in the confirma- known hPMCA genes, we have called hPMCA5. tion that true cDNAs were isolated because their This data is discrepant with that of Wang et al. homologies allowed the comparison of cDNAs (1994) who mapped hPMCA3 to Xq28. Because isolated by different methods. The clustering of at least four human Ca2+-ATPase isotypes have cDNAs, exon-trap products, and/or ESTs was fur- been identified (Wang et al. 1994) and hPMCA3

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TRANSCRIPT MAP OF DXS52 REGION IN Xo28

was mapped to Xq28 by in situ hybridization, it is DXS52 region, the bare patches mutation (Bpa), conceivable that the isoform in the DXS52 region which has been suggested as representing the is a novel isoform and was mapped previously as mouse homolog to the dominant form of chon- isoform 3 owing to a cross-hybridization of this drodysplasia punctata (CDPX2 or Happle syn- conserved sequence family. drome; Herman and Walton 1990; Chatterjee et As the DXS52 region lies within the candi- al. 1994), maps syntenically to the DXS52 region date region for various diseases it was necessary in both mouse and man. Although the BGN gene to consider that the genes mapping to this region had been implicated as a candidate for CDPX2 are possible disease-causing candidates. The cal- (Traupe et a1.1992), this was disproved because tractin gene product forms an essential compo- no mutations were found by SSCP analyses on nent of centrosomes and together with its ho- patients affected with the disease (Das et al. mology to the CDC31 yeast protein was impli- 1994). It thus remains feasible to consider all the cated as playing a major role in cell division (Lee novel genes as candidates for CDPX2. and Huang 1993). By inference, the caltractin The heterogeneity of gene content in Xq28 is gene cannot be a candidate for any of the diseases in agreement with the varied GC-content which linked to the DXS52 region because mutations is at a high density of >52% in the gene-rich re- would be lethal in germ-line cells (Tanaka et al. gion around the G6PD and color vision genes 1994). Rogner et al. (1995) already suggested that (Pilia et al. 1993; Palmieri et al. 1994) where the the MAGE genes, which are rarely expressed in gene density has been estimated at one gene ev- healthy tissues (De Plaen et al. 1994; De Smet et ery 15 kb (Sedlacek et al. 1993). In proximal al. 1994) but exhibit tumor-specific expression in Xq28, however, the GC-content has been esti- melanoma cells (van den Bruggen et al. 1991; mated at a moderate 40%, indicative of a lower Traversari et al. 1992), may be implicated in dys- gene density in the region around the DXS52 keratosis congenita because of the precancerous VNTR. In total, six known genes and seven new lesions of epithelial tissues that are frequently as- transcripts have been mapped to the 700-kb sociated with the disease (Addison and Rice DXS52 region, giving an approximate gene den- 1965). Of the novel genes that we mapped to the sity of one gene every 50 kb. This does not cor- DXS52 region, XAP103, although also expressed relate with the failure of Maestrini et al. (1992) to in brain and pancreas, was found to be expressed detect CpG islands in the region proximal to in mesodermal tissues such as heart and skeletal DXS15 but rather supports the findings of Pal- muscle. Because BTHS includes clinical features mieri et al. (1994) who found at least two CpG such as cardio- and skeletal myopathy (Adds et al. islands in close vicinity of the DXS52 and DXS15 1993), further analyses on XAP103 as a candidate loci. Interestingly, and in convergance with the for BTHS should be performed. XAPI04 has a data of Palmieri et al. (1994), the six novel genes conserved and significant homology to two types are also clustered in closest proximity to the of dehydrogenases. In view of the ubiquitous ex- DXS52 and DXS15 loci (Fig. 2). The correlation of pression of XAP104 and the ubiquitous cellular Ll-free DNA regions with a high gene density in functions of dehydrogenases, mutations in this the G6PD region has been discussed by Rogner et gene would possibly be lethal. Although it is dif- al. (1994). The 700-kb region around the DXS52 ficult, therefore, to consider this gene as a poten- loci that we examined, although not depleted of tial disease gene, disease causing mutations L1 elements, is interrupted by three short might be tolerated if XAP104 can be shown to be stretches of Ll-free regions (Fig. 2) possibly in- an X-linked gene producing a recessive disease dicative of gene-containing stretches. Indeed, the phenotype in females. Hence, because BTHS pa- MAGE gene cluster mapped to the first Ll-free tients may exhibit abnormal mitochondria at the region that lies proximal to the DXS52 polymor- microscopic level and although little is known phic loci, whereas the novel genes, caltractin, about the associated biochemical changes (Adds BGN, and hPMCA5 mapped to the second and et al. 1993), a role of XAPI04 in the etiology of third Ll-free regions. BTHS should not be excluded. By analogy, a role Because the DXS52 region has been consid- of the novel hPMCA5 gene in BTHS might also be ered as a stretch of DNA with a high recombi- considered. The gene order in the chromosomal national activity (Bell et al. 1989) also suggestive region Xq28 has been shown to be highly con- of a higher mutation rate, it would be inter- served between human and mouse (Chatterjee et esting to investigate further the novel genes in al. 1994). Pertaining more specifically to the light of this chromosomal structure and to make

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HEISS ET AL. correlations with respect to the diseases linked cDNA sublibrary. A library of 1536 clones were picked into to this region. microtitre plates and spotted onto filters.

METHODS RT-PCR Exon Trapping For RT-PCR, primers with an annealing temperature of ~>60~ were designed based on the sequenced cDNAs. PCR One pool of 26 cosmids representing a minimal overlap of was carried out with 0.2S units of Amplitaq per 25 i~1 re- the 700-kb DXS52 region was subjected to exon trapping. action and 2.5 ~1 of template oligo-dT-primed first-strand Two sublibraries of 8000-10,000 clones were generated by cDNAs of fetal brain, fetal liver, and adult skeletal muscle, EcoRI and BamHI cloning into the pSpl3b vector (Inte- diluted to a concentration of 0.5-1 ng/p~l. Cycling was car- grated Genetics) and electroporation into Escherichia coli ried out with an initial 2 min at 94~ followed by 30 DHSc~ cells. The clones were pooled and 2-3 ~g of the cycles of 94 ~ for 1 min, 60~ for I rain, 72~ for 1 min with purified DNA was transfected into COS-7 cells by lipofec- a final 10-min extension at 72~ Sequences of the primers tion (Lipofectamine, GIBCO). Transfections were carried used were as follows: out for S hr and total RNA was Trizol-extracted (GIBCO) 48-72 hr later. First-strand cDNA synthesis was carried out using the SA2 (ATC TCA GTG GTA TIT GTG AGC) primer. Primary PCR was performed with an initial denaturation XAP 103a: GAA GGT TGA CCT CTA CCA GGG GC for 2 rain at 94~ followed by 10 cycles at 94~ for 1 min, CCG TGG TCC ATC ATG TTC TGT CAG C 60~ for 1 rain, 72~ for 3 min, and a final 10-min exten- XAP 103b: TCC CTC TCT TCC CGT CAG AGT CC sion at 72~ using the SA2 and and SD6 (TCT GAG TCA TCC TGG AAG GAG CCT GAC CTT GG CCT GGA CAA CC) primers. Primary PCR products were XAP 104: GCC TGG AGT TCA GTG GGT GCA GC digested with SO units of BstXl at SS~ for 16 hr. Secondary CCA TCA CAA GCA GC__~ATA GCA GG PCR was carried out using the USD2 (CUA CUA CUA CUA XAP 105: CAA GGG AGT CAG GAC AC(AT) CTG AAC C GTG AAC TGC ACT GTG ACA AGC TGC) and USA4 (CUA GAA GGA TGT ACC CAC CAG GGT CG CUA CUA CUA CAC CTG AGG AGT GAA TTG GTC) prim- XAP 106: CTG GCA TCT GCT GGT CTT GTC TTG G ers using the same PCR conditions as described for the GAC ATC CTA TCC TTG GCA GCC TCC primary PCR except that the samples were cycled 25 times. XAP 107: GGG CAA AGA CAG TTC AGC AAC AGT GG Secondary PCR products were then either used as a com- CTG ATC CCT TGG AGT TCT GAG GC plex hybridization probe on the enriched cDNA sublibrary XAP 108: CAC CAA AAG TGG GAC AGA CTT GCA CC filters and on cosmid digest filters, or cloned into the GTA TTT AGT CCT CAG GTC TCT TCC pAmpl vector (GIBCO) to generate an exon-trap library. A library of 1536 clones were picked into microtitre plates and spotted onto filters. Probe Labeling, Competition, and Hybridization cDNA Selection Experiments DNA (S0-100 ng) was labeled to a specific activity of cDNA selection was carried out essentially as described in S x 106 to 10 x 106 cpm by random priming, isopropa- Korn et al. (1992). The genomic DNA that served as a tem- nol precipitated, and then subjected to i>2 hr of competi- plate for cDNA selection consisted of the same pool of 26 tion with 25 ~g of human Cot-1 DNA (GIBCO) at 65~ cosmids used for exon trapping. One microgram of tem- Hybridizations were carried out at 65~ for 16 hr in plate DNA was biotinylated with biotin-16-dUTP by nick Church solution (Church and Gilbert 1984) and filters translation and repetitive elements were then suppressed were washed twice at 65~ for 20 rain in 2 x SSC contain- by competing with 10 I~g of Cot-1 DNA (GIBCO) and 15 t~g ing 0.2% SDS. of lawrist vector DNA for i>2 hr at 65~ in 750 mM NaC1, SO mM sodium phosphate (pH 7.2), and 5 mM EDTA. Hy- bridization of the cDNA to cosmid DNA templates was Sequencing and Data Base Comparisons subsequently carried out in the same buffer for 16 hr at 65~ Washes were carried out 6• at 65~ in 0.1x SSC Plasmid DNA was extracted using the Bio Robot 9600 and after capturing the hybrids on streptavidin-coated mag- Qiawell Ultra System (Qiagen). Reactions were sequenced netic beads (Dynal). Finally, the hybrids were eluted in 10 with the Taq DyeDeoxy Terminator Cycle Sequencing Kit mM Tris-HCl, 1 mM EDTA (TE), size-selected on Chro- (Perkin Elmer, ABI) and run on the ABI 373A automated maspin 400 columns (Clontech), and subjected to PCR sequencer. Sequences were analyzed with the XGAP pro- with an initial 1 min denaturation step at 94~ followed by gram of the Staden package (Dear and Staden 1991), and 20 cycles consisting of 94~ for 30 sec, 65~ for 1 min, and BLASTN and BLASTX programs (Altschul et al. 1990) of the 72~ for 2 min with a final 10-min extension at 72~ The Heidelberg Unix Sequence Analysis Resources (HUSAR). primary PCR product was subjected to a second round of enrichment and PCR amplification as described for the first round of enrichment. Secondary PCR products were ACKNOWLEDGMENTS then either used as a complex hybridization probe on the exon-trap library and cosmid-digest filters, or cloned into We are indebted to Petra Wilgenbus for her diligent, reli- the pAmplO vector (GIBCO) to generate an enriched able, and extensive sequencing work. We thank Sabine

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TRANSCRIPT MAP OF DXS52 REGION IN Xo28

Klauck and Zdenek Sedlacek for supporting the work on Howard-Peebles, P. Murphy, W.R. Breg, H. Veenema, and the hPMCA isoforms and for critically reading the manu- N.J. Carpenter. 1988. Multilocus analysis of the fragile X script; Gall Herman for providing helpful suggestions, syndrome. Hum. Genet. 78" 201-205. sharing sequence information with us prior to submission, and for providing the mouse YACs from the DXS52 region; Buckler, A., D. Chang, S. Graw, D. Brook, D. Haber, P. Gregor Haberhauer and Renate Gaul for helping with the Sharp, and D. Housman. 1991. Exon amplification: A sequence analyses; Scott Burk for providing the hPMCA3 strategy to isolate mammalian genes based on RNA probe, and Jean-Louis Mandel for providing the St35.638 splicing. Proc. Natl. Acad. Sci. 88: 4005-4009. probe. This research was supported by grants from the DFG (Deutsche Forschungsgemeinschaft) and the Human Burk, S.E. and G.E. Shull. 1992. Structure of the rat Genome Analysis programs of the European Community. plasma membrane Ca2+-ATPase isoform 3 gene and The sequence data described in this paper have been sub- characterization of alternative splicing and transcription mitted to the EMBL data library under accession nos. products. J. Biol. Chem. 267: 19683-19690. X96619-X96625. The publication costs of this article were defrayed in Chatterjee, A., C.J. Faust, L. Molinari-Storey, P. Kioschis, part by payment of page charges. This article must there- A. Poustka, and G.E. Herman. 1994. A 2.3-Mb yeast fore be hereby marked "advertisement" in accordance artificial chromosome contig spanning from Gabra3 to with 18 USC section 1734 solely to indicate this fact. G6PD on the mouse X chromosome. Genomics 21: 49-57.

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Received January 23, 1996; accepted in revised form April 12, 1996.

GENOME RESEARCH~ 491 Downloaded from genome.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press

Transcription mapping in a 700-kb region around the DXS52 locus in Xq28: isolation of six novel transcripts and a novel ATPase isoform (hPMCA5).

N S Heiss, U C Rogner, P Kioschis, et al.

Genome Res. 1996 6: 478-491 Access the most recent version at doi:10.1101/gr.6.6.478

References This article cites 51 articles, 13 of which can be accessed free at: http://genome.cshlp.org/content/6/6/478.full.html#ref-list-1

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