An Integrated Physical and Gene Map of the 3.5-Mb Chromosome 3P21.3 (AP20) Region Implicated in Major Human Epithelial Malignancies1

[CANCER RESEARCH 63, 404–412, January 15, 2003] An Integrated Physical and Gene Map of the 3.5-Mb Chromosome 3p21.3 (AP20) Region Implicated in Major Human Epithelial Malignancies1

Alexei Protopopov,2, 3 Vladimir Kashuba,3 Veronika I. Zabarovska, Olga V. Muravenko, Michael I. Lerman, George Klein, and Eugene R. Zabarovsky2 Microbiology and Tumor Biology Center, Karolinska Institute, 171 77 Stockholm, Sweden [A. P., V. K., V. I. Z., O. V. M., G. K., E. R. Z.]; Center for Genomics and Bioinformatics, Karolinska Institute, 171 77 Stockholm, Sweden [E. R. Z.]; Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk, 630090, Russia [A. P.]; Institute of Molecular Biology and Genetics, Ukrainian Academy of Sciences, Kiev, 252627, Ukraine [V. K.]; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 117984, Russia [O. V. M., E. R. Z.]; and Cancer-Causing Genes Section, Laboratory of Immunobiology, National Cancer Institute Frederick, Frederick, Maryland, 21702 [M. I. L.]

ABSTRACT INTRODUCTION

To facilitate the identification of tumor suppressor genes in the Numerous studies have indicated the presence of distinct TSGs4 or chromosome 3p21.3-p22 AP20 subregion, we constructed a 3.5-Mb groups of genes in human chromosome 3p involved in the origin physical and gene map of this segment (between markers D3S4285 and and/or development of carcinomas of the lung, breast, cervix, kidney,

D3S3873) that spans the distance from 124.4cR3000 to 133.5 cR3000 of and head and neck. Detailed studies reviewed elsewhere (1) showed the GB4 genetic map. We used NotI-linking and -jumping clones, that these tumor suppressors could be of various types: classical, as sequence-tagged site PCR marker analysis, and multicolor and fiber RB1 or P53, haplo-insufficient; cancer specific or multiple, i.e., in- fluorescence in situ hybridization to confirm the sequence order and volved in several distinct cancers. Our and others’ studies (1–6) map orientation. An integrated clone contig composed of 5 yeast identified two most frequently rearranged regions in human chromo- artificial chromosome, 15 bacterial artificial chromosome, 5 P1 artifi- some 3p, namely LUCA or 3p21.3C (centromeric) and AP20 or cial chromosome, and 8 NotI-linking clones provided the physical base 3p21.3T (telomeric). These two regions are hot spots for loss of of the map. We unequivocally established the order of 28 sequence- heterozygosity and/or homozygous loss in major epithelial cancers tagged sites and 35 genes in the region. Gaps between published mentioned above. In fact, homozygous deletions at both locations bacterial artificial chromosome contigs were determined and covered have been reported in lung, breast, kidney, and cervical carcinomas (1, by our own sequence data. Furthermore, three new genes were isolated, 3, 6–9). namely the human homologue to the rat Golgi peripheral membrane protein p65, GOLPH5 (GORASP1), the gene for stress-inducible pro- The LUCA region was completely sequenced and several candidate tein, STI2, and the AP20-region gene 1, APRG1. TSGs were identified as a result of the major effort by the Interna- The tumor suppressor gene candidate APRG1 was positioned close to tional Lung Tumor Suppressor Gene Consortium (1, 3, 8, 10). the border of the homozygous deletion in a small cell lung cancer cell A homozygous deletion in 3p21.3T region was found in a SCLC line ACC-LC5. Expression analysis with a tissue-specific panel of ACC-LC5 cell line. Physical and gene maps covering this deletion cDNA revealed seven distinct tissue-specific splice variants (A–G)of were constructed and 14 genes were identified, however, none of them the message (size range, 1.0–1.8 kb). Although the gene was expressed demonstrated features of TSGs (11–14). at a low level in all tested tissues, comparatively higher expression was Several of our previous studies were associated with human NotI- detected in pancreas (splice forms B and D), kidney (A) and placenta (B jumping and -linking clones (13, 15–18). NotI-linking clones contain and C). The APRG1 gene encoded a predicted protein of 170 amino DNA fragments flanking a single NotI recognition site, whereas acids (isoform B), which had an NH2-terminal part conserved among NotI-jumping clones contain sequences adjacent to neighboring sites. members of the eukaryotic translation factor 6 gene family. A Prosite Such clones were shown to be tightly associated with CpG islands and pattern corresponding to the cell attachment sequence Arg-Gly-Asp genes (18). was also found. The presence of this domain raised the intriguing We have shown that our previously built NotI clone contig (AP20) possibility that APRG1B may be directly involved in membrane inter- overlapped with the homozygous deletion in ACC-LC5 (4, 13). The actions and cell adhesion. physical map constructed with NotI-jumping and -linking clones (13) We showed that the AP20 region was duplicated during mammalian revealed significant differences between our map and that of Daigo et evolution and homologous gene clusters were present in human chromo- al. (14) and the draft human genome sequence.5 Careful analysis of some 2 and syntenic mouse regions on chromosomes 1, 2, and 9. Interest- these differences resulted in the identification of several new genes ingly, the HYA22 gene (human ortholog of the yeast YA22 gene) was and alternative gene splicing forms that are currently under analysis. located at the borders of both breakpoints, evolutionarily conserved gene Here, we present an integrated 3.5-Mb physical and gene map that cluster and homozygous deletions detected in lung, kidney and other cancers. facilitates identification of multiple TSG(s) in the critical AP20 NotI digestion revealed that the AP20 region was frequently and ex- region. tensively methylated in renal carcinoma cell lines and tumor biopsies. MATERIALS AND METHODS

Cell Lines and General Methods. MCH/mouse (MCH939.2, MCH910.7, Received 7/23/02; accepted 11/13/02. and MCH924.4) and human MCH/rat (MCH429.11) microcell cell lines were The costs of publication of this article were defrayed in part by the payment of page generated by microcell-mediated chromosome transfer as described previously charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by research grants from the Swedish Cancer Society, the 4 The abbreviations used are: TSG, tumor suppressor gene; SCLC, small cell lung Swedish Research Council, Pharmacia Corporation, Åke Wiberg Foundation, STINT, and cancer; MCH, human monochromosome; RCC, renal cancer cell; PFGE, pulsed-field gel Karolinska Institute. electrophoresis; FISH, fluorescence in situ hybridization; YAC, yeast artificial chromo- 2 To whom requests for reprints should be addressed, at Microbiology and Tumor some; BAC, bacterial artificial chromosome; NCBI, National Center for Biotechnology Biology Center, Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden. Phone: Information; ORF, open reading frame; TPR, tetratricopeptide repeat; PAC, P1 artificial 46-8-728-67-37; Fax: 46-8-31-94-70; E-mail: [email protected] or Phone: 46-8-728-67-50; chromosome; EST, expressed sequence tag; ISREC, Swiss Institute for Experimental Fax: 46-8-31-94-70; E-mail: [email protected]. Cancer Research. 3 These authors contributed equally to this work. 5 Internet address: www.ncbi.nlm.nih.gov. 404

(19). The ACC-LC5 SCLC cell line that carried a deletion in 3p21.3 (11) was 50 mM EDTA. A small piece of block (5 ␮l) was placed at the end of kindly provided by Dr. Yusuke Nakamura (University of Tokyo, Tokyo, 3-aminopropyl-trimethoxysilane-coated glass microscope slide (Merck), 15 ␮l Japan). ACHN, HN51, and Caki-2 RCC cell lines were purchased from the of water were added onto the agarose, and the slide was heated at 95°C for American Type Culture Collection (Manassas, VA). Lymphoblastoid cell line 20–30 s. DNA extended on the slide that was then air dried, submerged in a CBMI-Ral-STO (20), as well as RCC cell lines KRC/Y, TK-164, TK-10, and 70% ethanol for 30 min at room temperature, cross-linked at 80°C for 30 min, KH-39, were obtained from Microbiology and Tumor Biology Center, Karo- and stored in the dark at Ϫ20°C. linska Institute (Stockholm, Sweden) cell lines collection (4). FISH was performed using standard procedures (22). Biotinylated probes All molecular biology and microbiology procedures were performed as were detected using Cy3-conjugated avidin (Amersham Pharmacia Biotech, described previously (15–17). Plasmid DNA was purified using REAL-Prep Piscataway, NJ), and the signal was amplified by biotinylated goat antiavidin kit (Qiagen, Valencia, CA). Sequencing was done using ABI 310 and ABI 377 (Vector Laboratories, Burlingame, CA) and another layer of Cy3-avidin. For Sequencers (Applied Biosystems, Foster City, CA) according to manufactur- digoxigenin-labeled probes, mouse antidigoxigenin (Roche Molecular Bio- er’s protocol. chemicals) and rabbit anti-FITC and fluorescein-conjugated swine antirabbit PFGE and Hybridization. Paired normal and renal cell carcinoma tissue (both from Dako A/S, Glostrup, Denmark) were used. Slides were counter- samples were obtained immediately after resection and stored at Ϫ80°C before stained with 5 ␮g/ml 4Ј,6-diamidino-2-phenylindole (Merck) and mounted in DNA extraction. Each tumor piece was examined histopathologically. Only Vectashield antifade medium (Vector Laboratories). clear cell type tumors were included. Preparation of DNA for PFGE, digestion Molecular Cloning of Human APRG1, GOLPH5, and STI2 Genes and with restriction enzymes, and PFGE were done using the Chef mapper (Bio- Expression Analysis. Gene fragments have been obtained by PCR from the Rad Laboratories, Hercules, CA) according to manufacturer’s protocols. Multiple Tissue cDNA panel no. K1421-1 (Clontech, Palo Alto, CA), using Southern transfer and hybridization were performed as described previously the following primer sets, according to manufacture’s manual: hP65–5 (16, 17). 5Ј-GAATCGAGCGCCGAGAGAGCGAGT-3Ј, hP65–35Ј-GTGAGGGCA- Molecular Probes. The construction of NotI-linking/jumping libraries was ACTTTGGGTCAGACT-3Ј; APRG1–55Ј-ATCTGTTATGTTCACTGGGG- described previously (15–17). The following clones were used for DNA CATCTCC-3Ј, APRG1–35Ј-AATGAAGTGCCATCATTTAGCCAGTCC- hybridizations: NotI-linking clones NL1-024 (D3S4258), AP40 (D3S1646), 3Ј; STI2–55Ј-GAGATGAGCAGCAATGACTCCTCCCTTAT-3Ј, STI2–35Ј- NL1-401 (D3S4581), NLJ-003 (D3S1642), AP20 (D3S4311), NL3-003 AATGTGTCATTTTCTGAATCCCTTCTCCA-3Ј. (D3S3872), and NL1-308 (D3S3873); and NotI-jumping clones J32-611 (con- PCR products were cloned by Topo TA cloning kit for sequencing (Invitro- necting NLJ-003 and AP20), J32-612 (connecting AP20 and NL3-003), and gen, Carlsbad, CA). J31-613 (connecting NL3-003 and NL1-308). NL3-019 (D3S4633) was cloned To determine expression pattern of GOLPH5 and STI2, the Northern hy- by PCR from normal lymphocytes DNA using PCR primers: NL3-019F bridization with human multiple tissue nos. 7760-1 and 7766-1 Northern blots 5Ј-GGATCCGGGATGGGGTATAC-3Ј; NL3-019R 5Ј-GGATCCTTAAAT- (Clontech) was performed. GCATAAGACCC-3Ј. Bioinformatics. DNA homology searches were performed using BLASTX D3S1611, D3S3880, D3S1298, WI-6058, WI-692, SGC30812, D3S1260, and BLASTN (23, 24) programs at the NCBI server. Sequence assembling was D3S3521, D3S2343, and WI-7900 STS PCR markers were selected from done using Dnasis (Hitachi-Pharmacia). The Beauty Post-Processor was used databases of Whitehead Institute and CHLC.6, 7 Additionally, new PCR probes with the BLASTP protein database searches provided by the Human Genome were designed: DLC1F 5Ј-GAGATACATGTTGCCTCACCAG-3Ј, DLC1R Sequencing Center (Houston, TX).8 Scanning of the Prosite and the PfamA 5Ј-CATACTGGTCTTCGCTATGCAC-3Ј; APRG1F 5Ј-TGTAAACTTTC- protein families and domains was performed at the server of the Swiss Institute CAGAACAGGCCCAGA-3Ј, APRG1R 5Ј-TTAATAAGGCTGTTACC- for Experimental Cancer Research and at the NCBI server (CD-Search).9 GTGTAAATGT-3Ј; NL1-024F 5Ј-GGGCTGGCAGAACAGGTAACG-3Ј, Transmembrane regions and their orientation/topology (TMpred prediction) NL1-024R 5Ј-GAGGCATCACTGGGTTCGCTG-3Ј; AP40F 5Ј-GGTA- were provided by the ISREC-server.10 GCTTTCGGGCTTCC-3Ј, AP40R 5Ј-TCTGCACCTAGATGGCTGTG-3Ј; Ј Ј Ј NL1-401F 5 -AAGAAGCCTGTTAGTGACGG-3 , NL1-401R 5 -CACA- RESULTS AND DISCUSSION AGCTCTGTACCACTGG-3Ј; NLJ3F 5Ј-GGGACACGAGGATGCCCTAA- 3Ј, NLJ3R 5Ј-CAGAGGCAGCCAGCCAATTT-3Ј; AP20F 5Ј-CTTCACCA- NotI Physical Map of the AP20 (3p21.3T) Region. We have CAGCTGGCCAC-3Ј, AP20R 5Ј-CCTATGGCATCGTGTGTCTG-3Ј. established the order of NotI-linking clones NLJ-003, AP20, and Probes for the FISH analysis were labeled using Nick translation kits NL3-003 and showed that the 3p21.3 breakpoint in MCH939.2 was (Roche Molecular Biochemicals, Indianapolis, IN) with either biotin-14-dATP located between NLJ-003 and AP20 clones (13). Moreover, this or digoxigenin-11-dUTP according to manufacturer’s protocol. region was missing in all other spontaneously deleted MCH cell lines Five YAC clones, 925e3, 938g7, 936c1, 803g5, and 790f3, were selected from Centre d’Etude du Polymorphisme Humain-Genethon integrated maps. that we analyzed previously (25–28): MCH910.7, MCH429.11, and Human PAC clones 167i15, 84h8, 38k3, and 296a3 were identified using MCH924.4. hybridization of NotI-linking clones with high-density-gridded filters (PAC To confirm that the breakpoint in the MCH939.2 cell line was Library RPC11, HGMP Resource Centre, Hinxton, Cambridge, United King- inside the homozygous deletion described by Murata et al. (11), we dom). P1 clone RDK3118, containing a full-length human mutL (Escherichia designed PCR primers, including NL1-024, AP40, NL1-401, NLJ- coli) homologue 1 (MLH1) gene, was kindly provided by Dr. Richard D. 003, and AP20. All primers except D3S1611, AP20, and D3S3521 Kolodner (Ludwig Institute for Cancer Research, La Jolla, CA). Human BAC yielded expected products with the YAC 936c1 DNA that completely clones were purchased from HGMP Resource Center. covered the homozygous deletion. This confirmed that the region FISH. Slides with metaphase spreads of normal male individuals were spontaneously deleted in MCH cell lines coincided with the homozy- supplied by Micro System Sweden AB (Stockholm, Sweden). gous deletion detected in SCLC and non-small cell lung cancer cell DNA fibers were prepared according to the technique that uses agarose- embedded high molecular weight DNA as a target for FISH, essentially as lines. This was additionally validated by PCR analysis that placed the described previously (21). In brief, peripheral blood lymphocytes were em- NLJ-003 linking clone into the homozygous deletion in ACC-LC5 bedded in a 1% low melting point agarose for the preparation of blocks cell line (Table 1). containing 108 cells/ml. Cells were lysed by incubation at 52°C in 1 mg/ml Using FISH and the deletion cell hybrid panel, eight NotI-linking proteinase K (Merck, Darmstadt, Germany) in 1ϫ 10 mM Tris (pH 8)-1 mM clones were mapped to the 3p21.3-p22 region (25) and connected by EDTA in the presence of 1% N-laurylsarcsine for 48 h. Agarose blocks were NotI-jumping clones and PFGE hybridization (Fig. 1; jumping clone washed five times in 1ϫ 10 mM Tris (pH 8)-1 mM EDTA overnight, treated J31-613 connecting NL3-003 and downstream located NL1-308 is not with 100 ␮g/ml RNase A in 2ϫ SSC at 52°C overnight, and stored at 4°Cin

8 Internet address: dot.imgen.bcm.tmc.edu: 9331. 6 Internet address: www-genome.wi.mit.edu. 9 Internet address: www.isrec.isb-sib.ch/software/PFSCAN_form.html. 7 Internet address: gai.nci.nih.gov/CHLC. 10 Internet address: www.ch.embnet.org. 405

Table 1 AP-20 region markers ordering according to PCR with YAC clones genetic maps released at various time points were rather different (see and ACC-LC5 for example Build 25 and Build 29), thus we can compare our map only with published previously (14, 29, 30). Because their maps overlapped partially with our map, only limited comparisons were possible. The contig orientation and hence the gene map between AP40 (D3S1646) and NIB1520 were in reversed order as compared with our data. Published positions of MYD88 and ACAA1 genes were not concordant with their real physical positions because we have assigned these genes by direct sequencing to a single PAC clone 296a3. The order of markers of internal 1.2-Mb region between NIB1520 and SGC38212 in our map was opposite to earlier published integrated map (14). Also there were two gaps between NCBI contigs in this region, close to NIB1520 and SGC38212. Thus, it is likely that the region between D3S1646 and SGC38212 was misplaced in earlier genetic and physical maps. To confirm the contig assembly of AP20 region by an independent method, several clones were mapped by multicolor FISH on metaphase chromosomes (see examples in Fig. 3, A–C). From Fig. 3, B a PCR positive (black) and C, where RDK3118 (containing MLH1 gene), 167i15 (containing clone NL1-401), and AP20 clones were used, it was clear that AP20 shown, as well as the PFGE data for NL1-024 and NL3-019). The marker was the most centromeric probe. To prove the physical dis- NotI map for the AP20 region (Fig. 2) was significantly different from tances and assignment of the borders of ACC-LC5 homozygous Gemmill et al. (29). For example, between MLH1 and NL3-003 we deletion, we performed Fiber-FISH with 5 PACs (167i15, 84h8, 38k3, found 4 NotI clones compared with 12 in this published map (29). 296a3, and RDK3118) and two NotI clones (AP40 and AP20). The Our map was also in contradiction with other published (12, 14) and length of the PAC signals and gaps was measured using AP20 (11.4 draft maps available online.11 The most striking difference is the kb) as a ruler (Fig. 3F). All PACs were end sequenced and fiber-FISH orientation of the central part between clones NL1-401 and AP20. confirmed their perfect integrity. For instance, the size of PAC 296a3 According to our data, the order of the clones is as follows: tel, was estimated by Fiber-FISH as 190 kb, compared with 190,764 bp, NL1-401–NLJ-003 - AP20–cen (Fig. 1B) and NotI-jumping clones obtained by end sequencing and alignment to published data J31-611 and J32-612 ultimately argue against order suggested in (AB026898). The data proved that PACs 167i15 and 296a3 were Ref.14, tel, AP20 - NL1-401–NLJ-003 (Fig. 1A) where such NotI- located at the borders of ACC-LC5 homozygous deletion, with the jumping clones could not exist (see construction of NotI jumping libraries in Ref. 16). This NotI map is in excellent agreement with our previously published data (13) and contains significantly more information, including physical distances between framework markers. Construction of an Ordered Contig of Overlapping Clones Using FISH. Similarly to the YACs in the LUCA 3p21.3C region (6), all YAC clones covering our AP20 region showed rearrangements and deletions (see Table 1). However, by combining published, internet data, and our own search for PAC and BAC clones from the region, we succeeded in creating a contig of overlapping PAC/BAC/NotI- linking clones completely covering 3.5 Mb in the region with only one gap of ϳ90 kb that was spanned by the SCN5A gene. We found that the 1-Mb centromeric segment was enriched by endogenous retrovirus sequences (between SGC38212 and D3S3521 and around WI2025). It, perhaps, created crucial difficulties for shotgun genome sequencing approaches and could be an explanation as to why maps published by Celera Genomics (Rockville, MD) and NCBI (Bethesda, MD) were not correct for this particular region. Many LINE repeats were present in the only remaining gap, and it was very unlikely that an unknown protein coding gene could be present in this gap. Therefore, the gene map shown in Fig. 2D is the most complete among available at present. The minimal contig of overlapping clones included 15 BACs, 5 PACs, and 8 NotI-linking clones (Fig. 2C). Gaps between NCBI bactigs were overlapped. To generate new sequence tags, direct sequencing of the PACs ends was performed. In total, 28 genetic markers were tested, including 9 derived by us, 16 by the Whitehead Institute, and 3 by the Cooperative Human Linkage Center. All these Fig. 1. A NotI physical map of the AP20 region. Two halves of NotI clones are designated by letters a–k. A, orientation of NLJ-003 and NL1-401 clones according to markers were mapped in separate genetic maps, and we were able to Daigo et al. (14). Black boxes show possible NotI-jumping clones. B, orientation of NotI integrate them onto our single physical map. Both NCBI physical and clones and physical distances between them established in this study. Possible NotI jumping clones are shown. Two of them (J31-611 and J32-612) have been isolated from libraries described previously (16, 17). C, representative PFGE hybridizations with 11 Internet address: www.ncbi.nlm.nih.gov/cgi-bin/Entrez/maps.cgi?org ϭ hum&chr ϭ 3. NotI-linking clones. 406

Fig. 2. An integrated map of the AP20 region. A, ideogram of the banding pattern of human chromosome 3. B, physical map (in Mb) of the region with NotI framework markers and sequence-tagged sites, including microsatellite markers. C, mini- mum tiling contig of BAC, PAC, and NotI-linking clones covering the region. The YAC 936c1 and homozygous deletion in ACC-LC5 are also shown. D, gene map. Orientations of transcription are designated by arrows. Putative pseudogenes and other in silico predicted genes for which no confirmed information was obtained are not shown. E, the cluster of genes orthologous to AP20 region on murine chromosome 9 (MMU9), 62–70 cM, in same orientation: from telomere to centromere. Solid line shows the region of continuous DNA homology. GenBank accession numbers are given for unnamed genes. F, paralogous genes on human chromosome 2 (red, HSA2q35, and green, HSA2q32, lines) and murine chromosomes 1 and 2 (MMU1 and MMU2, deep and light blue lines). The region of natural resistance/susceptibility to intracellular macrophages parasites (NRAMP1, gene SLC11A1) on the HSA2q35 is highlighted. The name for the murine ortholog of the human NLI-IF gene, Gip, is given according to GenBank accession no. AY028804.

distance between them of ϳ37 kb in this cell line compared with showed reduced mRNA levels or loss of expression in SCLC: MLH1, ϳ606 kb in normal lymphocytes. Interestingly, the AP40 marker was DLEC1, and AXUD1. However, no other data supporting their TSG rather close to MLH1 gene (30-kb gap between this clone and activity or involvement in lung or kidney carcinogenesis was pub- RDK3118 containing MLH1). lished (1). Gene Map of AP20 Region. First of all we tested which genes Three new genes found in the critical segment are described below. were linked to the studied NotI sites. We have already shown that The APRG1 gene (GenBank accession nos. AJ493599–AJ493605) NL1-308 contains the MOBP gene, and AP20 was a part of the was discovered by the analysis of EST clone H86663, assigned to SCN5A gene (13, 25). Additionally, clone NL1-401 was assigned to PAC 167i15. The gene occupied ϳ36.3 kb of genomic space and was the ITGA9 gene; NLJ-003 marked the HYA22 gene (human ortholog composed of at least seven exons. By screening of cDNA libraries and Ј of the yeast YA22 gene); NL3-019 contained 5 end of the ORCTL4 the Multiple Tissue cDNA panel with APRG1-5/APRG1-3 primers gene; and NL3-003 had the GOLPH5 gene. Clone NL1-024 possessed set, we have found four splice forms as 1.0–1.8 kb messages (Fig. 4): the OSBPL10 gene, and AP40 was tied to a gene with an unknown form A (coding exon 6), form B (coding exons 6 and 8␣␤), form C function (AW967589). We analyzed microsatellite markers from the (coding exons 4 and 8␤), and form D (coding exons 4 and 8␣␤). Two AP20 region used by us in earlier studies and found that all of them were associated with genes. D3S1611 was inside the MLH1 gene, the ESTs from heart cDNA library were available in public databases. villin-like gene connected to D3S1298, the XYLB gene contained The clone H86663 contained intron 6 because of incomplete splicing D3S1260, and the MYD88 gene contained STS SGC30812. (form E at Fig. 4; AJ493603), and clone AA593744 presented exons Ϫ In silico methods based on computational analysis of the ϳ3.5-Mb 2–4 only (form F; AJ493604). Promoter/enhancer site ( 778 bp) was sequence allowed us to discover and clone additional genes there by predicted upstream of exon 2. Numerous putative transcription factor increasing the total number of resident genes to 35 (Fig. 2D, Table 2) binding signals could be found throughout the first 500 bp, e.g., for compared with 14 genes mapped earlier (14). transcription factors Lyf-1, DeltaEF-1, Tcf-11, GATA, and others. Gene sets were analyzed extensively using manual experimental Two more ESTs, BM701622 (retina) and AW152277 (uterus), formed methods as well as web-based computational servers to assign, when rare splice variant G, including exon 1 (AJ493605). The predicted possible, protein functions. Three of the genes by Northern analysis promoter/enhancer site started at Ϫ215 bp. A predicted molecular 407

Fig. 3. Representative FISH images. A, two-color FISH assignment of PAC 167i15 (red) and AP20 (green) to 3p22-p21.3. B, ordering of PAC 167i15 (red) and AP20 (green) clones. Selected interphase nuclei (1) and metaphase chromosome images (2) probe show more centromeric localization of AP20 clone. C, ordering of RDK3118 (green), 167i15 (red), and AP20 (green) clones. Figure demonstrates that these clones are colocalized at one band of metaphase chromosome (2), but interphase FISH (1) reveals their order as following: RDK3118, 167i15, AP20. D, localization of PAC 296a3 (green) centromeric to RDK3118 (red) and 167i15 (red) clones. Interphase FISH was done with 167i15 and 296a3 (1), RDK3118 and 296a3 (2), and all three clones (3). E, localization of 296a3 (green) between 167i15 (red) and AP20 (red) clones. Examples of interphase FISH are shown (1). F, collection of high-resolution fiber-FISH images. The length of fiber-FISH hybridization signal is proportional to DNA length of the probe. Thus, an image of probe with known length could be used as a visual ruler to measure physical distances. G, evaluation of physical distance between AP40 (red) and RDK3118 (green). The gap in hybridization pattern corresponds to cirka 30-kb distance between these probes on physical map. H, fiber-FISH with PAC 167i15 (red) and PAC 84h5 (green). The second image is shown with artificial shift between green and red signals, whereas the first shows the real image, where the overlapping part is yellow. I, FISH with PAC 167i15 (red) and PAC 296a3 (green) using DNA fibers from ACC-LC5 SCLC cell line. The homozygous deletion drastically decreases distance between these two probes.

weight of the longest polypeptide, coding by isoform B, was Mr program. Prosite pattern corresponding to the cell attachment se- 18,500. The splice variants F and G did not present an extended ORF. quence, Arg-Gly-Asp, was found in the position 135–137. The pres- Northern hybridization did not result in any major specific signal ence of these domains raised the intriguing possibility that APRG1B with neither A nor B splice variants, indicating low abundance of may be directly involved in membrane interactions and cell adhesion APRG1 mRNA. We performed PCR analysis of APRG1 expression (Prosite: pdoc00016). Interestingly, the isoform E, containing the with the Multiple Tissue cDNA panel, using the APRG1–5/APRG1–3 same NH2-terminal domain structures, had no membrane helix or primers (exons 2, 8␤). The APRG1 mRNA was expressed at the Arg-Gly-Asp sequence. highest level in pancreas (splice forms B and D), kidney (A), and BLAST searches in the mouse and other EST databases revealed placenta (B and C). No PCR products were observed with cDNAs cDNA from Bos taurus (AW358963; similarity 93% over 190 bp; from heart, skeletal muscles, liver, brain, retina, and uterus. Therefore, exon 6). Only limited protein homologies to the Drosophila’s dah we hypothesized that the above-mentioned tissues expressed incom- gene product, microneme protein-1 (Plasmodium vivax) and related to plete splicing forms without either exon 2 or exon 8␤ of the APRG1. transforming growth factor ␤ receptor associated protein 1 (Neuros- By sequence analysis, the gene encoded an unknown protein (170 pora crassa) were found (similarity 52% at 57 aa overlap, 51% at 58 aa, isoform B) that has NH2 terminus conserved among eIF6 gene aa, and 44% at 87 aa, respectively). family (pfam01912 and pfam02697). This family included eukaryotic The GOLPH5 (AJ409349; latest NCBI name GORASP1) was dis- translation factor 6 as well as its presumed archaebacterial homo- covered by screening EST databases with NotI-linking clone NL3-003 logues. (D3S3872) sequence. The gene occupied ϳ11.0-kb of genomic space Using the TMpred program, the APRG1B protein was predicted to was composed of 8 exons and abundantly represented in EST data- span the membrane once at the COOH terminus (residues 148–166 bases. aa). In addition, weak matches of kinase phosphorylation sites, N- To determine the tissue distribution of GOLPH5 mRNA, a multiple myristoylation, and amidation sites were discovered by ProfileScan tissue Northern blot was probed with the hP65-5/hP65-3 PCR product, 408

Table 2 Genes identified in AP-20 region Gene symbol Name Transcript size (kb) Accession no. OSBPL10 Oxysterol binding protein-like 10 4.0 AF392451 KIAA0342 KIAA0342 gene product 9.0 AB002340 AW967589 cDNA Similar to PA domain containing protein ? AW967589 (LOC166003) RPL29 Ribosomal protein L29 1.3 U49083 KIAA0766 KIAA0766 gene product 4.2 AB018309 MLH1 Mismatch repair protein homologue 1 3.1 U07418 LRRFIP2 Leucine rich repeat (in FLII) interacting protein 2 2.8, 3.9 AF115509 TCEA1 Transcription elongation factor A (SII), 1 1.4 NM_006756 GOLGA4 Trans-Golgi p230 7.7 NM_002078 APRG1 AP20 region gene 1 2.4, 5.0 AJ493599 ITGA9 Integrin ␣ RLC 4.1, 9.5 NM_002207 HYA22 Human YA22 2.7, 4.6 NM_005808 VILL Villin-like 2.2 NM_015873 PLCD1 Phospholipase C ⌬ 1 3.8 NM_006225 DLEC1 Deleted in lung cancer 1 6.0, 8.0 NM_007338 ACAA1 Acetyl-coenzyme A acyltransferase 1 2.9 NM_001607 MYD88 Myeloid differentiation protein 2.2, 4.0 NM_002468 OSR1 Oxidative stress response 1 4.6 NM_005109 ORCTL3 Organic cation transporter-like 1 2.4, 2.6 NM_004256 ORCTL4 Organic cation transporter-like 2 2.3, 2.4 NM_004803 XYLB Xylulokinase-like protein 2.3, 1.8 NM_005108 ACVR2B Activin receptor typeIIB 2.5, 10 NM_001106 ENDOGL2 Endonuclease G-like 1.7, 2.1 XM_084146 SCN5A Cardiac tetrodotoxin-insensitive voltage-dependent 9.0 NM_000335 channel 5, ␣ subunit SCN10A Cardiac tetrodotoxin-insensitive voltage-dependent 6.0 NM_006514 channel 10, ␣ subunit SCN11A Cardiac tetrodotoxin-insensitive voltage-dependent 6.3 AF188679 channel 11, ␣ subunit KIAA1449 KIAA1449 gene product 2 NM_020839 GOLPH5 Golgi peripheral membrane protein p65 3.5 AJ409349 STI2 Stress-inducible protein 6.5 AJ487015 AXUD1 AXIN1 up-regulated 3.2 NM_033027 CX3CR1 Chemokine (C-X3-C) receptor 1 3.4 AY016370 CCR8 Chemokine (C-C) receptor 8 4.6 AY016370 FLJ20551 Hypothetical protein FLJ20551 ? NM_017875 LAMR1 Laminin receptor 1, ribosomal protein SA 1.7 NM_002295 MOBP Myelin-associated oligodendrocyte basic protein 2.6, 3.8 NM_006501 containing complete ORF of GOLPH5. GOLPH5 was expressed as a 3.5-kb mRNA in many normal human tissues (Fig. 5A). The transcript was underrepresented in lung and heart. By sequence analysis, the gene product of 440 aa was homologous (81% identity) to rat Golgi reassembly stacking protein, GRASP65 (NP_062258), and mouse hypothetical protein (82%, BC012251).

NH2-terminal end was discovered by CD-Search to have two PDZ domains (residues 2–71 aa and 78–131 aa). They may function in targeting signaling molecules to submembranous sites. COOH-terminal domain (288–410 aa) was very conserved with rat and mouse (91% similarity), but its function seemed to be unclear. The neighboring domain 206–288 aa was less homologous (62%) and detected as a Pro-rich sequence (PS50099) by ProfileScan program in human and in rodent orthologs. The rat ortholog was shown to be a Golgi- associated protein, targeted by mitotic kinases (31), and it is reason- able to suggest the same function for the GOLPH5. The STI2 (AJ487015) was discovered with the help of ESTs

Fig. 5. Analysis of expression of the GOLPH5 (A) and STI2 (B) in different tissues. Control hybridization with the same filter (Human Multiple Tissue Northern blots, Clontech, nos. 7765-1 and 7760-1) using ␤-actin probe is shown for comparison.

AC092053 assigned to BAC 331g2. The gene occupied ϳ27kb of genomic space, was composed of 27 exons, and abundantly represented in EST databases. To determine the tissue distribution of STI2 mRNA, multiple tissue Northern blots were probed with the STI2-5/ Fig. 4. Organization of the APRG1 gene. Boxes indicate APRG1 exons (numbers 1–8). STI2-3 PCR product, containing complete ORF of STI2. As shown in Two CpG islands with the transcription initiation sites are designated with black, thick lines. Seven APRG1 transcripts, A–G, are outlined and their structures are also presented Fig. 5B, STI2 was strongly expressed as a 6.5-kb mRNA only in testis. on the right. In the following tissues, pancreas, kidney, skeletal muscle, liver, lung, 409

interactor-interacting factor (NLI-IF), which is linked to SLC11A1, gene of natural resistance/susceptibility to intracellular macrophages parasites (NRAMP1 region; Refs. 32, 33). SLC11A1 encodes a bial- lelic (G169D) macrophage-restricted divalent-cation transporter and is implicated in iron regulation in vivo (33). Moreover, it was suggested that this region is involved in susceptibility to tuberculosis (34). The SLC11A1 gene spans 13604 bp and its sequence is highly enriched for DNA repeats. We performed careful search for the homologous gene in 3p22-p21.33 and have found only an untran- scribed sequence with weak homology to 3Ј-end of the SLC11A1. Thus, the nucleotide sequence overlapping HYA22 shares both breakpoints of evolutionary conserved gene cluster and homozygous deletion. This gene product contains a nuclear LIM interactor (NLI) interacting domain. NLI domain is a structural motif that has been well conserved throughout evolution from yeast to plants and known to play important regulatory roles in cellular development. Present evidence suggests that NLI facilitates long-range promoter enhancer interaction and is involved in mediating the cross-talk between transcriptional control elements. NLI may regulate the transcriptional activity of LIM homeodomain proteins by determining specific part- ner interactions. It is likely that NLI may function as an adapter protein to mediate the interaction between LIM domain transcription Fig. 6. Methylation analysis of AP20 region. A, Southern hybridization with DNA from RCC cell lines digested with XbaI (X) or simultaneously with XbaI and NotI (XN). The factors and a non-LIM factor (NLI-interacting factor). Thus it is Southern blot was hybridized with NL1-401 NotI-linking clone. CBMI-Ral-STO DNA possible that HYA22 may function as coordinator of transcriptional was used to show unmethylated status of NotI site. NotI digestion was not observed in all RCC cell lines. PFGE hybridization of telomeric AP20 (g) probe to cell lines (B)or activity via its interaction with NLI. normal (N) and RCC (T) biopsies (C). DNA from ACHN RCC cell line was also added Noteworthy, this gene was deleted during the construction of origin C for comparison. From these last data, it is clear that only normal kidney DNA samples inal cosmid contig covering ACC-LC5 deletion and yeast cells that contain cleavable NL3-019 NotI site located between AP20 and NLJ-003. lack YA22 lose viability (12). Furthermore, closely related OS4 (conserved gene amplified in osteosarcoma) gene is most likely involved placenta, brain, heart, the STI2, the transcript was hardly detectable by in the development of human sarcomas (35). Northern analysis (Fig. 5). Methylation status of AP20 region in RCC cell lines and biop- By sequence analysis, the gene product of 1320 aa was homologous sies. Aberrant de novo methylation and silencing of tumor suppressor (76% identity) to putative murine protein containing TPR domain genes may be an initiating event in carcinogenesis. Therefore, addi- (BAB29674 and AK015017) and stress-inducible protein STI1 of C. tional experiments were performed to study methylation status of elegans (29%, NP_498315). NH2-terminal end was found by CD- AP20 region. All this experiments exploited methylation sensitivity of Search to have 10 TPRs. The tetratricopeptide repeat of ϳ34 amino NotI restriction enzyme. acids was first described in the yeast cell cycle regulator Cdc23p and DNA from RCC cell lines was digested either with single methy- later was found to occur in a large number of proteins. This family has lation-insensitive restriction enzyme (XbaIorBamHI) or double di- been implicated in a wide variety of functions, including tumorigen- gested with one of these enzymes and NotI. Example of such exper- esis. It has been proposed that TPR proteins preferably interact with iment is shown in Fig. 6A. The NL1-401 NotI site is methylated in all WD-40 repeat proteins, but in many instances, several TPR proteins RCC cell lines in contrast to the control. Cell line CBMI-Ral-STO seemed to aggregate to multiprotein complexes (InterPro accession served as a control because of highly hypomethylated status of its no. IPR001440). DNA (20). The summary data from this experiment are shown in AP20 Region Was Duplicated during Mammalian Evolution. Table 3. As demonstrated by Southern blot analysis, the NL3–019 We have found the gene set paralogous to AP20 region in chromo- NotI site was methylated even in the control. The AP40, NL1-401, and some 2q. Its chromosomal location was assigned to 2q32 and 2q35 NL1-308 CpG islands remained methylated in all seven studied RCC (Fig. 2F). The protein homologies between chr.2 and chr.3 cluster cell lines. members were as follows: 60% positive for ITGA4/ITGA9, 76% In the second type of experiments, methylation was investigated by NLI-IF/HYA22, 63% VIL1/VILL, 73% PLCD4/PLCD1, 69% PFGE, using NotI clones as probes. Again it was found that AP20 SCN2A/SCN5A, 80% SCN9A/SCN10A, and 84% GRASP55/ region was methylated in all 11 studied RCC cell lines and eight GOLPH5. The linkage relationship within at least a 2-Mb region biopsies. Example of such hybridization with AP20 (g) fragment (Fig. indicates a single large-scale genomic duplication. We have also found that the integrin ITGA4 gene (paralog to ITGA9) is assigned on 2q more centromeric, to chromosomal band 2q32 (shown in green in Table 3 Methylation of sites from AP-20 region in cell cultures Fig. 2F) and, thus, is discontinuous with the other gene set (shown in CBMI HN51 Caki-2 ACHN KRC/Y TK-164 TK-10 KH-39 red). The comparison with orthologous linkage groups in the murine NL1-024 Ϫa ϪϪ Ϫ Ϫ Ϫ Ϫ Ϫ AP40 Ϫϩϩ ϩ ϩ ϩ ϩ ϩ genome revealed a similar organization (Fig. 2, E and F): single NL1-401 Ϫϩϩ ϩ ϩ ϩ ϩ ϩ cluster on MMU 9 (HSA 3p22–21.3) and two clusters on MMU1 (Vil NLJ-003 ϪϪϪ Ϫ Ϫ Ϫ Ϫ Ϫ and Plcd4) and MMU2 (Itga4 gene). This could be explained by NL3-019 ϩϩϩ ϩ ϩ ϩ ϩ ϩ AP20 ϪϪϮ Ϫ Ϫ Ϫ Ϫ Ϫ additional rearrangement of originally duplicated region that still NL3-003 ϪϪϮ Ϫ Ϫ Ϫ Ϫ Ϫ exists in its original form in human chromosome 3 and mouse chro- NL1-308 Ϫϩϩ ϩ ϩ ϩ ϩ ϩ mosome 9. The HYA22 shares a common ancestor with nuclear LIM a Unmethylated (Ϫ), methylated (ϩ), partially methylated (Ϯ). 410

1) is shown in Fig. 6, B and C. Only 0.9-Mb fragment was revealed, 2. Yamakawa, K., Takahashi, T., Horio, Y., Murata, Y., Takahashi, E., Hibi, K., thus DNA was not digested at the NL3-019 site. Interestingly, normal Yokoyama, S., Ueda, R., Takahashi, T., and Nakamura, Y. Frequent homozygous deletions in lung cancer cell lines detected by a DNA marker located at 3p21.3-p22. kidney tissue samples were hemizygously methylated in NL3-019 Oncogene, 8: 327–330, 1993. (Fig. 6C), the region corresponded to 5Ј end of the ORCTL4 gene. 3. Lerman, M. I., and Minna, J. D. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. The 630-kb lung cancer homozygous deletion In summary, our results implied that inactivation of the 3p22- region on human chromosome 3p21.3: identification and evaluation of the resident p21.33-specific putative TSG(s) might be caused not only by deletion candidate tumor suppressor genes. Cancer Res., 60: 6116–6133, 2000. but also by hypermethylation of this region. We and others have 4. Alimov, A., Kost-Alimova, M., Liu, J., Li, C., Bergerheim, U., Imreh, S., Klein, G., and Zabarovsky, E. R. Combined LOH/CGH analysis proves the existence of inter- shown that another critical 3p21.3 region, LUCA, contained several stitial 3p deletions in renal cell carcinoma. Oncogene, 19: 1392–1399, 2000. genes with functions that antagonized tumor growth and therefore 5. Braga, E., Sentchenko, V., Bazov, I., Loginov, W., Liu, J., Ermilova, V., Kazubskaya, could be considered as bona fide TSGs. Importantly, TSGs were T., Garkavtseva, R., Mazurenko, N., Kisseljov, F., Lerman, M. I., Kisselev, L. L., and Zabarovsky, E. R. Critical TSG regions on chromosome 3p in major human epithelial located in a 0.5-Mb region and were frequently hemizygously or malignancies: allelotyping and quantitative real-time PCR. Int. J. Cancer, 100: homozygously deleted. These deletions result in inactivation of one or 534–541, 2002. both copies of a gene. Hypermethylation, the second, may be an even 6. Kok, K., van den Berg, A., Veldhuis, P. M. J. F., van der Veen, A. Y., Franke, M., Schoenmakers, E. F. P. M., Hulsbeek, M. M. F., van der Hout, A. H., de Leij, L., van more important inactivating mechanism of TSGs in the LUCA region, de Ven, W., and Buys, C. H. C. M. A homozygous deletion in a small cell lung cancer leads to severe decrease or complete loss of gene expression (1). cell line involving a 3p21 region with a marked instability in yeast artificial chro- Our finding that the AP20 region is heavily methylated in all mosomes. Cancer Res., 54: 4183–4187, 1994. 7. Roche, J., Boldog, F., Robinson, M., Robinson, L., Varella-Garcia, M., Swanton, M., studied RCC cell lines suggests that hypermethylation of TSG(s) in Waggoner, B., Fishel, R., Franklin, W., Gemmill, R., and Drabkin, H. Distinct 3p21.3 this region may also play a critical role similar to the situation in the deletions in lung cancer and identification of a new human semaphorin. Oncogene, 12: 1289–1297, 1996. LUCA region. It is possible that this locus contains insulators that can 8. Wei, M-H., Latif, F., Bader, F., Kashuba, V., Chen, J-J., Duh, F-M., Sekido, Y., Lee, inactivate several genes simultaneously. The AP20 locus is frequently C-C., Geil, L., Kuzmin, I., Zabarovsky, E., Klein, G., Zbar, B., Minna, J. D., and affected in several cancers (1, 5) and even YAC clones in AP20 are Lerman, M. I. Construction of a 600 Kilobase cosmid clone contig and generation of a transcriptional map surrounding the lung cancer tumor suppressor gene (TSG) locus extremely unstable (Table 1). on human chromosome 3p21.3.: Progress toward the isolation of a lung cancer TSG. In this study, we have constructed the most complete gene map of Cancer Res., 56: 1487–1492, 1996. the AP20 region and provided a foundation for the additional testing 9. Kok, K., Naylor, S. L., and Buys, C. H. Deletions of the short arm of chromosome 3 in solid tumors and the search for suppressor genes. Adv. Cancer Res., 71: 27–92, of candidate TSGs. No obvious TSG candidate could be recognized at 1997. a first look. However, it was indeed shown that the MLH1 and MYD88 10. Angeloni, D., and Lerman, M. I. Human Lung cancer: Cancer-Causing Genes and genes have tumor antagonizing activity (9, 36). It is very likely that Environmental Factors. In: G. A. Peters and B. J. Peters (eds.), Sourcebook on Asbestos Diseases, pp. 169–209. 2001. the function of the ITGA9 gene is also important for growth regulation 11. Murata, Y., Tamari, M., Takahashi, T., Horio, Y., Hibi, K., Yokoyama, S., Inazawa, and could be involved in tumorigenesis (1). Moreover, many genes in J., Yamakawa, K., Ogawa, A., Takahashi, T., and Nakamura, Y. Characterization of an 800 kb region at 3p22–p21.3 that was homozygously deleted in a lung cancer cell the region have multiple splicing forms and both mutation or func- line. Hum. Mol. Genet., 3: 1341–1344, 1994. tional analyses of such genes represent a difficult task. For example, 12. Ishikawa, S., Kai, M., Tamari, M., Takei, Y., Takeuchi, K., Bandou, H., Yamane, Y., DLEC1 (DLC1; Ref. 37) consists of at least 37 exons with numerous Ogawa, M., and Nakamura, Y. Sequence analysis of a 685-kb genomic region on chromosome 3p22–p21.3 that is homozygously deleted in a lung carcinoma cell line. alternative splice forms. Introduction of the DLEC1 cDNA signifi- DNA Res., 28: 35–43, 1997. cantly suppressed the growth of different cancer cell lines. These 13. Kashuba, V., Szeles, A., Allikmets, R., Bergerheim, U., Modi, W., Grafodatsky, A., features are reminiscent of the situation with the putative tumor Dean, M., Stanbridge, E. J., Winberg, G., Klein, G., and Zabarovsky, E. R. A contig of NotI jumping and linking clones cover 2.5 MB in 3p21–p22 region suspected to suppressor FHIT gene (1). Other examples could be the APRG1 and contain tumor suppressor gene. Cancer Genet. Cytogenet., 81: 144–150, 1995. HYA22 genes. Both genes have several alternative splice forms that 14. Daigo, Y., Isomura, M., Nishiwaki, T., Tamari, M., Ishikawa, S., Kai, M., Murata, Y., were not analyzed earlier. Preliminary data showed that both APRG1 Takeuchi, K., Yamane, Y., Hayashi, R., Minami, M., Fujino, M. A., Hojo, Y., Uchiyama, I., Takagi, T., and Nakamura, Y. Characterization of a 1200-kb genomic and HYA22 suppressed growth of ACC-LC5 cells in vitro in a tetra- segment of chromosome 3p22–p21.3. 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Shot-gun sequencing strategy for long range genome mapping: first results. promoter regions, or splice-site signals. However, it was recently Genomics, 21: 495–500, 1994. 17. Zabarovsky, E. R., Boldog, F., Erlandsson, R., Allikmets, R. L., Kashuba, V. I., understood that genes coding for so-called ncRNA could have ex- Marcsek, Z., Stanbridge, E., Sumegi, J., Klein, G., and Winberg, G. A new strategy tremely important functions. Many of these ncRNAs have regulatory for mapping the human genome based on a novel procedure for constructing jumping functions that play an important role in gene silencing mechanisms libraries. Genomics, 11: 1030–1039, 1991. 18. Kutsenko, A. S., Gizatullin, R. Z., Al-Amin, A. N., Wang, F., Kvasha, S. M., (RNA interference, RNAi; small intermediate RNAs, siRNA; for Podowski, R. M., Matushkin, Y. G., Gyanchandani, A., Muravenko, O. V., Levitsky, example, see Ref. 40). Regulatory ncRNA molecules could originate V. G., Kolchanov, N. A., Protopopov, A. 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The annual meeting of the Radiation Research Socie S. Harris. (2) On Monday night, June 22, a lecture by ty will be held at the State University of Iowa, Iowa Dr. L. W. Alvarez on meson physics has been tentative City, on June 22â€”24,1953. The Society will be the guest ly scheduled. On Tuesday night, June 23, Dr. L. H. of the University, and all meetings will be held on the Gray of the Hammersmith Hospital, London, will speak campus. The program will consist of: (1) Two symposia, on a topic to be announced. Dr. Gray's lecture is spon one on â€œTheEffects of Rwliation on Aqueous Solu sored by the Iowa Branch of the American Cancer Soci tions,â€• which includes the following speakers: E. S. G. ety. Those desiring to report original research in radia Barren, Edwin J. Hart, Warren Garrison, J. L. Magee, tion effects, or interested in attending or desiring addi and A. 0. Allen. The second is â€œPhysicalMeasurements tional information, please contact the Secretary of the for Radiobiologyâ€•and companion talks by Ugo Fano, Society, Dr. A. Edelmann, Biology Department, Brook Burton J. Moyer, G. Failla, L. D. Marinelli, and Payne haven National Laboratory, Upton, L.I., New York.

ERRATUM The following correction should be made in the arti by the glucose utilized by 16 per cent in CLL. If the as cle by Beck and Valentine, â€œTheAerobic Carbohydrate sumption is made that, in this respect, the myeloid and Metabolism of Leukocytes in Health and Leukemia. I. lymphoid celLsof leukemia are similar to those of nor Glycolysis and Respiration,â€• November, 1952, page 821; ma! blood, it may be that the computed normal figure substitute for the last paragraph: represents a summation of the myeloid (M) and The data in Table 3 permit several interesting calcu lymphoid (L) cells that make up the normal leukocyte lations. If one compares the amount of glucose actually population. Thus, if M = +0.27 and L = â€”0.16 and disappearing with the sum of the amount equivalent to the normal differential is 65 per cent M and So per cent lactic acid produced plus that equivalent to 02 con L, then sumption, it is seen that the amount of glucose â€œcleav 0.65 (+0.27) + 0.35 (â€”0.16) = +0.12 age productsâ€•exceedsthe amount of glucose utilized b a figure identical to the observed +0.12 for normal 12 per cent in N and 27 per cent in CML and is exceeded leukocytes.

308 An Integrated Physical and Gene Map of the 3.5-Mb Chromosome 3p21.3 (AP20) Region Implicated in Major Human Epithelial Malignancies

Alexei Protopopov, Vladimir Kashuba, Veronika I. Zabarovska, et al.

Cancer Res 2003;63:404-412.

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