Structure and Methylation-Associated Silencing of a Gene within a Homozygously Deleted Region of Human Chromosome Band 8p22

GENOMICS

35, 55–65 (1996) 0322

ARTICLE NO.

Structure and Methylation-Associated Silencing of a Gene within a Homozygously Deleted Region of Human Chromosome Band 8p22 DONAL MACGROGAN,* ALINA LEVY,* G. STEVEN BOVA,† WILLIAM B. ISAACS,† AND ROBERT BOOKSTEIN*,1 *Canji, Inc., 3030 Science Park Road, San Diego, California 92121; and †Departments of Urology, Pathology, and Oncology, The Johns Hopkins University School of Medicine and Brady Urological Institute, Baltimore, Maryland 21287 Received December 13, 1995; accepted April 10, 1996

tion regions have been derived by analysis of multiple cases. A region near the macrophage scavenger receptor gene (MSR) in band 8p22 has emerged as a focus of allelic loss in prostate, colorectal, lung, and liver cancers (Emi et al., 1992, 1993; Fujiwara et al., 1993, 1994; Ohata et al., 1993; Bova et al., 1993; MacGrogan et al., 1994; Yaremko et al., 1994; Suzuki et al., 1995). In prostate cancer, for example, 8p is the most frequently lost chromosome arm (Bergerheim et al., 1991; Visakorpi et al., 1995), and MSR is the most frequently lost marker within 8p (Bova et al., 1993; MacGrogan et al., 1994). MSR and adjacent polymorphic markers have been placed on a physical map of megabase yeast artificial chromosomes (YACs) and radiation hybrids (Bookstein et al., 1994). Homozygous deletions are far less common than allelic deletions but are generally much smaller in size, suggesting closer proximity of the detecting probe and the putative target TSG. A human prostate cancer metastatic to a regional lymph node with complete absence of genetic material at the MSR locus has been described (Bova et al., 1993). Based on the possibility of deriving valuable positional information from this case, the boundaries of its homozygous deletion were localized within a refined, distance-based physical map by Southern blot analysis with a battery of specific probes (Bova et al., 1996). The deletion region was found to extend telomerically from MSR and measured between 730 and 970 kb. Recently, Kagan et al. (1995) reported a primary prostate tumor with homozygous deletion of another 8p22 locus, LPL, based on the lack of PCR amplification of a (CA)n polymorphism near this gene. MSR was the closest telomeric marker tested and was found to be retained in this tumor. Because MSR is expressed only in macrophages, it did not itself appear to be a plausible candidate TSG for epithelial neoplasms (Bova et al., 1993); instead, these findings suggested the existence of a different gene within the deletion region of Bova et al. (1996) that could function as a suppressor. We used hybrid selection with YAC DNA to isolate Ç40 cDNA frag-

The structure and expression pattern of a human gene located within a homozygously deleted region of a metastatic prostate cancer have been characterized. Multiple cDNA fragments of this gene were isolated by hybrid capture with yeast artificial chromosome clones covering the deletion region. Eleven coding exons spanned 205–220 kb of the 730- to 970-kb deletion. The predicted amino acid sequence was 43% identical to that of an anonymous Caenorhabditis elegans gene and 20% identical to an accessory or regulatory subunit of the oligosaccharyltransferase enzyme complex in Saccharomyces cerevisiae. Hydrophobicity profiles of all three gene products were similar and showed four putative membrane-spanning domains in the molecules’ C-terminal halves, suggesting a general conservation of function. The gene was expressed as an Ç1.5kb mRNA in most nonlymphoid human cells/tissues including prostate, lung, liver, and colon. Expression was detected in many epithelial tumor cell lines, but was undetectable by Northern blot or RT-PCR in 14 of 15 colorectal, 1 of 8 lung, and 1 of 4 liver cancer cell lines. Lack of expression in tumor cell lines was highly correlated with hypermethylation of a CpG island located at the gene’s 5* end. These findings form a basis for further work on this candidate tumor suppressor gene. q 1996 Academic Press, Inc.

INTRODUCTION

Several lines of evidence point to the short arm of chromosome 8 as the location for one or more tumor suppressor gene(s) (TSGs) involved in human oncogenesis. Allelic losses, which are indirect indicators of TSG mutation, have been examined in many tumor types. Although such alterations typically span large genetic distances in individual tumors, smaller consensus deleSequence data from this article have been deposited with the GenBank/EMBL Data Libraries under Accession Nos. U42349–U42360. 1 To whom correspondence should be addressed. Telephone: (619) 597-0177. Fax: (619) 597-0237. E-mail: [email protected].

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0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ments encoded by genes mapping within or adjacent to the deletion region. Fragments were triaged by sequence analysis and expression pattern on multiple tissue blots as described under Materials and Methods. One group of overlapping probes defined a gene, N33, that was expressed in most epithelial cells and tissues including prostate, colon, lung, and liver but not in a subset of cultured tumor cell lines, prompting the complete structural characterization described below. Lack of expression of this gene was found to be correlated with methylation of a CpG island at its 5* end. Reexpression was induced by the demethylating agent 5-aza-2*-deoxycytidine, indicating the involvement of methylation-based gene silencing. MATERIALS AND METHODS YAC clones and cultured cells. A contig of megabase YAC clones containing MSR was described previously (Bookstein et al., 1994). Tumor cell lines were obtained from Dr. John Isaacs (TSU-Pr1), Dr. Art Brothman (PPC-1), and the American Type Culture Collection (Rockville, MD). Cells were grown in DMEM with 10% fetal bovine serum. For demethylation/reexpression experiments, this medium was supplemented with 5-aza-2*-deoxycytidine (Sigma) at a concentration of 0.25 mM. Construction of PCR-amplifiable short-fragment cDNA libraries. RNA was isolated from tissues and cells using TriReagent (Molecular Research Center, Inc.) per the manufacturer’s instructions. Poly(A)/ RNA was selected from total RNA with biotinylated oligo(dT) primers and streptavidin-conjugated paramagnetic particles (PolyATtract kit, Promega). Double-stranded cDNA was made from poly(A)/ RNAs and one sample of total RNA per the manufacturer’s instructions with random primers and MMLV RT (RiboClone cDNA synthesis kit, Promega). A final step with T4 DNA polymerase yielded bluntended cDNAs. cDNA made from total RNA was set aside for use as a probe for ribosomal DNA (rDNA). Oligonucleotide SEL2 (5*CTCTAGTGGATCCTGTCACGCACA-3*) was 5*-phosphorylated with ATP and T4 kinase (377C, 30 min), heated to inactivate the enzyme, and annealed to equimolar amounts of SEL1 (5*-CGTGACAGGATCCACTAGAG-3*) to form a linker that was blunt and phosphorylated on one end and nonligatable on the other. Excess linker was ligated to the poly(A)/-derived cDNAs with T4 DNA ligase. cDNA (1–5 ml) was amplified in 100-ml reaction volumes containing 0.5 mM SEL1, 2.5 U Taq polymerase (Perkin–Elmer Cetus), 101 PCR buffer, 1.5 MgCl2 , and 200 mM dNTPs. Conditions were 957C, 30 s, then 957C, 15 s; 607C, 30 s; 727C, 2.5 min 1 20, and 727C, 10 min on a Perkin– Elmer GeneAmp 9600. PCR products were purified with Wizard PCR Prep spin columns (Promega) and eluted in 50 ml of 0.51 TE. Repetitive sequences were blocked by hybridizing purified amplified cDNAs (1–2 mg) with equal amounts (w/w) of Cot-1 DNA (GIBCO-BRL) at total DNA concentrations of 80 mg/ml in 120 mM NaPO4 buffer, pH 7. Reactions were overlaid with mineral oil, heated to 1007C for 5 min to denature, and then incubated at 607C for 20 h (Cot Å 20). Hybrid selection. The method of Morgan et al. (1992) was adapted with minor modifications. Purified YAC DNA was prepared by pulsed-field gel electrophoresis (PFGE) as described previously (Bookstein et al., 1994); 100–200 ng was biotin-labeled by random primer extension in the presence of biotinylated dATP (BioPrime kit, BRL) in 50-ml volumes according to kit instructions. Biotin-labeled YAC DNAs (10 ml of labeling reaction per hybridization) were heatdenatured, loaded into Centricon 100 filter units (Amicon) with blocked cDNAs (1 mg excluding Cot-1 DNA) and 2 ml of 1 mM NaPO4 , pH 7, and spun at 1000g for Ç25 min. The phosphate buffer wash was repeated once, and the retentate (60–80 ml) was collected into microfuge tubes. Volumes were reduced to Ç5 ml in vacuo, at which point the hybridization mixes were adjusted to 120 mM NaPO4 , pH

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7, 1 mM EDTA, pH 8, and DNA concentrations (excluding Cot-1) of Ç160 mg/ml. Reactions were overlaid with mineral oil and then incubated at 607C for 60 h (Cot Å 120). Streptavidin-conjugated paramagnetic particles (Promega) were prewashed twice with TE / 1 M NaCl and then incubated with completed hybridization reactions in 200 ml of TE / 1 M NaCl at room temperature for 15 min. Particles were collected magnetically, and supernatants were removed. Particles were washed five times with 15-min incubations in 200 ml of 0.11 SSC / 0.1% SDS, two at room temperature, then three at 657C, with magnetic collection between the washes. Bound cDNA was eluted from particles with 100 ml of 50 mM NaOH for 15 min, neutralized with 100 ml of 1 M Tris–HCl, pH 7.5, and transferred to clean tubes. Supernatants were desalted and concentrated using NaI and silica matrices (Geneclean kit, Bio 101) per themanufacturer’s instructions into 20-ml volumes of TE. These cDNAs were reamplified exactly as for the original libraries (see above) except that 5 ml of templates was used and PCR was carried out for 25–30 cycles. The resulting products were purified and blocked with Cot-1 DNA as above. Selection with YAC DNA was also carried out a second time as above. Second-round selected cDNAs were captured and amplified one more time. Final PCR products were cloned directly into pCRII vector (Invitrogen), transformants of which were plated onto Amp LB–agar plates with X-gal for blue–white selection per kit instructions. White colonies were picked, grown, and cryopreserved in 96-well master plates. A 96-pin replicating tool was used to inoculate nylon circles laid onto ampicillin–agar plates, which were grown overnight. Filters were lifted and processed as for filter colony hybridization screening (Sambrook et al., 1989) through 10% SDS, denaturation, neutralization, and 21 SSC. Filters were baked, prewashed, prehybridized in 0.051 BLOTTO, and then hybridized with nick-translated total human DNA (Alu probe) and random-primed rDNA (ribosomal DNA probe). After washing and autoradiography, hybridization-negative colonies were picked from the master plate for further characterization. Mini-scale DNA preps were analyzed by BamHI digestion and agarose gel electrophoresis. Inserts (250–1000 bp) were excised from low-melt agarose gels and radiolabeled by random priming. Probes were hybridized to filters containing HindIII-digested DNA from YAC clones and to triplets of human, chromosome 8 human–mouse hybrid, and mouse genomic DNA to identify singlecopy probes localized to human chromosome 8. cDNA library screening. Placenta and fetal brain lgt11 phage cDNA libraries (Clontech) were screened by hybridization with hybrid-selected cDNA N33 using standard methods (Sambrook et al., 1989). Clone inserts, e.g., N33C, were recovered by digestion with EcoRI and subcloned into pBluescript (Stratagene). Cloning of N33 exons. P1 and PAC (P1 artificial chromosome) clones were isolated by PCR- or hybridization-based screening of libraries (Genome Systems, Inc) with D8S549, E6, E31, IAY877-15, P25, and J28 (Bookstein et al., 1994; Bova et al., 1996). Six nonidentical clones were found to hybridize with N33C as probe. P1 or PAC DNA (1–2 mg) was digested with HindIII, purified using NaI and silica matrices (Geneclean kit, Bio-101), and subcloned into pBluescript vector (Stratagene). White colonies were picked and grown in 96-well plates and replicated onto filters as above. Filters were hybridized for 2–3 h in 0.051 BLOTTO at 507C with oligonucleotides designed per the cDNA sequence and end-labeled with T4 polynucleotide kinase (New England Biolabs) and [g-32P] or [g-33P]ATP (Sambrook et al., 1989). Alternatively, HindIII-digested P1 or PAC fragments were separated on 1% analytical agarose gel, blotted, and hybridized to oligonucleotide probes. Hybridizing fragments were purified from preparative gels (Geneclean, Bio-101) and directly cloned into pBluescript. The 9-kb HindIII fragment containing exon 7 was unclonable despite repeated attempts and instead had to be reduced to a 1.5-kb PstI–XbaI fragment for subcloning. Sequence analysis. Sequencing reactions were performed with dye terminator cycle sequencing or Sequenase kits (Applied Biosystems, Inc) and analyzed on an ABI 373A automated sequencer. Sequence analysis software included AutoAssembler 1.0 (Applied Bio-

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STRUCTURE AND EXPRESSION OF A TSG AT 8p22 systems, Inc) and MacDNASIS 2.0 (Hitachi). Sequence homologies were identified by BLAST searches in the nonredundant DNA and protein databases of the National Center for Biotechnology Information (NCBI). Other protein features were analyzed by the program PSORT (Nakai and Kanehisa, 1992). Physical mapping. Long-range restriction mapping was performed essentially as described (Bova et al., 1996). In brief, YAC 946_c_9 DNA was digested with infrequently cutting enzymes including SfiI, NruI, MluI, EagI, and BssHII and separated in 1% agarose gels (Pulsed Field Certified, Bio-Rad) in 0.51 TBE on the CHEF-DR III apparatus (Bio-Rad) at 50- to 90-s switch times ramped over 22 h at 6 V/cm and 1207 orientation. Southern analysis was then carried out with radiolabeled genomic probes as described (Bova et al., 1996). Northern analysis. Total RNA was prepared from tumor cell lines using TriReagent (Molecular Research Center, Inc.) per the manufacturer’s instructions. Poly(A)/ RNA was purified by magnetic bead capture using a polyATtract mRNA isolation kit (Promega). Approximately 3 mg of mRNA per lane was electrophoresed through 1% agarose gels containing 0.66 M formaldehyde (Sambrook et al., 1989) and blotted overnight onto Hybond-N filters (Amersham). Human multiple tissue Northern blots (2 mg mRNA per lane) were purchased from Clontech. Hybridizations were performed for 1 h at 687C in RapidHyb solution (Amersham) with random-primed N33C probe. Alternatively, an antisense probe was generated by in vitro transcription (Maxiscript, Ambion) of linearized N33C with T7 polymerase in the presence of [a-33P]UTP (New England Nuclear). Blots were washed in 0.11 SSC, 0.1% SDS at 687C and autoradiographed with phosphor imaging screens (Molecular Dynamics). Blots were then stripped in 0.11 SSC, 0.5% SDS at 1007C and reused with additional probes, including b-actin (Clontech) as a control for RNA loading. Reverse transcription–PCR and direct sequencing. Random hexamer-primed reverse transcription of mRNA was performed using a kit (Perkin–Elmer Cetus) according to the manufacturer’s protocol. Oligonucleotide primers for displaying alternative splicing were N33m6f (5*-GGACCTCCATATGCTCATAA-3*) and N33m11r (5*CACTTTATGCAAATCCCACT-3*) (exons 6–11). PCR conditions were 957C, 1 min, then 957C, 15 s; 557C, 30 s 1 30, and 727C, 5 min on a Perkin–Elmer GeneAmp 9600 thermocycler. Products were separated by polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. Products were cloned into pCRII (Invitrogen) and sorted by insert size. Primers for measuring N33 expression were N33m2f (5*-GTAGAGCAGCTGATGGAATG-3*) and N33m5r (5*-CCAGAGTAGTTGGGTGGT-3*) (exons 2–5, 442 bp). An Ç950-bp segment of Rb1 cDNA defined by two primers (5*-CCTCCCATGTTGCTCAAAG-3* and 5*-CTTGTCAAGTTGCCTTCTGC-3*) was used as an internal control for the RT-PCR amplification in cell lines. PCR conditions were 957C, 1 min, then 957C, 15 s; 587C, 30 s; 727C, 30 s 1 30, and 727C, 5 min. Products were separated on 1.5% agarose gels and DNA was visualized by ethidium bromide staining. Template for direct sequencing was made by amplifying reversetranscribed mRNA with primers N33m2f and N33m11r (exons 2–11) in 100-ml volumes under the latter PCR conditions, then purifying products with Wizard PCR Prep columns (Promega). A Taq polymerase-based cycle sequencing kit (Perkin–Elmer Cetus) with [33P]dATP incorporation labeling was employed in conjunction with various primers made according to the cDNA sequence. Termination reactions were electrophoresed in groups by nucleotide to allow rapid visual detection of differences among samples in autoradiograms. Methylation analysis. High-molecular-weight DNA was prepared according to standard procedures (Sambrook et al., 1989). Approximately 20 mg of DNA was digested overnight with excess HindIII restriction enzyme. Restriction digests were ethanol-precipitated, resuspended, and divided equally for 5- to 6-h restriction digestions with EagI or BssHII. Digests were analyzed by electrophoresis in 1% agarose gels and blotted onto nylon filters as described (Sambrook et al., 1989). N33E1, a 1.1-kb HindIII fragment containing the N33 promoter and exon 1, was radiolabeled by random priming. Hybrid-

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izations were performed in RapidHyb (Amersham) for 2–3 h. Final washes were in 0.11 SSC, 0.5% SDS at 687C.

RESULTS

Characterization of Hybrid-Selected cDNAs PCR-amplifiable cDNA libraries representing placenta, fetal brain, colonic mucosa, normal prostate, and two cultured cell lines were constructed as described under Materials and Methods and were selected with several YACs from within the 8p22 contig. Forty selected cDNA clones that were nonrepetitive and mapped to human chromosome 8 and to the YAC used for selection were isolated (Bookstein et al., 1994). The subset of clones mapping within the homozygous deletion region (Bova et al., 1996) was sequenced. Three clones were found to be segments of MSR cDNA, indicating successful selection of a known gene within the contig. One clone (J12) was 100% identical to a cDNA segment of the a subunit of human protein phosphatase type 2C (GenBank Accession No. S87759). Cloning and sequencing of the corresponding locus in the YAC contig revealed about 5% nucleotide mismatches with the cDNA, a frameshift, and a lack of introns, suggesting the presence of a pseudogene that selected for a fragment of the authentic gene (data not shown). Four selected cDNAs (N33 and others) overlapped by sequence and defined a partial open reading frame with homology to an anonymous gene in Caenorhabditis elegans. The remaining clones lacked long open reading frames, and BLASTX searches of nonredundant protein databases at NCBI did not reveal any significant homologies with other protein sequences. To prioritize the analysis of candidate genes further, Northern blots of mRNA from multiple human tissues were hybridized with cDNA probes mapping within the deletion region. Representative results with two probes are shown in Fig. 1. Genes such as N33 that were widely expressed in, e.g., prostate, lung, colon, and liver, the tissues of origin of cancers with allelic losses in 8p22, were favored over genes such as J2 that had a more restricted pattern of expression in ‘‘irrelevant’’ tissues. N33 was further distinguished from many other probes in Southern blots of human and mouse DNA, which suggested the presence of multiple exons in the human and evolutionarily conserved sequences in the mouse (data not shown). Based on these observations, N33 became a focus for further structural analysis. Complete Structural Characterization of N33 To obtain longer cDNA clones and to exclude PCRrelated sequence artifacts, two l phage cDNA libraries were screened by hybridization with N33. The longest N33-related phage clone, N33C, was subcloned and completely sequenced and was found to contain a 1342bp EcoRI–EcoRI insert (GenBank Accession No.

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FIG. 1. Expression patterns of two genes detected by hybridselected cDNA fragments in various human tissues. Striated muscle forms of the b-actin control are seen in some lanes. PBL, peripheral blood leukocytes.

U42349) with a long open reading frame of 1044 bp (348 amino acids) terminating at nt 1202. The gene was then characterized at the genomic level by subcloning all exon-containing fragments from P1 or PAC genomic clones as described under Materials and Methods. A series of oligonucleotides based on the cDNA sequence was synthesized in a stepwise fashion to prime sequencing reactions in genomic templates, which defined one boundary for each exon; complementary primers were then made to sequence back from the intron through the exon to define the opposite exonic boundary and intron. The splice donor and acceptor sequences and boundary positions for the 11 exons are shown in Table 1 (all exon and flanking intron sequences have been deposited with GenBank under Accession Nos. U42350 through U42360). Of note is a

nonconforming/gc splice donor site for exon 10, which may explain the alternative splicing of exon 10 described below. The first exon of N33 was found within a 1.1-kb HindIII fragment (N33E1) that was entirely sequenced (Fig. 2). The first base of the cDNA was at nucleotide 302 of N33E1, suggesting that transcriptional initiation occurred at or before this point. GC-rich sequences were present upstream, within, and downstream of exon 1, indicating the presence of a CpG island at the gene’s 5* end. Two infrequently cutting restriction sites, EagI and BssHII, clustered within Ç100 bp (Fig. 2). The presumptive N33 promoter contained consensus recognition sequences for Sp1 (Kadonaga et al., 1987), AP2 (Imagawa et al., 1987), and p53 (El-Deiry et al., 1992), but no TATA box. Sequences around the first methionine codon at nt 450–452 (nt 158–160 in N33C) conformed to the Kozak consensus (Kozak, 1984). An upstream, in-frame stop codon was found at nt 396–398. Exon 11 of N33 was present within another 1.1-kb HindIII fragment (N33E11) that was also entirely sequenced (data not shown). Identical sequences preceding an EcoRI site were found in both genomic and cDNA clones, suggesting that the 3* end of N33 cDNA was truncated by EcoRI digestion during library construction. This may account for the size disparity between N33C (1342 bp) and N33 mRNA (Ç1.5 kb) despite the presence of an apparently complete 5* end in the former. A consensus polyadenylation signal (AATAAA) was found approximately 170 bp downstream from the EcoRI site. If polyadenylated near this site and transcribed from nt 302 of N33E1, full-length N33 mRNA would have a predicted size of Ç1530 bp plus the poly(A) tail. Physical mapping of N33 exons was performed using PFGE and Southern blotting of YAC DNA according to methods described in Bova et al. (1996). The location of exon 1 was pinpointed on the basis of

TABLE 1 N33 Exon/Intron Organization Donor exon No.

Exon length (bp)

Donor sequence

1 2 3 4 5 6 7 8 9 10

ú286a 170 118 141 141 90 64 75 91 65

GAAAAAGGAG/gtagaatgga CTGTGTGCAG/gtaatttatg TTTTCAGCAG/gtaagagtta GGATGTTCAT/gtatgttttt GGTGTCTCTG/gtatgtaaat TGGACAAGTG/gtaagtgtaa CTGGTACTGA/gtatcctttt AAAAGACGGA/gtaagtctct ATCCTTATAG/gtaatatctt ATAACCTCAG/gcaagtcttt

Position in N33C of exon boundary (nt No.) 295/296 465/466 583/584 724/725 865/866 955/956 1019/1020 1094/1095 1185/1186 1250/1251

Acceptor sequence

Acceptor exon No.

ttaattgcag/AATCTTTTAG ttctcatcag/GCAAGCTAAT tgttttacag/CTCAACATGA atgtagggag/ATTCGGGTTT tgtttttcag/TGTATAGTCT tcttttatag/AGCTACATTC actactgcag/ATGCCGCTAT ctgtttctag/TAATTTGCCT tattggaaag/TGATCTGGAC tctttttcag/CTTTTTAATT

2 3 4 5 6 7 8 9 10 11

Note. Exonic sequences are in uppercase; intronic sequences are in lowercase. a The first nine nucleotides of N33C insert are derived from the phage vector.

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FIG. 2. Sequence of the 5* upstream region and exon 1 of the N33 gene. Nucleotides present in N33 cDNA clones are in uppercase letters; nucleotides in the 5* upstream region and intron 1 are in lowercase. Consensus recognition sequences for cis-acting regulatory elements in the putative promoter are double-underlined; breaks in the first p53 site indicate positions not conforming to the consensus 5*-PuPuPuC(A/T)(T/A)GPyPyPy-3* (Pu, purine; Py, pyrimidine). Predicted translation from the first ATG codon is shown in single-letter code. Selected restriction sites are singly underlined. An upstream, in-frame stop codon is marked by an asterisk.

its infrequent-cutting restriction sites (Fig. 3). The derived map showed that exon 11 was located 205 – 220 kb from exon 1, indicating that N33 occupied a substantial fraction of the homozygous deletion region. The transcriptional orientation was tel – 5* – N33 – 3 * – cen. RT-PCR analysis with oligonucleotide primers in exons 6 and 11 showed length heterogeneity in N33 RNA products, with three mRNA forms evident in most cells or tissues (Fig. 4A). The larger two transcripts, Forms 1 and 2, were generally found at abundance ratios of about 1:2, whereas the smallest transcript, Form 3, was least abundant and was most easily seen in placenta. Additional clones representing these three cDNA products were isolated as described under Materials and Methods. Sequence analysis showed that they were products of alternative splicing of exons 9 and 10 (Fig. 4B). Form 1 included all 11 exons as represented by N33C, Form 2 lacked exon 10, and Form 3 lacked exons 9 and 10. Alternative splicing resulted in minor variation at the C-termini of the predicted amino acid sequences (Fig. 4B). Form 1 products were predicted to contain 348 amino acids terminating at a stop codon in exon 10. Form 2 products were predicted to contain 347 amino acids terminating at a stop codon in exon 11. Form 3 products were predicted to contain 314

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amino acids terminating at another stop codon in exon 11. The predicted products of the two most abundant transcripts differed in only the last 4 to 5 amino acids. A BLASTP search with the N33 protein sequence revealed a highly significant homology (P Å 5.8 1 100100) to an anonymous gene in C. elegans randomly sequenced at both the genomic (cosmid ZK686) and cDNA levels (database entries: SWISS-PROT P34669; GenBank M88869, T01933, L17337; PIR S44911). A more distant relationship (P Å 1.0 1 10010) to the OST3 gene of Saccharomyces cerevisiae was also detected (Karaoglu et al., 1995). A gapped alignment of these sequences (Fig. 5) showed 43% identical residues between the human and C. elegans sequences extending throughout most of the length of both proteins, with local runs of 12 or 13 consecutive perfect matches and many conservative substitutions as well. The human and yeast sequences were only 20% identical, but matches were again scattered throughout most of the protein length and were clustered at regions of greatest conservation between human and worm. The C. elegans and S. cerevisiae sequences were 21% identical in this alignment (P Å 1.8 1 10012) (Fig. 5). The N33 sequence had 26 additional amino acids at its N-terminus compared to those of worm and yeast, whereas a C-terminal region from the worm protein had a 17-amino-acid insert compared to the human sequence. Hydrophobicity profiles for the three polypeptides were strikingly similar (Fig. 6), with four presumptive membrane-spanning domains in the C-terminal halves of the molecules. The Ntermini of the worm and yeast proteins included apparent signal sequences 19 and 22 residues in length, respec-

FIG. 3. Long-range restriction map of the N33 region. Rare-cutting restriction enzyme sites (A, AscI; B, BssHII; E, EagI; M, MluI; Nr, NruI; S, SalI; Sf, SfiI; X, XhoI) are indicated (the map is complete only for A, M, Nr, and Sf). Selected markers or exons were located in map segments (brackets) delimited by restriction sites or the ends of YAC (877_f_2, 932_e_9) or PAC (6M7) clones as described under Materials and Methods. Ends of clones are indicated by vertical bars. All markers shown here were within the reference homozygous deletion (Bova et al., 1996).

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FIG. 4. Alternative splicing of N33 exons. (A) Three splice variants were detected in most RNA samples by RT-PCR (see Materials and Methods). PCR with the same primers was also performed with cloned cDNA templates N33C, A4, A5, and N33-7. (B) Nucleotide sequences and predicted translation products of the three splice variants. Exon boundaries are indicated by shills, and stop codons by asterisks. Positions are numbered per N33C. Nucleotide 1252 was ‘‘C’’ in N33C and ‘‘T’’ in all other clones.

tively (Karaoglu et al., 1995; R. Gilmore, Worcester, MA, pers. comm. 1995). The human N-terminal sequence was predicted by the program PSORT to contain a transmembrane segment (Nakai and Kanehisa, 1992), although a possible signal peptide cleavage site at residue 40 was also detected, which if used would result in roughly similar positions for the N-termini of the three mature proteins. Expression of N33 in Tumor Cell Lines As shown in Fig. 1, N33 was expressed as an Ç1.5kb mRNA in most tissues including heart, placenta,

lung, liver, pancreas, prostate, testis, ovary, and colon. Expression in spleen, thymus, small intestine, and peripheral lymphocytes was not detected, implying that N33 was probably not a ubiquitous marker of immune, fibroblastic, or vascular cells. Similar Northern analyses of mRNA from a panel of tumor cell lines showed expression of N33 in 4 of 4 prostate cancer cell lines, 3 of 4 liver cancer cell lines, and 5 of 5 lung cancer cell lines, but in only 1 of 15 colorectal cancer cell lines (Fig. 7; Table 2). To specify further the expression of N33 in the precursor tissue of colon cancers, mRNA

FIG. 5. Alignment of the predicted protein sequences from N33 (human), ZK686.3 (C. elegans), and U25052 (S. cerevisiae). Identical residues are boxed; gaps (-) were introduced to optimize alignments. For N33, the Form 2 polypeptide sequence was used. The initiation codon of ZK686.3 was reassigned to add nine amino acids at the N-terminus (R. Gilmore, Worcester, MA, pers. comm., 1995).

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Of the 21 N33/ tumor cell lines listed in Table 2, 19 (not done: H596 and H522) were examined for nucleotide substitution mutations within N33 mRNA by direct cycle sequencing of RT-PCR products as described under Materials and Methods. Exon 1 was excluded from sequence analysis for technical reasons relating to its high G / C content, and alternative splicing of exon 10 prevented the reading of sequence data beyond exon 9. By these methods, cDNA sequences from all cell lines were identical to wildtype with one exception, HEP3B, which expressed N33 mRNA with both wildtype C and mutant A at nucleotide position 437 (data not shown). The relevant codon (94) encoded Gln as wildtype and Lys with the altered nucleotide. The heterozygous nature of this mutation suggested that it was probably not significant. We preliminarily concluded that expressed point mutations of N33 were uncommon in these epithelial tumor cell lines. Methylation-Associated Silencing of N33

FIG. 6. Kyte–Doolittle hydrophobicity profiles of predicted proteins from N33 (human), ZK686.3 (C. elegans), and U25052 (S. cerevisiae). N33 Form 2 polypeptide was used.

was extracted from colonic mucosa dissected from the colonic smooth muscle wall; N33 expression was again observed (Fig. 7). Many of the cell lines were also tested by RT-PCR, which confirmed the absence of N33 expression in most colorectal tumor lines and extended the analysis to additional cell lines, including a lung line (CALU-6) that tested as N330 (Table 2).

Aberrant hypermethylation of CpG islands near the 5* ends of some tumor suppressor genes has been proposed as a mechanism for their inactivation. To investigate this possibility with N33, we tested for digestion of tumor cell line DNA by methylation-sensitive restriction enzymes BssHII or EagI at their single recognition sites in exon 1. Codigestion with HindIII served to reduce exon 1-containing DNA fragments to 1.1 kb or less and allowed for their reliable detection in Southern blotting with the complementary probe N33E1. Detection of only the uncut 1.1kb band indicated completely methylated sites; if the DNA was partially or completely unmethylated, smaller fragment sizes were observed (Fig. 8). Application of this assay to the panel of tumor cell lines described above showed that lack of N33 expression was associated with complete methylation in most cases (the two exceptions are shaded in Table 2), whereas all N33/ lines were either partially or completely unmethylated. Colonic mucosa (N33/) was also unmethylated (Fig. 8). These observations suggested that unmethylated CpG islands were necessary but not sufficient for N33 expression in these tumor cell lines.

FIG. 7. Expression of N33 in cultured human tumor cell lines and normal colonic mucosa. Most colorectal tumor cell lines failed to express N33, as shown in these representative Northern blots and as summarized in Table 2. b-actin probe was used as a control for mRNA loading.

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TABLE 2 N33 Gene Expression and Methylation in Cell Lines Tissue

N33 expression

Cell line

PP PP PP PP

N33 gene methylation

Prostate

DU145 TSU-PR1 LNCaP PPC-1

P, P, P, P,

No No No No

Colon

nl.mucosa SW48 SW403 SW480 SW837 SW1417 HT-29 HCT-116 SK-CO-1 CACO-2 COLO-205 COLO-320 LS174T EB DLD-1 RKO

P, PP P, PP N, NN N, NN N, NN N, NN N, NN N, NN N, NN N, NN N, NN N, NN N, NN N, NN N, NN N, NN

No Partial No Yes Yes Yes Yes Yes Yes Yes ND Yes Partial Yes Yes Yes

Liver

HLE HEP3B SK-HEP-1 HEPG2

PP PP PP NN

Partial ND No Yes

Lung

WI-38 CALU-6 H460 H596 A549 SK-MES H522 H358

P N P, PP P, PP P P, PP PP PP

Partial Yes ND Partial No No No No

Breast

T47D MB468

PP PP

ND ND

Bladder

UMUC-3 5637 J28 HT1376

PP PP PP PP

No No ND No

FIG. 8. Methylation assay for restriction sites in N33 exon 1. Representative Southern blot analysis of HindIII/BssHII (Bs)- and HindIII/EagI (E)-digested DNAs from normal colonic mucosa and selected cell lines. Band sizes (in kilobases) detected by the N33E1 probe are indicated (right).

loss and infrequent homozygous deletion in prostate, lung, liver, and colon cancers and on its markedly decreased expression in colorectal and other cancer cell lines compared to corresponding normal tissues. Structural characterization showed that it was a previously undescribed human gene with distinct homologs only in distantly related organisms. We also found that the absence of N33 expression in many tumor cell lines was best explained by a mechanism of methylationassociated gene silencing, whereas N33/ tumor cell lines for the most part expressed the wildtype mRNA sequence. Alterations in patterns of DNA methylation are observed during both organismal development and carcinogenesis (Laird and Jaenisch, 1994). The latter process is associated with generalized hypomethylation of tumor cell DNA as well as with hypermethylation of

Note. ND, not done; P, positive/PCR; N, negative/PCR; PP, positive/ Northern blot; NN, negative/Northern blot.

Further evidence for the role of methylation in N33 expression was obtained by culturing an N330 colorectal tumor cell line, DLD-1, in the presence of the maintenance methylation inhibitor 5-aza-2*-deoxycytidine. Demethylation occurs in concert with cell division, during which the maintenance activity normally functions to methylate newly synthesized DNA strands. Reexpression of N33 was observed after 5 days in culture (Fig. 9). Decreased DNA methylation was therefore sufficient to reactivate the gene in this cell line. DISCUSSION

N33 was examined as a candidate tumor suppressor gene based on its location in a region of frequent allelic

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FIG. 9. Reexpression of N33 in a cultured cell line. RT-PCR was performed on RNA extracted from DLD-1 cells grown for 0 or 5 days in the presence of 0.25 mM 5-aza-2*-deoxycytidine. N33C was used as a control template. PCR products were separated in 1.5% agarose gels and visualized by ethidium bromide staining. A segment of the retinoblastoma gene (Rb) was amplified as an internal control for mRNA quality.

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STRUCTURE AND EXPRESSION OF A TSG AT 8p22

DNA within ‘‘CpG islands’’, i.e., G / C-rich regions that are present at the 5* ends of about 60% of human genes. In particular, hypermethylation of CpG islands upstream of tumor suppressor genes such as Rb1, VHL, and CDKN4 is associated with lack of expression and loss of gene function, much as are deletions or mutations of the same genes (Sakai et al., 1991; Herman et al., 1994, 1995). However, very little is known of the mechanisms leading to alterated methylation in cancer. It is not clear whether cancer-related hypermethylation of CpG islands is generalized or specific to certain genes or whether methylation is a primary cause or merely a secondary marker of gene silencing (Bird, 1992; Laird and Jaenisch, 1994). Furthermore, changes in methylation may occur as an artifact of cell culture (Bird, 1992), although we have observed similar methylation of the N33 5* end in DNA from primary colorectal tumors (D.M., M. Pegram, D. Slamon, and R.B., unpublished data). Because hypermethylation probably affects the expression of many cellular genes at once, this change may be intrinsically less specific than mutation for the purposes of identifying tumor suppressor genes. For example, a human gene called HIC-1 located in a CpG island in chromosome band 17p13 was found to be hypermethylated and silenced in many human cancer cells (Wales et al., 1995). Missense or nonsense point mutations, which might have provided structural evidence for the identification of HIC-1 as a tumor suppressor, were not reported, and these may not occur if methylation is a primary mechanism of inactivation. Instead, functional assays were performed to show that introduction of HIC-1 into cells lacking endogenous expression of this gene inhibits their growth in culture and in tumorigenicity assays (Wales et al., 1995). Because we did not uncover any specific, inactivating mutations of N33 in cultured tumor cell lines of various types, establishing N33 as a tumor suppressor may require functional studies of its replacement in N330 cell lines. Furthermore, the fact that absence of N33 expression and methylation was found primarily in colorectal tumor cell lines suggests that inactivation of this gene may not be as significant in the other cancer types. It is also possible that methylation of N33 is a manifestation of a regional methylation ‘‘hot spot’’ (Bustros et al., 1988), the actual target of which is a different, nearby gene. MSR is the only other well-characterized gene in the immediate vicinity, and its highly restricted natural tissue distribution would make it uninformative for such studies. Pairs of oppositely-transcribed genes may share CpG islands at their 5* ends, so a companion transcription unit of N33, if it exists, might be a good initial target for further investigation. Karaoglu et al. (1995) described the cloning of yeast OST3 (oligosaccharyltransferase 34-kDa subunit) and noted the sequence homology between its product (Ost3p) and an anonymous predicted open reading

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frame (ORF) from C. elegans. We had previously identified the C. elegans ORF as the most closely related gene to human N33 in the sequence databases (P Ç 100100) and found that N33 also displayed significant similarity to Ost3p. Dolichylpyrophosphoryloligosaccharide:polypeptide oligosaccharyltransferase (OST) (Das and Heath, 1980) catalyzes an early step of N-linked glycosylation of proteins in the endoplasmic reticulum, namely the en bloc transfer of a 14-subunit carbohydrate moiety onto asparagine residues within the sequence N-X-(S/T) (where X is any amino acid except proline). OST has been purified as a complex of three subunits in canine and avian cells and six subunits in S. cerevisiae (Kelleher and Gilmore, 1994). Products of the essential yeast genes OST1, SWP1, and WBP1 are homologs of the three OST vertebrate subunits ribophorin I, ribophorin II, and OST48, respectively (Silberstein et al., 1992, 1995; Kelleher and Gilmore, 1994). Unlike mutants of OST1, SWP1, and WBP1, yeast OST3 null mutants are fully viable regardless of culture conditions but show underglycosylation of soluble and membrane-bound glycoproteins, with selectively more severe effects on proteins in the latter category (Karaoglu et al., 1995). Ost3p thus has properties of a regulatory rather than a catalytic subunit. The percentage of sequence identity between Ost3p and N33 (20%) is somewhat less than the percentages of identity among the other OST subunits such as Ost1p/ribophorin I (28%), Swp1p/ribophorin II (22%), and Wbp1p/OST48 (25%) (Silberstein et al., 1992, 1995; Kelleher and Gilmore, 1994). The Ost3p/N33 sequence divergence is also slightly greater than expected based on the sequence divergence between human and C. elegans proteins (43%) and the times of descent from last common ancestors (Doolittle, 1993). Possible reasons include (i) nonuniform evolutionary rates, (ii) the existence of gene families in these organisms, other members of which are true orthologues, and (iii) unrecognized sequencing errors. Nevertheless, the similarities in primary and secondary structure between N33 and Ost3p suggest the existence of OST regulatory subunits in vertebrate cells. Although such additional subunits have not been observed in purified complexes from mammalian cells, their association with the catalytic subunits could be more easily disrupted during purification than in yeast. The significance of this homology with regard to carcinogenesis is uncertain. The glycosylation of proteins and other substrates is involved in numerous biological processes (Varki, 1993), some of which, such as cell:cell or cell:matrix interactions, have important roles in cancer. Altered glycosylation of tumor cell surface proteins has been observed repeatedly; most of the reported changes are of terminal glycosylation related to incomplete oligosaccharide processing or altered activities of various glycosyltransferases in the Golgi apparatus (Dennis, 1992). Glycosylation is generally perceived as facultative for important cancer cell functions; for ex-

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ample, increased branching and sialylation of N-linked oligosaccharides is associated with enhanced tumorigenicity and metastasis of tumor cells, and mutant CHO cells selected for various defects in glycosylation usually have reduced metastatic ability (Dennis, 1992). Although these features do not support the simple notion of an oncogenic glycosylation defect of rough endoplasmic reticulum, the complexity of glycosylation pathways allows for the speculative possibility that loss of a subtle regulatory activity could promote some aspect of the neoplastic phenotype. ACKNOWLEDGMENTS We thank Erika Rickel and Uyen-Chi Dang for technical assistance, Whei-Mei Huang and Vicki Carhart for DNA sequencing, Bei Shan, Art Mendoza, Mark Pegram, and Dennis Slamon for clinical samples, Art Brothman and John Isaacs for cell lines, and Yuh-Shan Jou, Rich Gregory, Michael Shepard, Russell Doolittle, and Reid Gilmore for advice. This work was supported by Grants R01 CA60358, SPORE CA58236, K11 CA59457, and R29 CA55231 from the National Cancer Institute.

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