A Homozygous Deletion Within the Carbonic Anhydrase-Like Domain of the Ptprg Gene in Murine L-Cells1

(CANCER RESEARCH 53. 14Â«)Â»-1502.April1. 1993|

Advances in Brief

A Homozygous Deletion within the Carbonic Anhydrase-like Domain of the Ptprg Gene in Murine L-Cells1

Kishore K. Wary, Zhuangwei Lou, Arthur M. Buchberg, Linda D. Siracusa, Teresa Druck, Sal LaForgia, and Kay Huebner2

Jefferson Cancer Instilare. Thomas Jefferson Medical College, Philadelphia. Pennsylvania Â¡9ÃŒ07

Abstract and murine predicted amino acid sequences for this isoform are greater than 90% identical: the Ptprg gene contains a single trans- Protein tyrosine phosphatases, on purely theoretical grounds, were sug membrane domain and two tandem tyrosine phosphatase domains. gested as possible tumor suppressor genes, and receptor protein tyrosine The extracellular region contains one fibronectin type repeat and. like phosphatase 7 (PTPRG ) has been proposed, on the basis of its location at the PTPRZ gene (7).' an NH2-terminal region of 266 amino acids with human chromosome region 3pl4.2, specifically as a tumor suppressor gene â€”¿30%sequence similarity to members of the carbonic anhydrase for renal cell carcinoma. We have isolated murine genomic and complementary DNA clones for enzyme family (8). analysis and mapping of the murine Ptprg locus; interspecific backcross During an investigation of the genomic organization of human and analysis showed that the Ptprg locus maps to the centramene region of mouse Ptprg loci, we noted extensive polymorphism of the murine mouse chromosome 14. We also observed a homozygous, intragenic dele PÃprglocus5 and a homozygous deletion of a portion of the CA-like tion in the Ptprg gene in all donai derivatives of the original I.-cell strain, domain in murine L-cell lines, which we undertook to describe in a methylcholanthrene-treated mouse connective tissue cell line which pro detail, since homozygous deletion of a gene is one of the hallmarks of duces sarcomas in syngeneic mice. The deletion begins in the second intron suppressor genes in tumors and tumor cell lines (for review, see Ref. of the carbonic anhydrase-like domain of the Ptprg gene and ends in the 9); at the same time we determined the chromosome location of the fourth intron of the carbonic anhydrase-like domain. At the genomic level, murine Ptprg locus. perhaps several hundred kilobases of DNA are deleted; at the complemen tary DNA level the 400 base pairs comprising exons 2, 3, and 4 of the carbonic anhydrase-like domain are deleted. By reverse transcription Materials and Methods polymerase chain reaction, an amplified fragment is produced from L-cell Cultured Cells. The TK- el-ID cell line is a clonal derivative of the inKN'A which is 400 base pairs shorter than the wild type gene product, parental L-strain (10). as are A9, LMTK-. and L929 cell lines. The parent suggesting that the deleted gene is transcribed and may produce a protein L-cells were established in culture from normal s.c. areolar and adipose tissue product. of a 100-day-old male C3H mouse after treatment with methylcholanlhrene Thus, mouse L-cells have lost one Ptprg alÃeleand sustained an in (10). The L929 and A9 cell lines were purchased from the American Type tragenic deletion in the other; such alÃeleloss and mutation frequently Culture Collection, while cl-ID (originally from Saul Kit) and LMTK- cell occur at tumor suppressor gene loci. lines were available in this laboratory, where they are routinely used in isola tion of somatic cell hybrids. IT22 cells are a TK- derivative of a Swiss 3T3 Introduction ftbroblast cell line. Mel cells were originally derived from the spleen of a virus-induced leukemic DBA/2J mouse (11). Mainly because of their potential to oppose or reverse effects of Ptprg cDNA and Genomic Clones. Approximately 3 x K)5 plaques of a tyrosine kinases (for reviews see Refs. 1 to 3), protein tyrosine phos B6/CBA mouse lung cDNA library (Stratagene) were screened by colony phatases were proposed as possible suppressor genes. In determining hybridization using the 5'-most 740 nucleotides of the human PTPRG cDNA the chromosomal location of several human receptor protein tyrosine as probe. cDNA clones obtained were subcloned into a pBluescript (SK+) phosphatase (PTPR) genes (4, 5),3 we noted that the PTPRG4 gene vector. A mouse genomic library (B6/CBA strain. ~2 X IO5 plaques in A Fix mapped to the 3p 14-21 chromosome region and might be a suppressor II vector; Stratagene) was screened with the same human probe; two Ptprg gene involved in kidney carcinomas (5). genomic clones, carrying a portion of the Ptprg CA domain 2nd exon and Recently, full length cDNA clones for at least one isoform of human adjacent intron. were mapped. A 1.5-kilobase unique fragment. pK.Wml.5B. and murine Ptprg have been sequenced and analyzed (6). The human was subcloned into a pGEM vector (Promega) and used in murine backcross analysis. DNA Sequence Analysis. Mouse Ptprg cDNA clones were sequenced us ing T-3 and T-7 primers and several synthetic oligonucleotides. Sequencing of Received 2/8/93; accepted 2/26/93. the double-stranded plasmids was performed with the Taq DyeDeoxy Termi The costs of publication of this article were defrayed in part by the payment of page nator Cycle Sequencing Kit (Applied Biosystems, Inc.): reaction products charges. This article musi therefore be hereby marked advertisement in accordance with were electrophoresed and recorded on the 373 DNA Sequencer (Applied Bio- 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported by USPHS Grant CA 21124 and by an American Cancer systems, Inc.). Sequence analyses were accomplished with GCG software. Society Junior Faculty Research Award (L. D. S-). PCR Probe Generation. The oligonucleotides for generating Ptprg probes 2 To whom requests for reprints should be addressed, at Jefferson Cancer Institute, were designed, based on the mouse Ptprg sequence (see Fig. I ), using the Thomas Jefferson Medical College, BLSB. Rm. 1008A. 233 S. 10th St., Philadelphia. PA 19107. computer program Oligo 4.0 (National Biosciences). For Southern blots, ' J. B. Levy, P. D. Canoll, O. Silvennoinen, G. Bamea, B. Morse, A. M. Honneger, J-T. probes A. B, C, D, and G (see Fig. 2A} were produced by PCR amplification Huang, L. A. Cannizzaro, S-H. Park. T. Druck, K. Huebner, J. Sup. M. Erhlich. J. M. using primers A-1 plus A-2, B-1 plus B-2. C-1 plus C-2. D-1 plus D-2. and G-1 Musacchio, and J. Schlessinger. The cloning of a receptor-type protein-tyrosine phos plus G-2, respectively. Primers R-l and R-2 were symhesi/.ed for RT-PCR; phatase that is expressed in specific regions of the central nervous system, J. Biol. Chem.. probe R. generated using primers R-3 and R-4. was used for detection of in press, 1993. 4 The abbreviations used are: PTPRG. receptor protein tyrosine phosphatase -y;cDNA, complementary DNA; PCR, polymerase chain reaction; RT, reverse transcription; CA, carbonic anhydrase; Mel. murine erythroleukemia; TK-. thymidine kinase deficient. ^ Unpublished data. 1498

M T V P H Q PtpraaÃ¶ ATTCCGGAACCAATTTCTCCCCTTTCCCTCCTCCCTCCTGGG ATG ACG GTG CCC CAC CAG PtprntÃ¶O

FVRGQERKKVYYNTLVKELG Ptpraa26 TTT GTA CGT GGG CAG GAG AGG AAG AAA GTC TAT TAT AAT ACA CTT GTA AAA GAG CTG GGG PtpTntl20

TFLGRNAPCPACPRLKAAVL PtpTaa46 ACC TTC CTC GGG AGG AAC GCT CCC TGT CCA GCT TGT CCA AGG CTG AAG GCT GCT GTG CTT PtpmtlSO A-l

HYVVCFPALTEGYVGTLQES PtpTaa66 CAT TAT GTG GTG TGC TTC CCG GCA TTG ACT GAA GGC TAT GTG GGG ACC CTG CAG GAG AGC PtpTnt240 (D-l) > R-1 > R-3 â€”¿ s H H w G CAaae RQDSSVQIRRRKASGDPYWA PtpTaa86 AGA CAG GAC AGC TCA GTG CAG ATC CGC AGA CGA AAG GCA TCC GGA GAC CCA TAC TGG GCG PtpTnt300

YSKHNGPENWHKDFPIANGD CAaa26 YSGAYGPEHWVTSSVSCGGS Ptpraal06 TAT TCT GGT GCC TAT GGT CCT GAG CAC TGG GTC ACA TCT AGT GTC AGC TGC GGA GGC AGT Ptpmt360 < A_2 l i 2

RQSPVDIDTATAHHDPALQP CAaa46 HQSPIDILDHHARVGDEYQE Ptpraal26 CAT CAG TCT CCT ATA GAC ATT TTA GAC CAC CAT GCT CGC GTT GGT GAT GAA TAC CAG GAG Ptprnt420 B-1 > < D-2 LLI -SYDKAASKS--IVMN1 CAaa63 LQLYGFDNESSNKTWMKNTG Ptpraal46 CTT CAA CTT TAT GGG TTT GAC AAC GAG TCC TCT AAC AAA ACC TGG ATG AAA AAT ACA GGG Ptprnt480

HSFNVEFD DSQDN YVLKGGPLSD CAaa86 KTVAILLKDDYFVSGAGLPG PtpTaal66 AAA ACA GTT GCC ATC CTG CTG AAA GAC GAT TAT2 TTT A 3 GTG AGT GGT GCC GGA CTG CCG GGC Ptprnt540 SYRLIQFHFHWG-SSDGQGS CAaalOS RFKAEKVEFHWGHSNGSAGS Ptpraal86 AGA TTC AAA GCT GAG AAG GTG GAG TTT CAC TGG GGG CAC AGC AAT GGC TCC GCT GGC TCA PtprntoOO < B_2

EHTVNKKKYAAELHLVHWN- CAaal25 EHSVNGRRFPVEMQIFFYNP Ptpraa206 GAG CAT AGT GTC AAT GGC CGG AGG TTT CCT GTG GAG ATG CAG ATT TTC TTT TAC AAC CCA PtpTnt660 314 G-l >

TKYGDFGKAVQQPDGLAVLG CAaal45 DDFDSFQTAISENRIIGAMA Ptpraa226 GAC GAC TTT GAC AGC TTT CAA ACG GCA ATT TCT GAG AAC AGA ATA ATT GGA GCT ATG GCC PtpTnt720 4 A5 YFLKIGP-ASQGLQKVLEAL CAaal64 IFFQVSPRDNSALDPIIHGL PtpTaa246 ATA TTT TTC CAA GTC AGT CCG AGG GAC AAT TCT GCA CTG GAT CCT ATT ATC CAC GGG CTG Ptprnt780 < G-2-(R-4) C-l >

HS IKTKGKRAAFANFDPCSL CAaal84 KGVVHHEKETFLDPFILRDL Ptpraa266 AAG GGC GTC GTA CAT CAC GAG AAG GAG ACT TTC CTG GAT CCT TTC ATT CTC CGA GAC CTC Ptprnt840 5 A 6

LPGNLD-YWTYPGSLTTPPL CAaa203 LPASLGSYYRYTGSLTTPPC Ptpraa286 CTG CCC GCA TCC CTG GGG AGT TAT TAC AGG TAC ACA GGC TCC TTG ACT ACG CCA CCC TGT Ptprnt900

LECVTWIVLRESITVSSEQM CAaa223 SEIVEWIVARRPVPISYHQV Ptpraa306 AGT GAA ATA GTG GAG TGG ATT GTC TTC CGG AGG CCC GTT CCC ATC TCC TAT CAC CAG GTA PtpTnt960 < C-2 6 i 7

Fig. 1. Partial sequence of the Ptprg CA domain aligned with mouse carbonic anhydrase II sequence. The nucleotide (nil sequence of the murine Plprg CA-like domain and some upstream sequence is shown. The deduced amino acid (aa) sequence in standard one-letter code is above the nucleotide sequence. The sequence of mouse carbonic anhydrase II ( 12) is aligned on the top. The approximate positions of the putative introns in the Ptprg sequence are shown by arrowheads, based on the structure of the CA II gene. The corresponding exon numbers of the mouse CA II gene are also indicated on either side of each arrowhead. The positions of oligonucleotides for generating probes or for reverse transcription are indicated with dashed lines and directional arrows.

1499

ods. Probes were labeled by random priming with [12P]dCTP (New England A Nuclear) and hybridized to membranes in 0.75 M NaCl-50% formamide at 42Â°Covernight. Final washes of membranes were in 0.1 x standard saline- Ptprg, Tpi-rs9 D D Â«DB citrate and 0.1% sodium dodecyl sulfate at 65Â°Cfor 30 min. RNA Extraction, Reverse Transcription, and RT-PCR Reaction. el-ID Plan n â€¢¿anÂ« mRNA was isolated from IT22 and el-ID cells after treatment with 4 M Psp-2 guanidinium isothiocyanate, followed by phenol-chloroform extraction and â€¢¿a â€¢¿nan isopropyl alcohol precipitation. Reverse transcription was performed in 20 ul final volume of 50 niM Tris-HCl (pH 8.3), 60 ITIMKCI, 1 HIMMgCl2, IO ITIM 33 37 O 14 5 dithiothreitol, I HIMdeoxynucleotide triphosphates, 0.5 ng oligodeoxythymidy- B late, murine leukemia virus-RT (200 units; BRL), RNasin (40 units; Promega), and 2 ug RNA at 37Â°Cfor 30 min. The RT-PCR reaction was carried out with primers R-l and R-2 under conditions as described above for PCR probe 3p24 generation. The products were run in 1.5% agarose gels, blotted to membranes, 3pl42 Tpi-rs9. Odc-rs9 n Â¿Â¡OJiOtf3-rs6.Tai and hybridized with appropriate probes. Murine Backcross Analysis. The interspecific mouse backcross used for DI4MU2 chromosome mapping was described previously (14). The pKWml.SB probe used to map the Plprg locus detected a 4.2-kilobase Hindlll fragment in 11.3 AEJ/Gn and a 4.0-kilobase Wwi/III fragment in Mus spretus DNA. The PCR 3pl43 Cchlla2 oligomers and simple sequence-length polymorphism detecting the Plini locus 10- D14MU3 I0q21-q24 -- were as described (15). The probes and restriction fragment length polymor Sftp-1 phisms detecting the Psp-2 and Tpi-rs9 loci were as described (16, 17). Psp-2 - - lOqll Bmp2b-l .Kbp-3 Results and Discussion DHMitS 4qll. 14qll Np-1. Np-2, Tcrd A probe representing the 5' â€”¿740nucleotidesof the human PTPRG Psp-2,D14Mit37.Rib-Â¡ I4qll Tcra gene was used to screen for murine cDNA and genomic Ptprg clones. . 14qll-ql3 Ctla-l.Mvhc 20- A 1.5-kilobase genomic fragment was used in linkage analysis of interspecific backcross mice to determine the chromosomal location of the murine gene. The results of the interspecific backcross analysis show that the Ptprg locus maps to the proximal portion of mouse Fig. 2. Summary of the results of the interspecific backcross. (A) Haplolype analysis chromosome 14 (Fig. 2). The localization oÃPtprg to mouse chromo of 80 NÂ»progeny from the interspecific backcross. Left, loci mapped in the backcross. some 14 is consistent with its position as determined by mapping of Each column represents the chromosome identified in the Ni offspring that was inherited from the AEJ/Gn X M. xpretus P\ parent. â€¢¿.AEJ/GnalÃele;D, M. xpretux alÃele.The the recombinant inbred strains (6). Specifically, Ptprg maps just prox number of Ni progeny carrying each type of chromosome is listed below the squares. (B) imal to Plan; Rarb has been shown to map proximal to Plan as well Genetic linkage map showing the chromosomal location of the Ptprg locus in the mouse. The chromosome on the left shows the loci typed in the interspecific backcross, with (18), suggesting close linkage between Ptprg and Rarb. The human distances between loci given in cM. The chromosome on the right shows a partial version homologues of Rarb and Ptprg reside on human chromosome 3p24 of the GBASE linkage map of mouse chromosome 14 with the distance from the cen and 3pl4.2, respectively. However, several human genes lie between tromere given in cM (13). The dashed line between the chromosomes indicates the reference locus used to align the maps. Loci mapped in human are underlined; gene RARB and PTPRG on human chromosome 3p, such as ACYÃŒ,MYL3, locations on human chromosomes are shown between the chromosomes. and GNAI2B; their mouse homologues (Acy-1, Myl3, and Gnai2b) are located on mouse chromosome 9 (19). In addition, CCHLÃ•A2maps to human chromosome 3pl4.3 and mouse chromosome 14 (Fig. 2). RT-PCR products. The sequences of all primers and their positions in the Ptprg Although Rarb, Ptprg, and Cchlla2 map to both mouse chromosome sequence were: 14 and human chromosome 3, current data indicate that they most likely represent three distinct homology segments, rather than defin A-l(D-l)A-2B-lB-2C-lC-2D-2G-lG-2'5'-ACCATAGGCACCAGAATA-3'5'-TGCTGTGCTTCATTATGT-3 ing a conserved linkage group. Directly determining the genetic dis '5'-ACCTTCTCAGCTTTGAATCT-3'5'-AGTCATCAGTCTCCTATAGA-3 tances between Rarb, Ptprg, and CcMla2 in the mouse would shed light on the evolutionary relationship of these regions on mouse chro '5'-GGG ACA ATTCTGCACTGG AT-3 mosome 14 and human chromosome 3. '5'-TATTCATCACCAACGCGAGC-3'5'-CTGCTTACCTGGTG ATAGGT-3 During analysis of the murine Ptprg locus in murine cell lines, we '5'-TTTCTTTTAC AACCC AG ACG-3 noted possible murine restriction fragment length polymorphisms. (R-4)R-lR-2R-31733183585607439654136457622018662315'5'-ATCC AGTGC AG AATTGTCCC-3 Thus, DNA from various cell lines, derived from different mouse '5'-TAATAACTCCCCAGGGATGC-3'5'-GCAGGAGAGCAGACAGGACA-3''-GGCATTG ACTG AAGGCTATG-3 strains, were more systematically investigated with control tissue DNAs from the respective parental mouse strains, using individual regions of the murine Ptprg cDNA. Results indicated that the mouse strains tested could be divided into two groups, based upon insertion/ PCR reactions were carried out in 50 ul final volume with 50 ng of cDNA deletion type polymorphic loci occurring in a number of introns of the template, 0.5 UMprimers, 10 ITIMTris-HCl (pH 8.3), 50 rriM KC1, O.I mg/ml Ptprg locus; BALB/c, AKR/J, C57BL/6, and Swiss were in one group gelatin, 15 ITIMMgCI2, 200 UMdeoxynucleotide triphosphates, and 2.5 units and DBA/2J, C3H/HeJ, 129/J, and BXH-2 were in the other group Taq polymerase (8MB). The amplifications were performed in a Perkin-Elmer Cetus thermal cycler for 30 cycles of 94Â°Cfor 1 min (for denaturation), 55Â°C (data not shown). When various L-cell derived clones such as A9, for I min (for annealing), and extending at 72Â°Cfor 1 min. The PCR products el-ID, LMTK-, and L929 were compared to C3H tissue DNA using were visualized Â¡nlowethidium bromide-stained low melting agarose gels. The human or mouse Ptprg cDNA probes, it was clear that a portion of the gene was entirely missing in all the L-cell lines examined (not shown). bands of amplified DNA were excised from gels for labeling. Genomic DNA Preparation and Southern Blot Hybridization. Cellular In order to determine the position and extent of the homozygous DNAs were isolated and Southern blots were prepared by conventional meth deletion, we positioned oligonucleotide primers along the length of 1500

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1993 American Association for Cancer Research. HOMOZYCOUS DELETION IN CA-LIKE DOMAIN OF the 5' end of the Ptprg cDNA, as shown in Fig. 1, for preparation of 6); filters were rehybridized to other murine probes to show equiva region-specific probes by PCR amplification, using the entire cDNA lent murine DNA in all lanes (not shown). Next, it was observed that as template. The probes generated using various primer pairs are probe C covering exons CA5 and CA6 was intact in el-ID DNA (see sketched in Fig. 3/4, which shows the relationship between the probes Fig. 3ÃŸ,Panel C, in which el-ID lanes are identical to Mel lanes). and exons of the Ptprg CA domain. The respective probes were Thus from Fig. 3B, Panels A, B, and C, using probes A, B, and C, we hybridized to filters carrying restriction enzyme-cleaved DNAs of the know that exon CAI and upstream exons are intact in L-cells, exons el-ID and Mel cell lines as shown in Fig. 3ÃŸ.Fig.3B, Panel A, shows CA2 and CA3 are at least partially missing (the portion covered by the DNAs. Mel and el-ID, in alternating lanes cut with BamtÃ¬Ã¬(Lanes probe B), and exons CA5 and CA6 are intact in L-cells. Similar results I and 2), EcoRl (Lanes 3 and 4), and Hind\\\ (Lanes 5 and 6), were obtained using DNA from A9 and L929 DNAs. To locate the 5' hybridized to probe A, which covers two exons. None of the three end of the deletion, probe D was hybridized to DNA from the two cell enzymes cut within either of the exons or the intervening intron; lines (see Fig. 3B, Panel D); probe D overlaps exon CAI and its therefore only one fragment is detected for each of the enzymes. The upstream exon (like probe A), but sees in addition exon CA2 (see Fig. Mel and cl-1D patterns are the same, showing that the region detected 3/4). Probe D (compare Panel D to Panel A) detects two bands in Mel by probe A is intact in L-cells. In Fig. 3B, Panel B, a similar filter was cells but only 1 in el-ID, showing that the 5' end of the deletion is hybridized to probe B, illustrating that this region of the Ptprg gene is within the first CA intron because none of the exons have recognition entirely missing in el-ID DNA (see Fig. 3B, Panel B, Lanes 2, 4, and sites for the enzymes used. To find the 3' end of the deletion, probe

CA I CA 2 CA 3 CA A CA 5 CA 6

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A B C D G Fig. 3. Analysis of the homozygous deletion within the Ptprg gene in the cl-ID cell line. (A) The putative exons of the CA-like domain of mouse Ptprg (CA I-CA 6) are shown by boxes, based on the mouse CA II gene structure; thin lines indicate introns but are not to scale. Probes A. B. C. D, G, and R are shown below the exons, indicating the regions of Ptprg sequence covered. Probe A covers exon CA 1 plus some of the upstream exon. Probe B includes a portion of CA2 and CA3. Probe C covers most of CA5 and the full CA6. Probe D overlaps probe A and carries the 5' end of CA2. Probe G covers most of CA4 and a portion of 5' CA5. Probe R covers CA 1 through CA6. Probes were generated from cDNA and contain no intron sequence, (ÃŸIHybridizations illustrating the extent of deletion using probes A. B, C, D. and G. respectively, are shown in the five blots. The 5 probes cover different regions of the Ptprg CA-like domain as described in A. Each pair of lanes contained restriction enzyme-digested DNAs from Mel and cl-ID cell lines. Lane I, ÃŸÂ«mHI-Mel;Lane2, BamHI-cl-lD; Lane j, EcoRI-Mel; Lane 4. Â£roRI-cl-lD; Lane 5. //im/III-Mel: Lane 6, Hind\ll-c\-\D. Probe A detected the same bands with each enzyme in Mel and cl-ID (Panel A ). Probe C also detected the same fragments in both cell lines (Panel C). In Panel B, each cl-1D lane shows that probe B, covering exons 2 and 3. is deleted. Probe D, crossing exons 1 and 2. detected the presence of exon CAI and absence of exon CA2 in cl-ID (Panel D). Probe G. spanning exons CA4 and CA5, detected presence of exon 5 and absence of exon CA4 (Panel C). Position of DNA size markers (kilobase pairs) are indicated on the right of Panel G. bp. base pairs. 1501

G was hybridized as shown in Fig. 3fi, Panel G; this panel shows that more than 200 kilobase pairs. In nearly 50 years of tissue culture, exon CA4 is missing in L-cells. The 3' end of the deletion is, there this deletion has remained the same in all L-cell lines, an unlikely fore, in intron 4 of the CA domain, leaving the 5' end and regulatory occurrence if the gene did not serve some function. We also specu regions for this gene intact in L-cells. late that this deletion could be involved in the tumorigenicity of the To determine if the partially deleted gene was expressed at the L-cell clones and could have arisen due to methylcholanthrene treat mRNA level and might, therefore, encode a functionally abnormal ment during establishment of the original L-cell strain. Interestingly, protein, RT-PCR was performed using mRNA from L-cells. IT22 Pathak et ai (20) have reported that structural abnormalities in mu cells, and Mel cells. RT-PCR products were size fractionated on rine chromosome 14, leading to deletion near the Plan locus, are as agarose gels, blotted, and hybridized to probe R or B (see Fig. 3A and sociated with high metastatic potential of K-1735 melanoma cells. It Fig. 4A for position of probes). Results demonstrate (see Fig. 4ÃŸ)that will be important to determine if the Ptprg locus could be the target a novel transcript is present in L-cell mRNA which is 400 bases of the deletion in this tumor model. shorter than the IT22 product (Fig. 4B, Lanes 1 and 2); the Mel Acknowledgments product and the product from control cDNA appeared identical to the IT22 product (not shown). The el-ID RT-PCR product is not detected We thank K. K. Nelson and J. J. Moskow for technical assistance. by probe B (Fig. 4B, Lane 3), which detects exons CA2 and CA3 in the IT22 RT-PCR product. These results confirm that the L-cell Ptprg References gene is expressed and could, therefore, code for an aberrant protein !. Alexander. D. R. The role of phosphutases in signal transduction. New Biologist. 2: product. Since the CA domain of the extracellular portion of this 1049-1062. 1990. 2. Saito, H., and Streuli, M. Molecular characterization of protein tyrosine phosphatases. receptor tyrosine phosphatase gene is very likely involved in ligand Cell Growth & Differ.. 2: 59-65. 1991. binding (6) and the L-cell deletion removes amino acids 12-145, 3. Hunter. T. Protein tyrosine phosphatases: the other side of the coin. Cell. 5K: 1013- including the putative active site of this CA domain (6), the L-cell 1016. 1989. 4. Kaplan. R.. Morse, B.. Huebner. K.. Croce. C.. Howk. R.. Ravera. M., Ricca, G., Jaye, Ptprg gene product could not transmit signals normally. M., and Schlessinger. J. Cloning of three human tyrosine phosphatases reveals a It is interesting that the deletion end points are within introns; in multigene family of receptor-linked protein [yrosine phosphatases in brain. Proc. Nati. fact, the actual DNA break points must be very far away from the Acad. Sci. USA. 87: 7(XX)-7(X)4. 1990. 5. LaForgia. S.. Morse. B.. Levy. J.. Bamea, G.. Cannizzaro. L. A.. Li. F.. Nowell. P. C. exons because we have never seen a DNA rearrangement using Boghosian-Sell. L., Click. J.. Weston. A.. Harris. C. C.. Drabkin. H.. Patterson, D., cDNA-derived probes and a large battery of standard restriction en Schlessinger. J.. Croce, C. M., and Huebner. K. Receptor protein-tyrosine phosphatase 7 is a candidate tumor suppressor gene in human chromosome region 3p2l. Proc. zymes; we are currently using infrequently cutting enzymes and Nati. Acad. Sci. USA. 81: 5036-5040. 1991. pulsed field gel electrophoresis to detect the rearrangement and plan 6. Bamea. G., Silvennoinen. O.. Shaanan, B., Honegger, A. M., D'Eustachio. P.. Morse. its cloning. Preliminary results indicate that the deletion involves B.. Levy. J. B., LaForgia, S.. Huebner. K.. Sap, J.. and Schlessinger, J. Localization of a unique carbonic-anhydrase-like domain in the extracellular region of receptor tyrosine phosphatase y. Mol. Cell. BioL /.): 1497-1506. 1993. 7. Krueger. N., and Saito. H. A human transmembrane protein-tyrosine phosphatase. PTPÂ£,is expressed in brain and has an N-terminal receptor domain homologous to carbonic anhydrase. Proc. Nati. Acad. Sci. USA, 89: 7417-7421, 1992. 8. Tashian, R. E. The carbonic anhydrases: widening perspectives on their evolution, I Ptp Y CA domain expression and function. Bioassays. 10: 186-192. 1989. 9. Marshall. C. J. Tumor suppressor genes. Cell. 64: 313-326, 1991. 10. Sanford. K. K.. Earle. W. R.. and Likely. G D. The growth in vitrn of single isolated tissue culture cells. J. Nati. Cancer Insi.. 9: 229-246. 1948. 11. Friend. C.. Patuleja. M. C., and dcHarven. E. Erythrocytic maturation in vitro of murine (Friend) virus-induced leukemic cells. Nati. Cancer Inst. Monogr., 22: 505- 522. 1966. 12. Patrick, J. V., Jeffry, C. M.. David. H. E., Karin, W., and Richard. E. T. Structure and B 1234 exon to protein domain relationships of the mouse carbonic anhydrase II gene. J. Biol. Chem.. 260: 12130-12135. 1985. 13. GBASE. The on line genomic database of the mouse maintained at The Jackson Laboratory (Bar Harbor. Maine) by M. T. Davisson. T. H. Roderick, D. P. Doolittle. A. L. Hillyard, L. U. Maltais, and J. N. Guidi, 1993. - 600 bp 14. Marini. J. C., Nelson, K. K., Battey. J.. and Siracusa. L. D. The pituitary hormones, arginine vasopressin-neurophysin II and oxytocin-neurophysin I. show close linkage with interleukin-1 on mouse chromosome 2. Genomics, in press, 1993. - 200 bp 15. Dietrich, W.. Katz. H., Lincoln. S. E.. Shin. H., Friedman, J.. Dracopoli, N. C., and â€¢¿ Lander. E. S. A genetic map of the mouse suitable for typing intraspecific crosses. Genetics, 131: 423^147, 1992. 16. Siracusa, L. D.. Buchberg, A. M., Copeland. N. G.. and Jenkins, N. A. Recomhinant Fig. 4. Transcription of Ptprg mRNA in cl-ID. (A) RT-PCR scheme showing the inbred strain and interspecific backcross analysis of molecular markers flanking the relationship of the primers used in amplification of the RT product and the probes used in murine agouti coat color locus. Genetics, 722: 669-679, 1989. detection of the product, (fi) Total RNAs from cl-ID and IT22 cells were reverse- 17. Siracusa, L. D., Jenkins, N. A., and Copeland, N. G. Identification and applications of transcribed with oligothymidylate primers. The region of cDNA covering CAI through repetitive probes for gene mapping in the mouse. Genetics, 127: 169-179. 1991. CA6 was amplified with primers R-l and R-2 using the reverse transcribed product as 18. Ceci. J. D., Kingsley, D. M.. Silan. C. M., Copeland. N. G.. and Jenkins, N. A. An template. Amplified products were electrophoresed and transferred to nylon filters. The interspecific backcross linkage map of mouse chromosome 14. Genomics. 6: 673- amplified DNAs were detected by probe R (made from primers R3 and R4. illustrated in 678. 1990. Fig. 3/\ ). The expected 6K)-base pair (bp) product was detected in IT22 (Lane 2); cl-ID 19. Nadeau, J. H., Davisson, M. T.. Doolittle, D. P., Grant, P.. Hillyard, A. L.. Kosowsky, (Ijme I ) mRNA produced a 200-base pair product. 4(X) base pairs smaller than the IT22 M. R., and Roderick, T. H. Comparative map for mice and humans. Mammalian transcript, indicating that the deleted Ptprg gene in cl-ID was transcribed. The blot was Genome,.?: 480-536, 1992. also hybridized with probe B. which is within the deleted region in the cl-ID locus; the 20. Pathak, S.. Staroselsky, A. N., and Fidler, I. J. Specific chromosomal defects associ IT22 transcription product was delected (Urne 4). but the cl-ID product was completely ated with metastatic potential in K-1735 melanoma clones: involvement of chromo negative (Ltine .f ). confirming transcription of the deleted cl-ID Pipr% gene. somes 4 and 14. Cancer Genet. Cytogenet., 56: 209-221. 1991.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1993 American Association for Cancer Research. A Homozygous Deletion within the Carbonic Anhydrase-like Domain of the Ptprg Gene in Murine L-Cells

Kishore K. Wary, Zhuangwei Lou, Arthur M. Buchberg, et al.

Cancer Res 1993;53:1498-1502.

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