SHORT COMMUNICATION Chromosomal Localization of an SHZ-Containing Tyrosine Phosphatase (PTPNG) 1. PLuTzKY,*-t B. G. NEEL,+’ R. D. RoSEbmERG,~~t R. L. EDDY,~ M. G. BYERS,~ S. JANI-SAIT,~ AND T. B. SHOW@ SMolecular Medicine Unit, Beth Israel Hospital, Boston, Massachusetts 022 15; *Division of Cardiology, Department of Medicine, The Brigham and Women’s Hospital, Boston, Massachusetts; tDepartment of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; and §Department of Human Genetics, Roswell Park Memorial Institute, Buffalo, New York 14263 Received
December
26, 1991;
revised
March
4, 1992
for the function and control of a specific PTP as well as for signal transduction in general. Interestingly, PTPNG is expressed at high levels predominantly in hematopoietic cells (12). Disregulated activity of PTKs in hematopoietic cells, due to chromosomal abnormalities [e.g., the t(9;22) translocation in chronic myelogenous leukemia resulting in the bcr-abl gene fusion] or retroviral gene transduction (e.g., the v-erbB oncogene), can contribute to leukemogenesis. Similarly, it is conceivable that deranged control of a PTP with a key role in signal transduction could have similarly deleterious effects. Therefore, we determined the localization of PTPNG on human chromosomes using somatic cell hybrid panels and fluorescent in situ hybridization (FISH). DNA was prepared from 32 somatic cell hybrids inPhosphorylation of proteins on tyrosyl residues is a volving 15 unrelated human and 4 mouse cell lines (16key mechanism by which important cellular processes 18). The hybrids were characterized by karyotyping and such as growth, differentiation, and the cell cycle are with mapped enzyme markers (l&16,18); their propercontrolled (2, 7). The steady-state level of phosphorylation of any given tyrosyl residue is set by the opposing ties are summarized in Table 1. A “t” in the table indicates that only the translocation (indicated in the colactions of protein tyrosine kinases (PTKs) and protein umn labeled “Translocations”) but no intact chromotyrosine phosphatases (PTPs). PTKs catalyze the phossome is present. Bands corresponding to the human phorylation of themselves and/or other cellular pro(6.7/6.4/2.8/1.2 kb) and mouse (5.1/4.1 kb) PTPNG teins. Many of these tyrosyl-phosphorylated proteins genes can easily be distinguished on Southern blots of interact with secondary signaling molecules that contain EcoRI-digested DNA (data not shown). Accordingly, SH2 (src-homology-2) domains, which are approxihybrid cell DNA was digested with EcoRI, electrophomately lOO-amino-acid stretches of sequence similarity resed on agarose gels, and transferred to nylon memfound in such disparate molecules as GTPase-activating branes for Southern blotting as described elsewhere protein, phospholipase-Cy, the regulatory subunit of (10). The 2.2-kb full-length PTPNG cDNA was labeled phosphatidylinositol-3 kinase, the src-family of nonwith [a-32P]dCTP using the random primers method (6) transmembrane tyrosine kinases, and the cytoskeletal and hybridized to the blot. Each hybrid DNA was scored protein tensin [reviewed in (9)]. for the presence (+) or absence (-) of human bands. The Although much progress has been made in determinresults of this analysis were compared to the known presing the structure of PTPs, less is known about their role in signal transduction. Recently, we (12) and others (14) ence or absence of specific human chromosomes in each hybrid. As shown in Table 1, there was 0% discordancy identified a novel member of the PTP gene family (PTPNG) that contains two SH2 domains. Since SH2 between the presence of human PTPNG bands by domains function to direct signaling molecules to spe- Southern analysis and the presence of human chromosome 12 in a given hybrid. Thus, PTPNG maps unambigcific tyrosyl phosphoproteins, the discovery of an SH2containing PTP has obvious potential implications both uously to chromosome 12 by somatic cell hybrid analysis. To determine regional localization on chromosome 12, ’ To whomcorrespondence shouldbe addressed. we performed FISH on metaphase chromosomes. ChroWe have used panels of somatic cell hybrids and tluorescent in situ hybridization to determine the chromosomal localization of the novel nontransmembrane tyrosine phosphatase PTPNG (protein tyrosine phosphatase, nonreceptor type 6), which contains two SH2 domains. PTPNG maps to 12~13, a region commonly involved in leukemia-associated chromosomal abnormalities. Since PTPNG is expressed at high levels in hematopoietic cells of all lineages and its expression is induced early in hematopoietic differentiation, altered expression and/or structure of PTPNG may play a role in leukemogenesis. 0 1992 Academic Press, Inc.
869
GENOMICS Copyright 0 1992 All rights of reproduction
13,869-872
(19%)
0888.7543/92 $5.00 by Academic Press, Inc. in any form reserved.
870
SHORT
COMMUNICATION
TABLE Segregation
of PTPNG
with
Human
Chromosomes
1
in EcoRI-Digested Human
DNA No. 660 233 197 859 186 1185 389 402 187 830 1146 192 42 184 254 394 1162 390 643 644 395 407 212 425 424 347 25 534 555 1107 332 57
Hybrid ATR- 13 DUA-SBSAGA DUA-5BSAGA DUA-6 DUM-13 GAR-1 JSR-2 JSR-14 JWR-26C KER-3 NSL-9 NSL-16 REW-11 REX-11BSAgB REX-11BSHF REX-26 RSR-3 SIR-11 TSL-1 TSL-2 VTL-6 VTL-17 WIL-2 WIL-6 WIL-8X WIL-14 WIL-15 XOL-6 XOL-13 XOL-21 XTR-2 XTR-3BSAgB
Concordant No. of hybrids (+/+) t-/-j Discordant No. of hybrids (+/-) (-/+) % discordancy:
PTPNG
+ + + + + + + + + + + + + + + + + + +
1
2
3
4
5
6
7
8
9
10
Human-Mouse
Cell Hybrid
chromosome
11
12
13
14
+++++++t++-+t++++++--+t -+----++----++~-f--~---+-+-----+---+--++--+ -+-++---------+--++---+ + + + -+t+--+++-+ --f-+--t-+-++-++---+--+ ++-~+-----++------~-+ -+++++-----++-p-+-p++-+ t++++++-++++-+++++-++-+
15
16
17
18
19
20
21
22
X
tt++++++t
x/15,15x
w 1719 17/9
~ + + + + + - --22/x 22/x
17/3
l/X X/l
t---+++--+++-t--+-++-+t t--++-+--+++-A--+p-+--+ +
-
-
-
ISO7p 3/X 3/x, lOq-
t+++++-+--++-+--+
t-+--+-+-+++---+-++-t -t----~+t-+-~---~-++-t
4 13
7 11 8 8
9 8
13 9
7 11 11 6
12 12 5 10 6 12 05554273077069337534726 41 53 34 47 31 44
Translocations 5/x
+-++-+-------+ -+--+t+ -+++++++-+++~ + + + - ++t+-+-+++++-++-+ -~ +---+ + + - - f--+-----+ - --+ + - +------+---+---+---tt ++++-++++++-++++++-+tt +--+p-++--+-++---+p+ +--~---+-----~---+++ -+++-----++-+--+++-++-- + t -++-+-+-+-++-t+-++-+ -+---+++-++---t-+-+++++-+---+t-++--+--++-+--f-+---t-+---+-+ - + - +++++-++--+--+-+++-+ -+++-++-+++-+--+++++-+ +-+-+-++-+-f-++-+-----+ -+++-++--++++++-++-++-+ ~
DNA
11 10
5 13
7
8
12
45
34
40
16 6
5 15 10 9
9 6
19 13
7 7
13 4
10 10
8 10
14 6
11 8
2 10
0
12
6
8
11
4
8
14
4
0 56
47
35
44
35
41
53
25
29
53
13 6
5 10
11 6
6 13
2
41
50
32
Note. DNA from 32 somatic cell hybrids as described in the text was subjected to Southern blot analysis with PTPNG cDNA probe. The chromosome content of each hybrid is indicated. A “t” in the table indicates that the translocation indicated in the “Translocation” column is present with no intact chromosome present. Each hybrid was scored for the presence (+) or absence (-) of human PTPNG bands. A 0% discordancy indicates matched segregation of the probe with a particular chromosome. As indicated, PTPNG maps to chromosome 12.
mosome spreads were prepared from &bromodeoxyuridine-synchronized lymphocyte cultures. A full-length PTPNG cDNA insert was biotinylated and hybridized to the chromosome spreads. Following appropriate washing, hybridization was detected by reaction with fluorescein-conjugated avidin (Vector Labs). Chromosome identification was achieved by means of Q banding (DAPI counterstaining) and R banding (propidium iodide counterstaining) (4,5). Slides were evaluated using a Nikon fluorescence microscope. Thirty metaphases were examined. A fluorescent sig-
nal was detected at 12~13 on both chromatids of at least a single chromosome 12 in 26 of the 30 metaphase spreads examined (86%; Fig. 1). In 2 of the 26 spreads, signals were detected at band pl3 on both chromatids of both chromosomes 12, whereas 6 of the 26 spreads had an additional single signal on one chromatid of the other chromosome 12. No other chromosome displayed significant hybridization. These data conclusively localize PTPNG to 12~13. The localization of PTPNG at 12~13 is potentially of great interest, since the distal short arm of chromosome
SHORT COMMUNICATION 13.3
IJ.,
13.2 A.9 a i-l
l oeoooooooeoo l oooooeooo@oo
12.3
cell differentiation
(J.P., B.G.N.,
R.D.R.,
Bing Lim, and
Chaker Adra; unpublished data). It is easy to imagine how mutations in an SHB-containing PTP might perturb normal hematopoietic growth or differentiation. Thus, PTPNG is a good potential candidate for the gene targeted by leukemia-associated I2p chromosome abnormalities. ACKNOWLEDGMENTS The DNA and protein sequence of PTPNG have been submitted previously to GenBank (M77273) and the chromosomal localization has been reported to the Genome DataBase. We thank Pamela Beahm for technical assistance. This work was supported by NIH Grant R-Ol-CA 49152 to B.G.N. and NIH Grants HG00333, HD05196, and ACS CD62 to T.B.S.
15 9
21.1 21.2 21.3
#:# H
24.1 24.2
Note added in proof. After this paper was accepted, Yi et al. (19a) independently reported the molecular cloning of PTPNG (which they denote “HC-PTP”) and its localization by FISH to 12p12-P13, in agreement with the somatic cell hybrid and FISH results reported here.
REFERENCES
12
FIG. bution 86% of matids
1. Localization of PTPNG by FISH. Idiogram of the distriof fluorescence signals on both chromatids of chromosome 12. the metaphases examined had signals at 12~13 on both chroof at least a single chromosome 12 (see text).
12 is involved in chromosomal abnormalities in several forms of leukemia. Approximately 10% of pediatric acute lymphoblastic leukemia (ALL) cases display abnormalities in the 12p12-p13 region; these include interstitial and terminal deletions and translocations (3, 13). Several different chromosomes participate with 12~ in these translocations, suggesting that a 12p gene(s) is the target for these rearrangements. An intriguing exception to this theme is the dicentric translocation [tdic(9;12)(pll;pl2)] found in about 1% of pediatric ALLs (3). Deletions of the 9pll region are found independently in ALL (ll), raising the tantalizing possibility that the dicentric translocation simultaneously targets 9p and 12p genes. Adult leukemias, of both lymphoid and myeloid origin, also display 12p abnormalities, although at much lower frequency (1,8,19,20). Such abnormalities may be particularly common in leukemias associated with eosinophilia (8). Since involvement of 12~12 has been implicated in some studies and 12~13 in others, it is not yet clear whether all of these leukemiaassociated 12p abnormalities are targeting the same gene. However, given the difficulty of precisely specifying chromosomal breakpoints in clinical specimens, a single 12p target cannot be excluded. Its ubiquitous expression in hematopoietic cells of all lineages and differentiation stages suggests that PTPNG plays an important role in hematopoietic cell signal transduction. This notion is supported by our recent finding that PTPNG expression is induced at an early stage of hematopoietic
1.
Berger, R., Flandrin. G., Bernheim, A., Le Coniat, M., Vecchione, D., Pacot, A., Derre, J., Daniel, M.-T., Valensi, F., Sigaux, F., and Ochoa-Noguera, M. (1987). Cytogenetic studies on 519 consecutive de novo acute nonlymphocytic leukemias. Cancer Genet. Cytogenet. 29: 9-21.
2.
Cantley, L., Auger, K., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. (1991). Oncogenes and signal transduction. Cell 64: 281-302. Carroll, A., Raimondi, S., Williams, D., Behm, F., Borowitz, M., Castleberry, R., Harris, M., Patterson, R., Pullen, D., and Crist, W. (1987). tdic (9;12): A nonrandom chromosome abnormality in childhood B-cell precursor acute lymphoblastic leukemia: A pediatric oncology group study. Blood 70: 196221965.
3.
4.
5.
Cherif, D., Bernard, O., and Berger, R. (1989). Detection of single-copy genes by nonisotopic in situ hybridization on human chromosomes. Hum. Genet. 81: 358-362. Fan, Y.-S., Davis, L., and Shows, T. (1990). Mapping small DNA sequences by fluorescence in situ hybridization directly on banded metaphase chromosomes. Proc. Natl. Acad. Sci. USA 87: 6223-6227.
6.
7.
Feinberg, A., and Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. Hunter, T., and Cooper, J. (1985). Protein tyrosine kinases. Annu. Rev. Biochem. 54: 897-930.
8.
Keene, P., Mendelow, B., Pinto, M., Bezwoda, W., MacDougall, L., Falkson, G., Ruff, P., and Bernstein, R. (1987). Abnormalities of chromosome 12~13 and malignant proliferation of eosinophils: A nonrandom association. Br. J. Haematol. 67: 25-31.
9.
Koch, C., Anderson, D., Moran, M., Ellis, C., and Pawson, T. (1991). SH2 and SH3 domains: Elements that control interactions of cytoplasmic signaling proteins. Science 252: 668-672.
10.
Naylor, S., Sakaguchi, A., Shows, T., Law, M., Goeddel, D., and Gray, P. (1983). Human immune interferon gene is located on chromosome 12. J. Exp. Med. 57: 1020-1027. Pellet, P., Hillion, J., Carroll, A., Crist, W., Brouet, J., and Tsapis, A. (1991). Heterogeneity of the breakpoint localization in malignant cells with a 9pll chromosomal abnormality. Leukemia 5:468-472.
11.
SHORT COMMUNICATION
872 12
Plutzky, J., Neel, B., and Rosenberg, R. (1992). novel SRC homology 2 (SHZ) containing tyrosine Proc. Natl. Acad. Sci. USA 89: 1123-1127.
13.
Raimondi, S., Williams, D., Callihan, T., Peiper, S., Rivera, G., and Murphy, S. (1986). Nonrandom involvement of the 12~12 breakpoint in chromosome abnormalities of childhood acute lymphoblastoma leukemia. Blood 68: 69-75.
Isolation of a phosphatase.
14.
Shen, S.-H., protein-tyrosine SH2 domain 739.
Bastien, L., Posner, B., and Chretien, P. (1991). A phosphatase with sequence similarity to the of the protein-tyrosine kinases. Nature 352: 736-
15.
Shows, T. (1983). In “Isozymes: Current Topics in Biological and Medical Research” (M. C. Rattazzi, J. G. Scandalios, and G. S. Whitt), Vol. 10, pp. 323-339, A. R. Liss, New York.
16.
Shows, T., Brown, J., Haley, L., Byers, M., Eddy, R., Cooper, and Goggin, A. (1978). Assignment of the beta-glucuronidase structural gene to the pter leads to q22 region of chromosome man. Cytogenet. Cell. &net. 21: 99-104.
E., 7 in
17.
Shows, T., Eddy, R., Haley, L., Byers, M., Henry, M., Fujita, T., Matsui, H.. and Taniguci, T. (1984). Interleukin 2 (IL2) is assigned to human chromosome 4. Somat. Cell Mol. &net. 10: 315-318.
18.
Shows, T., Sakaguchi, A., and Naylor, S. (1982). In “Advances in Human Genetics” (H. Harris and K. Hirschhorn), Vol. 12, pp. 341-452, Plenum, New York/London.
19.
Wilmoth, D., Feder, M., Finan, J., and Nowell, P. (1985). Preleukemia and leukemia with 12p- and 19q+ chromosome alterations following alkeran therapy. Cancer Genet. C’ytogenet. 15: 95-98.
19a.
Yi, T., Cleveland, J. L., and Ihle, J. N. (1992). Protein tyrosine phosphatase containing SH2 domains: Characterization, preferential expression in hematopoietic cells and localization to chromosome 12 p 12-13. Mol. Cell. Biol. 12: 836-846. Zaccaria,A., Rosti, G., Testoni, N., andTura, S. (1985). Chromosome 12 rearrangement with breakage at the pll level in hematologic disorders: Report of four cases. Cancer Genet. C’ytogenet. 15: 309-314.
20.
December
26, 1991;
revised
March
4, 1992
for the function and control of a specific PTP as well as for signal transduction in general. Interestingly, PTPNG is expressed at high levels predominantly in hematopoietic cells (12). Disregulated activity of PTKs in hematopoietic cells, due to chromosomal abnormalities [e.g., the t(9;22) translocation in chronic myelogenous leukemia resulting in the bcr-abl gene fusion] or retroviral gene transduction (e.g., the v-erbB oncogene), can contribute to leukemogenesis. Similarly, it is conceivable that deranged control of a PTP with a key role in signal transduction could have similarly deleterious effects. Therefore, we determined the localization of PTPNG on human chromosomes using somatic cell hybrid panels and fluorescent in situ hybridization (FISH). DNA was prepared from 32 somatic cell hybrids inPhosphorylation of proteins on tyrosyl residues is a volving 15 unrelated human and 4 mouse cell lines (16key mechanism by which important cellular processes 18). The hybrids were characterized by karyotyping and such as growth, differentiation, and the cell cycle are with mapped enzyme markers (l&16,18); their propercontrolled (2, 7). The steady-state level of phosphorylation of any given tyrosyl residue is set by the opposing ties are summarized in Table 1. A “t” in the table indicates that only the translocation (indicated in the colactions of protein tyrosine kinases (PTKs) and protein umn labeled “Translocations”) but no intact chromotyrosine phosphatases (PTPs). PTKs catalyze the phossome is present. Bands corresponding to the human phorylation of themselves and/or other cellular pro(6.7/6.4/2.8/1.2 kb) and mouse (5.1/4.1 kb) PTPNG teins. Many of these tyrosyl-phosphorylated proteins genes can easily be distinguished on Southern blots of interact with secondary signaling molecules that contain EcoRI-digested DNA (data not shown). Accordingly, SH2 (src-homology-2) domains, which are approxihybrid cell DNA was digested with EcoRI, electrophomately lOO-amino-acid stretches of sequence similarity resed on agarose gels, and transferred to nylon memfound in such disparate molecules as GTPase-activating branes for Southern blotting as described elsewhere protein, phospholipase-Cy, the regulatory subunit of (10). The 2.2-kb full-length PTPNG cDNA was labeled phosphatidylinositol-3 kinase, the src-family of nonwith [a-32P]dCTP using the random primers method (6) transmembrane tyrosine kinases, and the cytoskeletal and hybridized to the blot. Each hybrid DNA was scored protein tensin [reviewed in (9)]. for the presence (+) or absence (-) of human bands. The Although much progress has been made in determinresults of this analysis were compared to the known presing the structure of PTPs, less is known about their role in signal transduction. Recently, we (12) and others (14) ence or absence of specific human chromosomes in each hybrid. As shown in Table 1, there was 0% discordancy identified a novel member of the PTP gene family (PTPNG) that contains two SH2 domains. Since SH2 between the presence of human PTPNG bands by domains function to direct signaling molecules to spe- Southern analysis and the presence of human chromosome 12 in a given hybrid. Thus, PTPNG maps unambigcific tyrosyl phosphoproteins, the discovery of an SH2containing PTP has obvious potential implications both uously to chromosome 12 by somatic cell hybrid analysis. To determine regional localization on chromosome 12, ’ To whomcorrespondence shouldbe addressed. we performed FISH on metaphase chromosomes. ChroWe have used panels of somatic cell hybrids and tluorescent in situ hybridization to determine the chromosomal localization of the novel nontransmembrane tyrosine phosphatase PTPNG (protein tyrosine phosphatase, nonreceptor type 6), which contains two SH2 domains. PTPNG maps to 12~13, a region commonly involved in leukemia-associated chromosomal abnormalities. Since PTPNG is expressed at high levels in hematopoietic cells of all lineages and its expression is induced early in hematopoietic differentiation, altered expression and/or structure of PTPNG may play a role in leukemogenesis. 0 1992 Academic Press, Inc.
869
GENOMICS Copyright 0 1992 All rights of reproduction
13,869-872
(19%)
0888.7543/92 $5.00 by Academic Press, Inc. in any form reserved.
870
SHORT
COMMUNICATION
TABLE Segregation
of PTPNG
with
Human
Chromosomes
1
in EcoRI-Digested Human
DNA No. 660 233 197 859 186 1185 389 402 187 830 1146 192 42 184 254 394 1162 390 643 644 395 407 212 425 424 347 25 534 555 1107 332 57
Hybrid ATR- 13 DUA-SBSAGA DUA-5BSAGA DUA-6 DUM-13 GAR-1 JSR-2 JSR-14 JWR-26C KER-3 NSL-9 NSL-16 REW-11 REX-11BSAgB REX-11BSHF REX-26 RSR-3 SIR-11 TSL-1 TSL-2 VTL-6 VTL-17 WIL-2 WIL-6 WIL-8X WIL-14 WIL-15 XOL-6 XOL-13 XOL-21 XTR-2 XTR-3BSAgB
Concordant No. of hybrids (+/+) t-/-j Discordant No. of hybrids (+/-) (-/+) % discordancy:
PTPNG
+ + + + + + + + + + + + + + + + + + +
1
2
3
4
5
6
7
8
9
10
Human-Mouse
Cell Hybrid
chromosome
11
12
13
14
+++++++t++-+t++++++--+t -+----++----++~-f--~---+-+-----+---+--++--+ -+-++---------+--++---+ + + + -+t+--+++-+ --f-+--t-+-++-++---+--+ ++-~+-----++------~-+ -+++++-----++-p-+-p++-+ t++++++-++++-+++++-++-+
15
16
17
18
19
20
21
22
X
tt++++++t
x/15,15x
w 1719 17/9
~ + + + + + - --22/x 22/x
17/3
l/X X/l
t---+++--+++-t--+-++-+t t--++-+--+++-A--+p-+--+ +
-
-
-
ISO7p 3/X 3/x, lOq-
t+++++-+--++-+--+
t-+--+-+-+++---+-++-t -t----~+t-+-~---~-++-t
4 13
7 11 8 8
9 8
13 9
7 11 11 6
12 12 5 10 6 12 05554273077069337534726 41 53 34 47 31 44
Translocations 5/x
+-++-+-------+ -+--+t+ -+++++++-+++~ + + + - ++t+-+-+++++-++-+ -~ +---+ + + - - f--+-----+ - --+ + - +------+---+---+---tt ++++-++++++-++++++-+tt +--+p-++--+-++---+p+ +--~---+-----~---+++ -+++-----++-+--+++-++-- + t -++-+-+-+-++-t+-++-+ -+---+++-++---t-+-+++++-+---+t-++--+--++-+--f-+---t-+---+-+ - + - +++++-++--+--+-+++-+ -+++-++-+++-+--+++++-+ +-+-+-++-+-f-++-+-----+ -+++-++--++++++-++-++-+ ~
DNA
11 10
5 13
7
8
12
45
34
40
16 6
5 15 10 9
9 6
19 13
7 7
13 4
10 10
8 10
14 6
11 8
2 10
0
12
6
8
11
4
8
14
4
0 56
47
35
44
35
41
53
25
29
53
13 6
5 10
11 6
6 13
2
41
50
32
Note. DNA from 32 somatic cell hybrids as described in the text was subjected to Southern blot analysis with PTPNG cDNA probe. The chromosome content of each hybrid is indicated. A “t” in the table indicates that the translocation indicated in the “Translocation” column is present with no intact chromosome present. Each hybrid was scored for the presence (+) or absence (-) of human PTPNG bands. A 0% discordancy indicates matched segregation of the probe with a particular chromosome. As indicated, PTPNG maps to chromosome 12.
mosome spreads were prepared from &bromodeoxyuridine-synchronized lymphocyte cultures. A full-length PTPNG cDNA insert was biotinylated and hybridized to the chromosome spreads. Following appropriate washing, hybridization was detected by reaction with fluorescein-conjugated avidin (Vector Labs). Chromosome identification was achieved by means of Q banding (DAPI counterstaining) and R banding (propidium iodide counterstaining) (4,5). Slides were evaluated using a Nikon fluorescence microscope. Thirty metaphases were examined. A fluorescent sig-
nal was detected at 12~13 on both chromatids of at least a single chromosome 12 in 26 of the 30 metaphase spreads examined (86%; Fig. 1). In 2 of the 26 spreads, signals were detected at band pl3 on both chromatids of both chromosomes 12, whereas 6 of the 26 spreads had an additional single signal on one chromatid of the other chromosome 12. No other chromosome displayed significant hybridization. These data conclusively localize PTPNG to 12~13. The localization of PTPNG at 12~13 is potentially of great interest, since the distal short arm of chromosome
SHORT COMMUNICATION 13.3
IJ.,
13.2 A.9 a i-l
l oeoooooooeoo l oooooeooo@oo
12.3
cell differentiation
(J.P., B.G.N.,
R.D.R.,
Bing Lim, and
Chaker Adra; unpublished data). It is easy to imagine how mutations in an SHB-containing PTP might perturb normal hematopoietic growth or differentiation. Thus, PTPNG is a good potential candidate for the gene targeted by leukemia-associated I2p chromosome abnormalities. ACKNOWLEDGMENTS The DNA and protein sequence of PTPNG have been submitted previously to GenBank (M77273) and the chromosomal localization has been reported to the Genome DataBase. We thank Pamela Beahm for technical assistance. This work was supported by NIH Grant R-Ol-CA 49152 to B.G.N. and NIH Grants HG00333, HD05196, and ACS CD62 to T.B.S.
15 9
21.1 21.2 21.3
#:# H
24.1 24.2
Note added in proof. After this paper was accepted, Yi et al. (19a) independently reported the molecular cloning of PTPNG (which they denote “HC-PTP”) and its localization by FISH to 12p12-P13, in agreement with the somatic cell hybrid and FISH results reported here.
REFERENCES
12
FIG. bution 86% of matids
1. Localization of PTPNG by FISH. Idiogram of the distriof fluorescence signals on both chromatids of chromosome 12. the metaphases examined had signals at 12~13 on both chroof at least a single chromosome 12 (see text).
12 is involved in chromosomal abnormalities in several forms of leukemia. Approximately 10% of pediatric acute lymphoblastic leukemia (ALL) cases display abnormalities in the 12p12-p13 region; these include interstitial and terminal deletions and translocations (3, 13). Several different chromosomes participate with 12~ in these translocations, suggesting that a 12p gene(s) is the target for these rearrangements. An intriguing exception to this theme is the dicentric translocation [tdic(9;12)(pll;pl2)] found in about 1% of pediatric ALLs (3). Deletions of the 9pll region are found independently in ALL (ll), raising the tantalizing possibility that the dicentric translocation simultaneously targets 9p and 12p genes. Adult leukemias, of both lymphoid and myeloid origin, also display 12p abnormalities, although at much lower frequency (1,8,19,20). Such abnormalities may be particularly common in leukemias associated with eosinophilia (8). Since involvement of 12~12 has been implicated in some studies and 12~13 in others, it is not yet clear whether all of these leukemiaassociated 12p abnormalities are targeting the same gene. However, given the difficulty of precisely specifying chromosomal breakpoints in clinical specimens, a single 12p target cannot be excluded. Its ubiquitous expression in hematopoietic cells of all lineages and differentiation stages suggests that PTPNG plays an important role in hematopoietic cell signal transduction. This notion is supported by our recent finding that PTPNG expression is induced at an early stage of hematopoietic
1.
Berger, R., Flandrin. G., Bernheim, A., Le Coniat, M., Vecchione, D., Pacot, A., Derre, J., Daniel, M.-T., Valensi, F., Sigaux, F., and Ochoa-Noguera, M. (1987). Cytogenetic studies on 519 consecutive de novo acute nonlymphocytic leukemias. Cancer Genet. Cytogenet. 29: 9-21.
2.
Cantley, L., Auger, K., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. (1991). Oncogenes and signal transduction. Cell 64: 281-302. Carroll, A., Raimondi, S., Williams, D., Behm, F., Borowitz, M., Castleberry, R., Harris, M., Patterson, R., Pullen, D., and Crist, W. (1987). tdic (9;12): A nonrandom chromosome abnormality in childhood B-cell precursor acute lymphoblastic leukemia: A pediatric oncology group study. Blood 70: 196221965.
3.
4.
5.
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