- Email: [email protected]
Chromosomal mapping of the human proteinase inhibitor 6 (PI6) gene to 6p25 by fluorescence in situ hybridization
BRIEF REPORTS corded and graphically merged by computer using the software program COMOS (Bio-Rad). We analyzed over 100 metaphase spreads and consistently found distinct fluorescent signals on chromosome 12. A representative F I S H image is presented in Fig. 1, clearly showing t h a t the Pmel 17 gene maps to band q13 close to q14 of chromosome 12. Pmel 17 protein is thought to play an important role in melanin biosynthesis as a catalyst at a step distal to tyrosinase and m a y act as a positive regulator for melanin biosynthesis (3). In our studies, Pmel 17 (BA7) m R N A is expressed only in retina among the tissues t h a t we have tested (unpublished data). It will be interesting to investigate whether Pme117 mRNA is expressed in a pigment epithelium of retina or somewhere else. To date, there are no retina diseases mapped to chromosome 12, but the Pmel 17 gene can be a candidate for a yet-unveiled degenerative retina disease, including retinitis pigmentosa, which involves intraretinal pigmentation, or ocular albinism, which involves a defect in melanin biosynthesis.
ACKNOWLEDGMENTS R.K is the recipient of a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. This work was supported by a Grant-in-Aid for Creative Basic Research (Human Genome Program) from the Ministry of Education, Science and Culture of Japan.
REFERENCES 1. Craig, I. W., Gedde-Dahl, T., Jr., and Kucherlapati, R. (1994). " H u m a n Gene Mapping 1993," The Johns Hopkins Univ. Press, Baltimore, MD. 2. Fukuyama, R., and Shimizu, N. (1992). Detection of variation in the ribosomal R N A gene clusters by a modified fluorescence in situ hybridization method. Jpn. J. H u m . Genet. 37: 139-143. 3. Kwon, B. S. (1993). Pigmentation genes: The tyrosinase gene family and the pmel 17 gene family. J. Invest. Derrnatol. 100: 134S-140S. 4. Kwon, B. S., Chintamaneni, C., Kozak, C. A., Copeland, N. G., Gilbert, D. J., Jenkins, N., Barton, D., Francke, U., Kobayashi, Y., and Kim, I~ I~ (1991). A melanocyte-specific gene, Pmel 17, maps near the silver coat color locus on mouse chromosome 10 and is in a syntenic region on h u m a n chromosome 12. Proc. Natl. Acad. Sci. U S A 88: 9228-9232. 5. Kwon, B. S., Halaban, R., Kim, G. S., Usack, L., Pomerants, S. H., and Haq, A. K. (1987). A melanocyte-specific c D N A clone whose expression is inducible by M S H and IBMX. Mol. Biol. Med. 4: 339-355. 6. Kwon, B. S., Haq, A. K, Pomerantz, S. H., and Halaban, R. (1987). Isolation and sequence ofa cDNA clone for human tyrosinasoe that maps at the mouse c-albino locus [published erratum appears in Proc. Natl. Acad. Sci. USA (1988) 85: 6352]. Proc. Natl. Acad. Sci. USA 84: 7473-7477. 7. Wang, Y., Minoshima, S., and Shimizu, N. (1993). Precise mapping of the EGF receptor gene on the human chromosome 7p12 using an improved FISH technique. Jpn. J. Hum. Genet. 38: 399-406.
431
Chromosomal Mapping of the Human Proteinase Inhibitor 6 (PI6) Gene to 6p25 by Fluorescence in 5itu Hybridization Paul Coughlin,* Jillian Nicholl,t Jiuru Sun,* Hatem Salem,* Phillip Bird, *'1 and Grant R. Sutherlandt *Department of Medicine, Monash Medical School, Clive Ward Centre, Box Hill Hospital, Box Hill 3128, Australia; and tCentre for Medical Genetics, Department of Cytogenetics and Molecular Genetics, Women's and Children's Hospital, Adelaide, South Australia 5006, Australia
ReceivedSeptember30, 1994;January9, 1995
Proteinase inhibitor (PI6), also known as placental thrombin inhibitor, is a recently identified protein originally purified from h u m a n placentas t h a t belongs to the serpin superfamily of proteins (3). The amino acid sequence of PI6 (2) suggests t h a t it is a member of a subfamily known as "ovalbumin serpins" (7). Characteristic features of this subfamily are: (a) they lack N-terminal and C-terminal sequences common in other serpins, (b) they do not have a classical Nterminal signal sequence for secretion, and (c) they display a similar gene organization, with seven exons and eight introns. Interest in ovalbumin serpins has been stimulated by the identification of a serpin called maspin, expressed in murine m a m m a r y epithelium, which m a y be a tumor suppressor (9). Maspin is down-regulated in transformed m a m m a r y epithelial cells. Furthermore, reintroduction of maspin into transformed cells appears to inhibit tumor growth and ability to metastasize. PI6 differs from most of the other ovalbumin serpins in t h a t it appears to be entirely intracellular. In addition, it has oxidation-sensitive residues adjacent to the reactive site, making it unlikely t h a t functional activity would be possible in the extracellular environment (2). The function of PI6 is uncertain. It is unlikely to be a thrombin inhibitor given its intracellular location and is more likely to interact with one or more intracellular proteinases. The pattern of expression of PI6 does not give any clues as to a function because Northern blot data show PI6 mRNA in a wide variety of h u m a n tissues (2). While the physiological role of intracellular serpins is unclear, recent developments indicate their potential importance. The cowpox virus protein c r m A has been shown to confer virulence by virtue of its ability to inhibit the proinflammatory interleukin-lfl-converting enzyme (ICE) (6). Subsequent studies suggest t h a t ICE m a y be pivotal in the biochemical events leading up to apoptosis (4, 5, 8). Thus, it is quite likely t h a t there exists a mammalian, intracellular serpin responsible for regulation of ICE. The probe used for chromosomal localization was plasmid 1 To whom correspondence should be addressed. Telephone: 03 895 0315. Fax: 03 895 0332. GENOMICS26, 431-433 (1995) 0888-7543/95 $6.00 Copyright © 1995 by AcademicPress, Inc. All rights of reproduction in any form reserved.
432
BRIEF REPORTS
FIG. 1° Metaphase showing FISH with the PTI probe. (A) Normal male chromosomes stained with propidium iodide. Hybridization sites on chromosome 6 are indicated by arrows. (B) The same metaphases as A stained with DAPI for chromosome identification.
pSVTf-PTIIP, which contains a full-length 1300-bp PTI cDNA (2). It was nick-translated with biotin-14-dATP and hybridized in situ at a final concentration of 30 ng/#l to metaphases from two normal males. The fluorescence in situ hybridization (FISH) method was modified from that previously described (1) in t h a t chromosomes were stained before analysis with both propidium iodide (as counterstain) and DAPI (for chromosome identification). Images of metaphase preparations were captured by a CCD camera and computer enhanced. Twenty-five metaphases from the first normal male were examined for fluorescent signal. Twenty-four of these metaphases showed signal on one (50%) or both (50%) chromatids of chromosome 6 in the region 6p24-p25; 93% of this signal was at the extreme distal end of the chromosome arm, t h a t
is, 6p25 (Fig. 1). There was a total of 21 nonspecific background dots observed in these 25 metaphases. A similar result was obtained from hybridization of the probe to 20 metaphases from the second normal male (data not shown). Considerable experience in the localization of single-copy genes by FISH, using small (800-1500 bp) probes, has shown that signal is often present on only one chromatid in a significant proportion of metaphases. In the presence of a very high signal to background ratio, the results are unequivocal. The data presented in this paper localize PI6 to chromosome 6p25. The only other ovalbumin serpin for which a chromosomal mapping has been reported, PAI-2, is located at 18q21-q23. Therefore, on the basis of the currently available data there is no evidence of a clustering of this subfamily of proteins.
BRIEF REPORTS ACKNOWLEDGMENTS This work was supported by the National Health and Medical Research Council of Australia and an International Research Scholar's award to G.R.S. from the Howard Hughes Medical Institute. REFERENCES
1. Callen, D. F., Baker, E., Eyre, H. J., Chernos, J. E., Bell, J. A., and Sutherland, G. R. (1990). Reassessment of two apparent deletions of chromosome 16p to an ins(11; 16) and a t(1; 16) by chromosome painting. Ann. Genet. 33" 219-221. 2. Coughlin, P., Sun, J., Cerruti, L., Salem, H. H., and Bird, P. (1993). Cloning and molecular characterization of a human intracellular proteinase inhibitor. Proc. Natl. Acad. Sci. USA 90: 9417-9421. 3. Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993). Identification and purification of a novel serine proteinase inhibitor. J. Biol. Chem. 268: 9541-9547. 4. Gagliardini, V., Fernandez, P.-A., Lee, R. K. K., Drexler, H. C. A., Rotello, R. J., Fishman, M. C., and Yuan, J. (1994). Prevention of vertebrate neuronal cell death by the crmA gene. Science 263: 826-828. 5. Miura, M., Zhu, H., Rotello, R., Hartwieg, E. A., and Yuan, J. (1993). Induction of apoptosis in fibroblasts by IL-1/3-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell 75: 653-660. 6. Ray, C. A., Black, R. A., Kronheim, S. R., Greenstreet, T. A., Sleath, P. R., Salvesen, G. S., and Pickup, D. J. (1992). Viral inhibition of inflammation: Cowpox virus encodes an inhibitor of the interleukin-1/3-converting enzyme. Cell 69: 597-604. 7. Remold-O'Donnell, E. (1993). The ovalbumin family of serpins. FEBS Lett. 315" 105-108. 8. Yuan, J., Shasham, S., Ledoux, S., Ellis, H. M., and Horvitz, H. R. (1993). The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1/3-converting enzyme. Cell 75: 641-652. 9. Zou, Z., Anisowicz, A., Hendrix, M. J. C., Thor, A., Neveu, M., Sheng, S., Rafidi, K., Seftor, E., and Sager, R. (1994). Maspin, a serpin with tumor-suppressing activity in mammary epithelial cells. Science 263: 526-529.
The DAD1 Protein, Whose Defect Causes Apoptotic Cell Death, Maps to Human Chromosome 14 Isik G. Ylug,* Chee Gee See, t and Elizabeth M. C. Fisher *'1 *Department of Biochemistry and Molecular Genetics, St. Mary's Hospital Medical School, Imperial College, Norfolk Place, London W2 1PG, United Kingdom, and t The Galton Laboratory, Department of Genetics and Biometry, University College, London NWI 2HE, United Kindgom
Received October 31, 1994; accepted December 23, 1994
1 To whom correspondence should be addressed. Telephone: 44 1 71 723 1252. Fax: 44 71 706 3272. GENOMICS 26, 4 3 3 - 4 3 5 (1995) 0888-7543/95 $6.00 Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
433
P r o g r a m m e d cell d e a t h (PCD) is an i m p o r t a n t process in t h e development of a n i m a l embryos. Once this m e c h a n i s m t a k e s over, cells die by a process known as apoptosis in which the cells undergo a characteristic set of changes including cell shriveling a n d DNA b r e a k d o w n (2, 13). I n t h e biological s y s t e m s in which PCD h a s been investigated, it a p p e a r s t h a t specific p a t h w a y s t h a t l e a d to apoptosis exist. Some of the genes whose products act in these p a t h w a y s have been characterized extensively in Caenorhabditis elegans (such as the gene ced-9) a n d in m a m m a l i a n cells (such as Bcl2). I f C. elegans ced-9 is inactivated, cells t h a t would n o r m a l l y survive die (3, 4). Similarly, in m a n y cell lines, the B c l 2 protein a p p e a r s to protect cells from apoptosis (6, 10, 12). Cellular p a t h w a y s t h a t r e s u l t in PCD involve complex interactions between a n u m b e r of proteins, a n d t h e cell biology of apoptosis r e m a i n s to be understood. One gene whose protein product probably acts in a PCD p a t h w a y is the recently identified DAD1 gene (8), which was isolated by N a k a s h i m a a n d colleagues in a screen for sequences t h a t would correct a t e m p e r a t u r e - s e n s i t i v e m u t a t i o n in a h a m s t e r cell line, tsBN7 (9). At nonpermissive t e m p e r a tures tsBN7 cells r o u n d up a n d float off tissue culture surfaces, the c h r o m a t i n is condensed, and t h e cells lose t h e i r viability. Electron microscopy of these cells indicates t h a t t h e y have the characteristic f e a t u r e s of apoptosis. N a k a s h i m a a n d colleagues t r a n s f e c t e d tsBN7 cells with h u m a n cDNAs a n d rescued the t e m p e r a t u r e - s e n s i t i v e phenotype with one cDNA clone, which t h e y n a m e d DAD1 (defender a g a i n s t apoptotic cell death). The DAD1 gene was shown to have a point m u t a t i o n in tsBN7 cells t h a t r e s u l t s in loss of DAD1 function a t the n o n p e r m i s s i v e t e m p e r a t u r e . The DAD1 gene encodes a 12.5-kDa protein t h a t is expressed t h r o u g h o u t t h e body a t high levels (8). No k n o w n proteins have a n y simil a r i t y to DAD1. PCD is of profound i m p o r t a n c e to the developing h u m a n embryo, a n d m u t a t i o n s in t h e proteins t h a t e i t h e r protect from or lead to apoptosis could have significant effects on cell growth. To d e t e r m i n e w h e t h e r DAD1 m a p s to the site of a n y known m u t a t i o n in h u m a n s , we have localized this gene within the h u m a n genome. To m a p t h e DAD1 gene we designed p o l y m e r a s e chain reaction (PCR) p r i m e r s to t h e 3 ' u n t r a n s l a t e d region (UTR) of the h u m a n DAD1 cDNA sequence (Accession No. $65756, Fig. 1). The 193-bp 3 ' UTR product was amplified in a h u m a n monochromosomal somatic cell h y b r i d m a p p i n g panel as described by H e r n a n d e z a n d colleagues (5). The amplification was human-specific a n d did not produce f r a g m e n t s in a n y of t h e r o d e n t control lanes. Only two lanes gave amplification products: total h u m a n a n d cell line GM10479, which contains h u m a n chromosome 14 only on a mouse b a c k g r o u n d (7). This amplification r e s u l t indicated t h a t DAD1 gene m a p s to chromosome 14. To determine a regional m a p p i n g position for DAD1, we isolated D A D l - c o n t a i n i n g h u m a n YACs for fluorescence in situ hybridization (FISH) m a p p i n g by amplifying the 193-bp 3 ' UTR within YAC pools from the ICI total h u m a n YAC l i b r a r y (1). This l i b r a r y h a s a n average YAC i n s e r t size of 350 kb. The 3 'UTR f r a g m e n t amplified in a n initial set of 4 p r i m a r y positives in the 40 p r i m a r y pools and t h e n in 4 secondary positives in the 9 secondary pools. Finally, 11 t e r t i a r y positives amplified in the 20 rows a n d columns. E a c h t e r t i a r y positive was s t r e a k e d onto A H C - - plates (5 g y e a s t nitrogen b a s e
ACKNOWLEDGMENTS R.K is the recipient of a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. This work was supported by a Grant-in-Aid for Creative Basic Research (Human Genome Program) from the Ministry of Education, Science and Culture of Japan.
REFERENCES 1. Craig, I. W., Gedde-Dahl, T., Jr., and Kucherlapati, R. (1994). " H u m a n Gene Mapping 1993," The Johns Hopkins Univ. Press, Baltimore, MD. 2. Fukuyama, R., and Shimizu, N. (1992). Detection of variation in the ribosomal R N A gene clusters by a modified fluorescence in situ hybridization method. Jpn. J. H u m . Genet. 37: 139-143. 3. Kwon, B. S. (1993). Pigmentation genes: The tyrosinase gene family and the pmel 17 gene family. J. Invest. Derrnatol. 100: 134S-140S. 4. Kwon, B. S., Chintamaneni, C., Kozak, C. A., Copeland, N. G., Gilbert, D. J., Jenkins, N., Barton, D., Francke, U., Kobayashi, Y., and Kim, I~ I~ (1991). A melanocyte-specific gene, Pmel 17, maps near the silver coat color locus on mouse chromosome 10 and is in a syntenic region on h u m a n chromosome 12. Proc. Natl. Acad. Sci. U S A 88: 9228-9232. 5. Kwon, B. S., Halaban, R., Kim, G. S., Usack, L., Pomerants, S. H., and Haq, A. K. (1987). A melanocyte-specific c D N A clone whose expression is inducible by M S H and IBMX. Mol. Biol. Med. 4: 339-355. 6. Kwon, B. S., Haq, A. K, Pomerantz, S. H., and Halaban, R. (1987). Isolation and sequence ofa cDNA clone for human tyrosinasoe that maps at the mouse c-albino locus [published erratum appears in Proc. Natl. Acad. Sci. USA (1988) 85: 6352]. Proc. Natl. Acad. Sci. USA 84: 7473-7477. 7. Wang, Y., Minoshima, S., and Shimizu, N. (1993). Precise mapping of the EGF receptor gene on the human chromosome 7p12 using an improved FISH technique. Jpn. J. Hum. Genet. 38: 399-406.
431
Chromosomal Mapping of the Human Proteinase Inhibitor 6 (PI6) Gene to 6p25 by Fluorescence in 5itu Hybridization Paul Coughlin,* Jillian Nicholl,t Jiuru Sun,* Hatem Salem,* Phillip Bird, *'1 and Grant R. Sutherlandt *Department of Medicine, Monash Medical School, Clive Ward Centre, Box Hill Hospital, Box Hill 3128, Australia; and tCentre for Medical Genetics, Department of Cytogenetics and Molecular Genetics, Women's and Children's Hospital, Adelaide, South Australia 5006, Australia
ReceivedSeptember30, 1994;January9, 1995
Proteinase inhibitor (PI6), also known as placental thrombin inhibitor, is a recently identified protein originally purified from h u m a n placentas t h a t belongs to the serpin superfamily of proteins (3). The amino acid sequence of PI6 (2) suggests t h a t it is a member of a subfamily known as "ovalbumin serpins" (7). Characteristic features of this subfamily are: (a) they lack N-terminal and C-terminal sequences common in other serpins, (b) they do not have a classical Nterminal signal sequence for secretion, and (c) they display a similar gene organization, with seven exons and eight introns. Interest in ovalbumin serpins has been stimulated by the identification of a serpin called maspin, expressed in murine m a m m a r y epithelium, which m a y be a tumor suppressor (9). Maspin is down-regulated in transformed m a m m a r y epithelial cells. Furthermore, reintroduction of maspin into transformed cells appears to inhibit tumor growth and ability to metastasize. PI6 differs from most of the other ovalbumin serpins in t h a t it appears to be entirely intracellular. In addition, it has oxidation-sensitive residues adjacent to the reactive site, making it unlikely t h a t functional activity would be possible in the extracellular environment (2). The function of PI6 is uncertain. It is unlikely to be a thrombin inhibitor given its intracellular location and is more likely to interact with one or more intracellular proteinases. The pattern of expression of PI6 does not give any clues as to a function because Northern blot data show PI6 mRNA in a wide variety of h u m a n tissues (2). While the physiological role of intracellular serpins is unclear, recent developments indicate their potential importance. The cowpox virus protein c r m A has been shown to confer virulence by virtue of its ability to inhibit the proinflammatory interleukin-lfl-converting enzyme (ICE) (6). Subsequent studies suggest t h a t ICE m a y be pivotal in the biochemical events leading up to apoptosis (4, 5, 8). Thus, it is quite likely t h a t there exists a mammalian, intracellular serpin responsible for regulation of ICE. The probe used for chromosomal localization was plasmid 1 To whom correspondence should be addressed. Telephone: 03 895 0315. Fax: 03 895 0332. GENOMICS26, 431-433 (1995) 0888-7543/95 $6.00 Copyright © 1995 by AcademicPress, Inc. All rights of reproduction in any form reserved.
432
BRIEF REPORTS
FIG. 1° Metaphase showing FISH with the PTI probe. (A) Normal male chromosomes stained with propidium iodide. Hybridization sites on chromosome 6 are indicated by arrows. (B) The same metaphases as A stained with DAPI for chromosome identification.
pSVTf-PTIIP, which contains a full-length 1300-bp PTI cDNA (2). It was nick-translated with biotin-14-dATP and hybridized in situ at a final concentration of 30 ng/#l to metaphases from two normal males. The fluorescence in situ hybridization (FISH) method was modified from that previously described (1) in t h a t chromosomes were stained before analysis with both propidium iodide (as counterstain) and DAPI (for chromosome identification). Images of metaphase preparations were captured by a CCD camera and computer enhanced. Twenty-five metaphases from the first normal male were examined for fluorescent signal. Twenty-four of these metaphases showed signal on one (50%) or both (50%) chromatids of chromosome 6 in the region 6p24-p25; 93% of this signal was at the extreme distal end of the chromosome arm, t h a t
is, 6p25 (Fig. 1). There was a total of 21 nonspecific background dots observed in these 25 metaphases. A similar result was obtained from hybridization of the probe to 20 metaphases from the second normal male (data not shown). Considerable experience in the localization of single-copy genes by FISH, using small (800-1500 bp) probes, has shown that signal is often present on only one chromatid in a significant proportion of metaphases. In the presence of a very high signal to background ratio, the results are unequivocal. The data presented in this paper localize PI6 to chromosome 6p25. The only other ovalbumin serpin for which a chromosomal mapping has been reported, PAI-2, is located at 18q21-q23. Therefore, on the basis of the currently available data there is no evidence of a clustering of this subfamily of proteins.
BRIEF REPORTS ACKNOWLEDGMENTS This work was supported by the National Health and Medical Research Council of Australia and an International Research Scholar's award to G.R.S. from the Howard Hughes Medical Institute. REFERENCES
1. Callen, D. F., Baker, E., Eyre, H. J., Chernos, J. E., Bell, J. A., and Sutherland, G. R. (1990). Reassessment of two apparent deletions of chromosome 16p to an ins(11; 16) and a t(1; 16) by chromosome painting. Ann. Genet. 33" 219-221. 2. Coughlin, P., Sun, J., Cerruti, L., Salem, H. H., and Bird, P. (1993). Cloning and molecular characterization of a human intracellular proteinase inhibitor. Proc. Natl. Acad. Sci. USA 90: 9417-9421. 3. Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993). Identification and purification of a novel serine proteinase inhibitor. J. Biol. Chem. 268: 9541-9547. 4. Gagliardini, V., Fernandez, P.-A., Lee, R. K. K., Drexler, H. C. A., Rotello, R. J., Fishman, M. C., and Yuan, J. (1994). Prevention of vertebrate neuronal cell death by the crmA gene. Science 263: 826-828. 5. Miura, M., Zhu, H., Rotello, R., Hartwieg, E. A., and Yuan, J. (1993). Induction of apoptosis in fibroblasts by IL-1/3-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell 75: 653-660. 6. Ray, C. A., Black, R. A., Kronheim, S. R., Greenstreet, T. A., Sleath, P. R., Salvesen, G. S., and Pickup, D. J. (1992). Viral inhibition of inflammation: Cowpox virus encodes an inhibitor of the interleukin-1/3-converting enzyme. Cell 69: 597-604. 7. Remold-O'Donnell, E. (1993). The ovalbumin family of serpins. FEBS Lett. 315" 105-108. 8. Yuan, J., Shasham, S., Ledoux, S., Ellis, H. M., and Horvitz, H. R. (1993). The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1/3-converting enzyme. Cell 75: 641-652. 9. Zou, Z., Anisowicz, A., Hendrix, M. J. C., Thor, A., Neveu, M., Sheng, S., Rafidi, K., Seftor, E., and Sager, R. (1994). Maspin, a serpin with tumor-suppressing activity in mammary epithelial cells. Science 263: 526-529.
The DAD1 Protein, Whose Defect Causes Apoptotic Cell Death, Maps to Human Chromosome 14 Isik G. Ylug,* Chee Gee See, t and Elizabeth M. C. Fisher *'1 *Department of Biochemistry and Molecular Genetics, St. Mary's Hospital Medical School, Imperial College, Norfolk Place, London W2 1PG, United Kingdom, and t The Galton Laboratory, Department of Genetics and Biometry, University College, London NWI 2HE, United Kindgom
Received October 31, 1994; accepted December 23, 1994
1 To whom correspondence should be addressed. Telephone: 44 1 71 723 1252. Fax: 44 71 706 3272. GENOMICS 26, 4 3 3 - 4 3 5 (1995) 0888-7543/95 $6.00 Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
433
P r o g r a m m e d cell d e a t h (PCD) is an i m p o r t a n t process in t h e development of a n i m a l embryos. Once this m e c h a n i s m t a k e s over, cells die by a process known as apoptosis in which the cells undergo a characteristic set of changes including cell shriveling a n d DNA b r e a k d o w n (2, 13). I n t h e biological s y s t e m s in which PCD h a s been investigated, it a p p e a r s t h a t specific p a t h w a y s t h a t l e a d to apoptosis exist. Some of the genes whose products act in these p a t h w a y s have been characterized extensively in Caenorhabditis elegans (such as the gene ced-9) a n d in m a m m a l i a n cells (such as Bcl2). I f C. elegans ced-9 is inactivated, cells t h a t would n o r m a l l y survive die (3, 4). Similarly, in m a n y cell lines, the B c l 2 protein a p p e a r s to protect cells from apoptosis (6, 10, 12). Cellular p a t h w a y s t h a t r e s u l t in PCD involve complex interactions between a n u m b e r of proteins, a n d t h e cell biology of apoptosis r e m a i n s to be understood. One gene whose protein product probably acts in a PCD p a t h w a y is the recently identified DAD1 gene (8), which was isolated by N a k a s h i m a a n d colleagues in a screen for sequences t h a t would correct a t e m p e r a t u r e - s e n s i t i v e m u t a t i o n in a h a m s t e r cell line, tsBN7 (9). At nonpermissive t e m p e r a tures tsBN7 cells r o u n d up a n d float off tissue culture surfaces, the c h r o m a t i n is condensed, and t h e cells lose t h e i r viability. Electron microscopy of these cells indicates t h a t t h e y have the characteristic f e a t u r e s of apoptosis. N a k a s h i m a a n d colleagues t r a n s f e c t e d tsBN7 cells with h u m a n cDNAs a n d rescued the t e m p e r a t u r e - s e n s i t i v e phenotype with one cDNA clone, which t h e y n a m e d DAD1 (defender a g a i n s t apoptotic cell death). The DAD1 gene was shown to have a point m u t a t i o n in tsBN7 cells t h a t r e s u l t s in loss of DAD1 function a t the n o n p e r m i s s i v e t e m p e r a t u r e . The DAD1 gene encodes a 12.5-kDa protein t h a t is expressed t h r o u g h o u t t h e body a t high levels (8). No k n o w n proteins have a n y simil a r i t y to DAD1. PCD is of profound i m p o r t a n c e to the developing h u m a n embryo, a n d m u t a t i o n s in t h e proteins t h a t e i t h e r protect from or lead to apoptosis could have significant effects on cell growth. To d e t e r m i n e w h e t h e r DAD1 m a p s to the site of a n y known m u t a t i o n in h u m a n s , we have localized this gene within the h u m a n genome. To m a p t h e DAD1 gene we designed p o l y m e r a s e chain reaction (PCR) p r i m e r s to t h e 3 ' u n t r a n s l a t e d region (UTR) of the h u m a n DAD1 cDNA sequence (Accession No. $65756, Fig. 1). The 193-bp 3 ' UTR product was amplified in a h u m a n monochromosomal somatic cell h y b r i d m a p p i n g panel as described by H e r n a n d e z a n d colleagues (5). The amplification was human-specific a n d did not produce f r a g m e n t s in a n y of t h e r o d e n t control lanes. Only two lanes gave amplification products: total h u m a n a n d cell line GM10479, which contains h u m a n chromosome 14 only on a mouse b a c k g r o u n d (7). This amplification r e s u l t indicated t h a t DAD1 gene m a p s to chromosome 14. To determine a regional m a p p i n g position for DAD1, we isolated D A D l - c o n t a i n i n g h u m a n YACs for fluorescence in situ hybridization (FISH) m a p p i n g by amplifying the 193-bp 3 ' UTR within YAC pools from the ICI total h u m a n YAC l i b r a r y (1). This l i b r a r y h a s a n average YAC i n s e r t size of 350 kb. The 3 'UTR f r a g m e n t amplified in a n initial set of 4 p r i m a r y positives in the 40 p r i m a r y pools and t h e n in 4 secondary positives in the 9 secondary pools. Finally, 11 t e r t i a r y positives amplified in the 20 rows a n d columns. E a c h t e r t i a r y positive was s t r e a k e d onto A H C - - plates (5 g y e a s t nitrogen b a s e