- Email: [email protected]
Assignment of Human 17β-Hydroxysteroid Dehydrogenase IV to Chromosome 5q2 by Fluorescencein SituHybridization
BRIEF REPORTS Assignment of Human 17b-Hydroxysteroid Dehydrogenase IV to Chromosome 5q2 by Fluorescence in Situ Hybridization Frauke Leenders,*,1 Gabriele Prescher,†,1 Vincent Dolez,* Agne`s Begue,* Yvan de Launoit,* and Jerzy Adamski‡,2 *Unite´ d’Oncologie Mole´culaire, Institut Pasteur de Lille, CNRS URA 1160, 1 rue Calmette, 59019 Lille, France; ‡Max-Planck-Institut fu¨r experimentelle Endokrinologie, Postfach 610309, 30603 Hannover, Germany; and †Innere Klinik und Poliklinik (Tumorforschung), Universita¨tsklinikum Essen, Hufelandstrasse 55, 45122 Essen, Germany Received March 5, 1996; accepted August 13, 1996
The redox reactions at position C17 of the steroid molecule are a key step in the synthesis and inactivation of androgens and estrogens. Until now, four different human 17b-hydroxysteroid dehydrogenases (17b-HSD) have been characterized (2). We have identified a cDNA of human (gene name HSD17B4) 17b-HSD IV that inactivates hormones very efficiently (1). A 3.0-kb mRNA codes for a peroxisomal 80-kDa (736 amino acids) protein featuring domains that are not present in the other 17b-hydroxysteroid dehydrogenases. Its N-terminal domain (amino acids 1 – 323) is able to perform the dehydrogenase reaction not only with steroids at the C17 position but also with 3-hydroxyacyl-CoA. The central part (amino acids 324 – 596) catalyzes with high efficiency one of the steps in the peroxisomal b-oxidation of fatty acids: the 2-enoyl-acyl-CoA hydratase reaction. The C-terminal domain (amino acids 597– 736) is similar to sterol carrier protein 2 and facilitates the transfer of 7-dehydrocholesterol and phosphatidylcholine between membranes in vitro (6). The unique multidomain structure of the 80-kDa protein allows for the catalysis of several reactions thus far thought to be performed by complexes of different enzymes. Steroid inactivation in breast cancer tissues and cell lines has been reported to be decreased (3). Deficiencies in peroxisomal enzymes are associated with severe disorders of neuronal and renal development such as Zellweger syndrome and adrenoleukodystrophy (5). Zellweger syndrome patients reveal deficits in steroid and bile acid metabolism assigning peroxisomes to steroidogenesis. To facilitate the study of the gene’s involvement in human disease states, we have mapped the gene of 17b-HSD IV. Human genomic Lambda Dash library was screened with fragments of human cDNA (nucleotides 1029 – 1787 corresponding to the central domain and 1788 – 2211 correspond1
These authors contributed equally to this work. To whom correspondence should be addressed at present address: GSF-Forschungszentrum fu¨r Umwelt und Gesundheit, Institut fu¨r Sa¨ugetieregenetik, Neuherberg, Postfach 1129, D-85758 Oberschleißheim, Germany. Telephone: /49-89-3187-4110. Fax: /49-893187-3099. E-mail: [email protected]. 2
ing to the C-terminal domain) of the 17b-HSD IV (1). Two independent phages, FL6 and FL1 (identified with the central and C-terminal probes, respectively), with inserts of about 14 kb showing different patterns after digestion with XbaI and EcoRI were used for in situ hybridization. After the digestion of the phages FL6 and FL1 with EcoRI, the fragments that hybridized on Southern blot with a 17b-HSD IV cDNA 32 P-labeled probe (3 and 7 kb, respectively) were subcloned in pGem-3Zf and pBluescript vectors to confirm sequence identity with the 17b-HSD IV cDNA. The phage FL6 contains DNA coding for at least one exon corresponding to amino acids 560 – 590 (numbering according to Ref. 1). The FL1 phage codes for at least two exons. The first contains amino acids 665 – 707, and the second begins at amino acid 707 and includes the stop codon and the 3 *-nontranslated region. Verified FL6 and FL1 phages were labeled by standard nick-translation with biotin-16 –dUTP or digoxigenin-11– dUTP (Boehringer Mannheim), respectively. Slides with normal lymphocyte metaphases were prepared by standard protocols from PHA-stimulated lymphocyte cultures. Slides were pretreated with RNase and pepsin and fixed with 1% formalin as described (10). Hybridization and detection were performed as described (7). Detection of the biotin labeling was performed with fluorescein isothiocyanate (FITC) conjugated to avidin DCS (Vector Laboratories) and detection of digoxigenin was performed with anti-digoxigenin rhodamine-conjugated antibodies from sheep (Boehringer Mannheim). For signal amplification biotinylated anti-avidin (Vector Laboratories) and rhodamine-conjugated anti-sheep IgG (Jackson Immuno Research Laboratories) were used. Slides were counterstained with 4*,6-diamidino-2-phenylindole (DAPI) to enable the identification of the chromosomes by a G-bandinglike pattern and were mounted with antifade solution (Vector Laboratories). Digital gray-scale images of each fluorochrome were obtained separately with a cooled charge-coupled device camera (Photometrics, Tucson, AZ). After inversion of the gray-scale pattern of the DAPI image, the images were combined using the Gene Join software package (Yale University). The signals of both clones mapped overlapping in the same region on chromosome 5q2 (Fig. 1), as could be seen by the banding pattern. This region was confirmed by calculating the positions of the signals on 12 chromatids in relation to the whole chromosome length and comparing them with the idiogram (4) of chromosome 5. The chromosome designation was confirmed (not shown) by an additional cohybridization of the biotin-labeled clone FL1 and digoxigenin-labeled chromosome 5 library DNA from a mapping panel (mapping panel 2, consisting of DNA from human/rodent somatic cell hybrids, Repository No. NA 10114, Coriell Institute, Camden, NJ). The signal frequency for both clones was examined on 25 metaphases. For clone FL1, 20 cells showed signals on all four chromatids, 2 cells showed signals on three chromatids, and 3 cells showed signals on two chromatids. For clone FL6, 12 cells showed signals on four chromatids, 4 cells showed signals on three chromatids, 7 cells showed signals on two chromatids, one cell showed signal on one chromatid, and one methaphase showed no signal. Hybridization efficiency GENOMICS
403
37, 403–404 (1996) ARTICLE NO. 0578
0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AID Genom BREP
/
6r20$$381x
10-04-96 16:39:42
gnmbr
AP: Genomics
404
BRIEF REPORTS
4. 5.
6.
7.
8. 9.
10. FIG. 1. Localization of the human 17b-HSD IV gene. Digital image of clone FL1 (digoxigenin-labeled) hybridized on human chromosomes. Specific signals are seen at chromosome 5q2 (arrowheads). 11.
was independent of the type of label as checked by exchanging the labels of both clones (not shown). The efficient inactivation of steroids by 17b-HSD IV could be distorted by mutations in the HSD17B4 gene. The increased intracellular levels of estrogens could then result in the development of estrogen-sensitive tumors. Indeed, studies for loss of heterozygosity at tumor suppressor loci involved in sporadic breast cancer and in colorectal cancer (9, 11) revealed allele loss at 5q21. In addition to colorectal and breast cancer genes, other important genes involved in carcinogenesis, such as those participating in leukemogenesis, are associated with the long arm of chromosome 5q (8).
human breast tumours and in normal breast tissue. Clin. Endocrinol. 19: 727– 739. ISCN (1995). ‘‘An International System for Human Cytogenetic Nomenclature’’ (F. Mitelman, Ed.), Krager, Basel. Lazarow, P. B., and Moser, H. W. (1995). Disorders of peroxisome biogenesis. In ‘‘The Metabolic and Molecular Basis of Inherited Disease’’ (C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, Eds.), pp. 2287 –2316. McGraw –Hill, New York. Leenders, F., Tesdorpf, J. G., Markus, M., Engel, T., Seedorf, U., and Adamski, J. (1996). Porcine 80 kDa protein reveals intrinsic 17b-hydroxysteroid dehydrogenase, fatty acyl-CoA-hydratase/dehydrogenase and sterol transfer activities. J. Biol. Chem. 271: 5438 – 5442. Lichter, P., and Cremer, T. (1992). Chromosome analysis by non-isotopic in situ hybridization. In ‘‘Human Cytogenetics’’ (D. E. Rooney and B. H. Czepulkowski, Eds.), Vol. I, pp. 157– 192. IRL, Oxford/New York/Tokyo. Mitelman, F. (1991). ‘‘Catalog of Chromosome Aberrations in Cancer.’’ Wiley – Liss, New York. Nishisho, I., Nakamura, Y., Miyoshi, Y., Miki, Y., Ando, H., Horii, A., Koyama, K., Utsunomiya, J., Baba, S., and Hedge, P. (1991). Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients. Science 253: 665– 669. Ried, T., Lengauer, C., Cremer, T., Wiegant, J., Raap, A. K., van der Ploeg, M., Groitl, P., and Lipp, M. (1992). Specific metaphase and interphase detection of the breakpoint region in 8q24 of Burkitt lymphoma cells by triple-color fluorescence in situ hybridization. Genes Chromosomes Cancer 4: 69– 74. Thomson, A. M., Morris, R. G., Wallace, M., Wyllie, A. H., Steel, C. M., and Carter, D. C. (1993). Allele loss from 5q21 (APC/ MCC) and 18q21 (DCC) and DCC mRNA expression in breast cancer. Br. J. Cancer 68: 61 –68.
Assignment of Human Inhibitor of Apoptosis Protein (IAP) Genes xiap, hiap-1, and hiap-2 to Chromosomes Xq25 and 11q22 –q23 by Fluorescence in Situ Hybridization
ACKNOWLEDGMENTS We are thankful to Prof. Dr. B. Horsthemke, Institut fu¨r Humangenetik, Univerista¨tsklinikum, Essen, Germany, for his help and discussions. Part of this work was supported by DFG Grant 127/11 to J.A., and by ‘‘Institut Pasteur de Lille’’ (France) and CNRS (France).
Evica Rajcan-Separovic,* Peter Liston,*,† Charles Lefebvre,*,† and Robert G. Korneluk* ,†,1 *Department of Genetics, Children’s Hospital of Eastern Ontario, Ottawa, Ontario K1H 8L1, Canada; and †ApoptoGen, Inc., University of Ottawa, Ontario, K1H 8L1, Canada Received April 24, 1996; accepted July 16, 1996
REFERENCES 1. Adamski, J., Normand, T., Leenders, F., Monte, D., Begue, A., Stehelin, D., Jungblut, P. W., and de Launoit, Y. (1995). Molecular cloning of a novel videly expressed human 80 kDa 17bhydroxysteroid dehydrogenase IV. Biochem. J. 311: 437–443. 2. Andersson, S. (1995). 17b-hydroxysteroid dehydrogenases: Isozymes and mutations. J. Endocrinol. 146: 197–200. 3. Bonney, R. C., Reed, M. J., Davidson, K., Beranek, P. A., and James, V. H. T. (1983). The relationship between 17b-hydroxysteroid dehydrogenase activity and oestrogen concentrations in
xiap, hiap-1, and hiap-2 are members of a human multigene family that shows extensive homology to baculovirus IAPs (inhibitor of apoptosis proteins) and that encodes 1 To whom correspondence should be addressed at Children’s Hospital of Eastern Ontario, Solange Gautier Karsh Molecular Genetics, Research Laboratory, Ottawa, Ontario K1H 8L1. Telephone: (613) 738-3281. Fax: (613) 738-4833. E-mail: [email protected].
GENOMICS
37, 404–406 (1996) ARTICLE NO. 0579
0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AID Genom BREP
/
6r20$$381x
10-04-96 16:39:42
gnmbr
AP: Genomics
The redox reactions at position C17 of the steroid molecule are a key step in the synthesis and inactivation of androgens and estrogens. Until now, four different human 17b-hydroxysteroid dehydrogenases (17b-HSD) have been characterized (2). We have identified a cDNA of human (gene name HSD17B4) 17b-HSD IV that inactivates hormones very efficiently (1). A 3.0-kb mRNA codes for a peroxisomal 80-kDa (736 amino acids) protein featuring domains that are not present in the other 17b-hydroxysteroid dehydrogenases. Its N-terminal domain (amino acids 1 – 323) is able to perform the dehydrogenase reaction not only with steroids at the C17 position but also with 3-hydroxyacyl-CoA. The central part (amino acids 324 – 596) catalyzes with high efficiency one of the steps in the peroxisomal b-oxidation of fatty acids: the 2-enoyl-acyl-CoA hydratase reaction. The C-terminal domain (amino acids 597– 736) is similar to sterol carrier protein 2 and facilitates the transfer of 7-dehydrocholesterol and phosphatidylcholine between membranes in vitro (6). The unique multidomain structure of the 80-kDa protein allows for the catalysis of several reactions thus far thought to be performed by complexes of different enzymes. Steroid inactivation in breast cancer tissues and cell lines has been reported to be decreased (3). Deficiencies in peroxisomal enzymes are associated with severe disorders of neuronal and renal development such as Zellweger syndrome and adrenoleukodystrophy (5). Zellweger syndrome patients reveal deficits in steroid and bile acid metabolism assigning peroxisomes to steroidogenesis. To facilitate the study of the gene’s involvement in human disease states, we have mapped the gene of 17b-HSD IV. Human genomic Lambda Dash library was screened with fragments of human cDNA (nucleotides 1029 – 1787 corresponding to the central domain and 1788 – 2211 correspond1
These authors contributed equally to this work. To whom correspondence should be addressed at present address: GSF-Forschungszentrum fu¨r Umwelt und Gesundheit, Institut fu¨r Sa¨ugetieregenetik, Neuherberg, Postfach 1129, D-85758 Oberschleißheim, Germany. Telephone: /49-89-3187-4110. Fax: /49-893187-3099. E-mail: [email protected]. 2
ing to the C-terminal domain) of the 17b-HSD IV (1). Two independent phages, FL6 and FL1 (identified with the central and C-terminal probes, respectively), with inserts of about 14 kb showing different patterns after digestion with XbaI and EcoRI were used for in situ hybridization. After the digestion of the phages FL6 and FL1 with EcoRI, the fragments that hybridized on Southern blot with a 17b-HSD IV cDNA 32 P-labeled probe (3 and 7 kb, respectively) were subcloned in pGem-3Zf and pBluescript vectors to confirm sequence identity with the 17b-HSD IV cDNA. The phage FL6 contains DNA coding for at least one exon corresponding to amino acids 560 – 590 (numbering according to Ref. 1). The FL1 phage codes for at least two exons. The first contains amino acids 665 – 707, and the second begins at amino acid 707 and includes the stop codon and the 3 *-nontranslated region. Verified FL6 and FL1 phages were labeled by standard nick-translation with biotin-16 –dUTP or digoxigenin-11– dUTP (Boehringer Mannheim), respectively. Slides with normal lymphocyte metaphases were prepared by standard protocols from PHA-stimulated lymphocyte cultures. Slides were pretreated with RNase and pepsin and fixed with 1% formalin as described (10). Hybridization and detection were performed as described (7). Detection of the biotin labeling was performed with fluorescein isothiocyanate (FITC) conjugated to avidin DCS (Vector Laboratories) and detection of digoxigenin was performed with anti-digoxigenin rhodamine-conjugated antibodies from sheep (Boehringer Mannheim). For signal amplification biotinylated anti-avidin (Vector Laboratories) and rhodamine-conjugated anti-sheep IgG (Jackson Immuno Research Laboratories) were used. Slides were counterstained with 4*,6-diamidino-2-phenylindole (DAPI) to enable the identification of the chromosomes by a G-bandinglike pattern and were mounted with antifade solution (Vector Laboratories). Digital gray-scale images of each fluorochrome were obtained separately with a cooled charge-coupled device camera (Photometrics, Tucson, AZ). After inversion of the gray-scale pattern of the DAPI image, the images were combined using the Gene Join software package (Yale University). The signals of both clones mapped overlapping in the same region on chromosome 5q2 (Fig. 1), as could be seen by the banding pattern. This region was confirmed by calculating the positions of the signals on 12 chromatids in relation to the whole chromosome length and comparing them with the idiogram (4) of chromosome 5. The chromosome designation was confirmed (not shown) by an additional cohybridization of the biotin-labeled clone FL1 and digoxigenin-labeled chromosome 5 library DNA from a mapping panel (mapping panel 2, consisting of DNA from human/rodent somatic cell hybrids, Repository No. NA 10114, Coriell Institute, Camden, NJ). The signal frequency for both clones was examined on 25 metaphases. For clone FL1, 20 cells showed signals on all four chromatids, 2 cells showed signals on three chromatids, and 3 cells showed signals on two chromatids. For clone FL6, 12 cells showed signals on four chromatids, 4 cells showed signals on three chromatids, 7 cells showed signals on two chromatids, one cell showed signal on one chromatid, and one methaphase showed no signal. Hybridization efficiency GENOMICS
403
37, 403–404 (1996) ARTICLE NO. 0578
0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AID Genom BREP
/
6r20$$381x
10-04-96 16:39:42
gnmbr
AP: Genomics
404
BRIEF REPORTS
4. 5.
6.
7.
8. 9.
10. FIG. 1. Localization of the human 17b-HSD IV gene. Digital image of clone FL1 (digoxigenin-labeled) hybridized on human chromosomes. Specific signals are seen at chromosome 5q2 (arrowheads). 11.
was independent of the type of label as checked by exchanging the labels of both clones (not shown). The efficient inactivation of steroids by 17b-HSD IV could be distorted by mutations in the HSD17B4 gene. The increased intracellular levels of estrogens could then result in the development of estrogen-sensitive tumors. Indeed, studies for loss of heterozygosity at tumor suppressor loci involved in sporadic breast cancer and in colorectal cancer (9, 11) revealed allele loss at 5q21. In addition to colorectal and breast cancer genes, other important genes involved in carcinogenesis, such as those participating in leukemogenesis, are associated with the long arm of chromosome 5q (8).
human breast tumours and in normal breast tissue. Clin. Endocrinol. 19: 727– 739. ISCN (1995). ‘‘An International System for Human Cytogenetic Nomenclature’’ (F. Mitelman, Ed.), Krager, Basel. Lazarow, P. B., and Moser, H. W. (1995). Disorders of peroxisome biogenesis. In ‘‘The Metabolic and Molecular Basis of Inherited Disease’’ (C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, Eds.), pp. 2287 –2316. McGraw –Hill, New York. Leenders, F., Tesdorpf, J. G., Markus, M., Engel, T., Seedorf, U., and Adamski, J. (1996). Porcine 80 kDa protein reveals intrinsic 17b-hydroxysteroid dehydrogenase, fatty acyl-CoA-hydratase/dehydrogenase and sterol transfer activities. J. Biol. Chem. 271: 5438 – 5442. Lichter, P., and Cremer, T. (1992). Chromosome analysis by non-isotopic in situ hybridization. In ‘‘Human Cytogenetics’’ (D. E. Rooney and B. H. Czepulkowski, Eds.), Vol. I, pp. 157– 192. IRL, Oxford/New York/Tokyo. Mitelman, F. (1991). ‘‘Catalog of Chromosome Aberrations in Cancer.’’ Wiley – Liss, New York. Nishisho, I., Nakamura, Y., Miyoshi, Y., Miki, Y., Ando, H., Horii, A., Koyama, K., Utsunomiya, J., Baba, S., and Hedge, P. (1991). Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients. Science 253: 665– 669. Ried, T., Lengauer, C., Cremer, T., Wiegant, J., Raap, A. K., van der Ploeg, M., Groitl, P., and Lipp, M. (1992). Specific metaphase and interphase detection of the breakpoint region in 8q24 of Burkitt lymphoma cells by triple-color fluorescence in situ hybridization. Genes Chromosomes Cancer 4: 69– 74. Thomson, A. M., Morris, R. G., Wallace, M., Wyllie, A. H., Steel, C. M., and Carter, D. C. (1993). Allele loss from 5q21 (APC/ MCC) and 18q21 (DCC) and DCC mRNA expression in breast cancer. Br. J. Cancer 68: 61 –68.
Assignment of Human Inhibitor of Apoptosis Protein (IAP) Genes xiap, hiap-1, and hiap-2 to Chromosomes Xq25 and 11q22 –q23 by Fluorescence in Situ Hybridization
ACKNOWLEDGMENTS We are thankful to Prof. Dr. B. Horsthemke, Institut fu¨r Humangenetik, Univerista¨tsklinikum, Essen, Germany, for his help and discussions. Part of this work was supported by DFG Grant 127/11 to J.A., and by ‘‘Institut Pasteur de Lille’’ (France) and CNRS (France).
Evica Rajcan-Separovic,* Peter Liston,*,† Charles Lefebvre,*,† and Robert G. Korneluk* ,†,1 *Department of Genetics, Children’s Hospital of Eastern Ontario, Ottawa, Ontario K1H 8L1, Canada; and †ApoptoGen, Inc., University of Ottawa, Ontario, K1H 8L1, Canada Received April 24, 1996; accepted July 16, 1996
REFERENCES 1. Adamski, J., Normand, T., Leenders, F., Monte, D., Begue, A., Stehelin, D., Jungblut, P. W., and de Launoit, Y. (1995). Molecular cloning of a novel videly expressed human 80 kDa 17bhydroxysteroid dehydrogenase IV. Biochem. J. 311: 437–443. 2. Andersson, S. (1995). 17b-hydroxysteroid dehydrogenases: Isozymes and mutations. J. Endocrinol. 146: 197–200. 3. Bonney, R. C., Reed, M. J., Davidson, K., Beranek, P. A., and James, V. H. T. (1983). The relationship between 17b-hydroxysteroid dehydrogenase activity and oestrogen concentrations in
xiap, hiap-1, and hiap-2 are members of a human multigene family that shows extensive homology to baculovirus IAPs (inhibitor of apoptosis proteins) and that encodes 1 To whom correspondence should be addressed at Children’s Hospital of Eastern Ontario, Solange Gautier Karsh Molecular Genetics, Research Laboratory, Ottawa, Ontario K1H 8L1. Telephone: (613) 738-3281. Fax: (613) 738-4833. E-mail: [email protected].
GENOMICS
37, 404–406 (1996) ARTICLE NO. 0579
0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AID Genom BREP
/
6r20$$381x
10-04-96 16:39:42
gnmbr
AP: Genomics