© 1992 S. Kargcr AG. Basel 0301-0171/92/0613-017852.75/0
Cvtogenet Cell Genet 61:178-179 ( 1992)
Regional localization of the gene for clusterin (SP-40,40; gene symbol CLI to human chromosome 8pl2—>p21 E. D ietzsc h ,1 B.F. M u rp h y ,2 L. K irszb au m ,3 1.D. W alker,3 and O .M . G a rso n 4 1University of Melbourne Department of Medicine, St. Vincent's Hospital. Fitzroy, Victoria. 2University of Melbourne Department of Medicine and Renal Unit. St. Vincent's Hospital. Fitzroy. Victoria,3University of Melbourne Department of Veterinary Sciences. Parkville. Victoria, and 4 Department of Cytogenetics. St. Vincent's Hospital. Fitzroy, Victoria (Australia)
Abstract. The human clusterin (SP-40,40) gene, designated CLI (complement lysis inhibitor) by the Human Gene Nomen clature Committee, has previously been assigned to chromo
some 8. In situ hybridization allowed us to map the locus at 8p12—>p 2 1.
Clusterin is an apparently multifunctional glycoprotein present in the blood and semen of many species (O'Bryan et al., 1990). Human clusterin (SP-40.40) has been demonstrated to be a regulator of the terminal complement pathway (Murphy et al., 1989). associated with lipoproteins in blood plasma (de Silva et al., 1990), upregulated in the brains of patients with Alzheimer disease (Duguid et al., 1989), and present in very high concen trations in seminal plasma (O'Bryan et al., 1990). In this study, using a somatic cell hybrid mapping panel, we have confirmed the chromosomal location of SP-40,40 (Adams et al., 1991; Purrello et al.. 1991; Tobe et al., 1991). Further more. by means of in situ hybridization, we have regionally mapped this gene to 8pl2-»p21.
Southern transfer, and hybridization were performed as described in Dietzsch et al. (1991). Final washes were in 1 x SSC. 0.5 % SDS at 65 ° C. In situ hybridization. Human metaphase chromosome spreads were pre pared from PHA-stimulatcd peripheral blood lymphocyte cultures synchro nized with BrdU (Zabel et al.. 1983). The probe was labeled by random priming (Boehringer) using [3H]dATP. [3H]dCTP, and [3H]dTTP (Amersham) to a specific activity of 2.25 x 108 cpm/pg DNA. The probe was added to the hybridization solution containing 50% forntamide, 10% (wt/vol) dextran sulphate, 5 x SSPE (pH 7.4), SDS to a final concentration of 33 pg/ml, and salmon sperm DNA at a concentration 500 times that of the probe. The technique of in situ hybridization was a modification of the procedures of Harper and Saunders (1981) and Choo et al. (1990). RNase treatment of the chromosome preparations and denaturation of the chromosomal and probe DNAs were as described. Probe mixes of 5. I, and 0.3 ng DNA per slide were hybridized to metaphase chromosomes overnight at 42 °C. The chro mosome preparations were washed at 44 °C in four changes each of 50% formamide in 2 x SSC and 2 x SSC (all pH 7.0). followed by dehydrating in ethanol. The slides were dipped in RPN 41 emulsion (Amersham) diluted 1:1 with distilled HyO and exposed for 5-43 d at 4°C . Autoradiographs were developed for 5.5 min in Kodak D -19 (diluted 1:1 with distilled HjO). fixed (Agfa), transferred to 10% NaySOj for 25 min. and rinsed under run ning tap HjO. Chromosomes were replication G-bandcd according to Choo et al. (1990).
Probe. An PcoRl cDNA fragment encompassing nucleotides 206-869 from the sequence of the clone LK.107 (Kirszbaum et al.. 1989) was isolated from the recombinant plasmid pUCI 8 by agarose gel electrophoresis. Southern analysis. DMA isolated from a panel of 18 human x rodent somatic cell hybrid lines was obtained from thcCoriell Institute. Camden. NJ. The DNA was digested with £coRI (Boehringer). Agarose gel electrophoresis,
E.D. and O.M.G. were in receipt of a gram from the Anti-Cancer Council of Victoria. O M .G . and B.F.M. were members of the stall'of St. Vincent's Hospital. Received 16 March 1992: accepted 5 May 1992.
Reprint requests from Mrs. Erin Dietzsch. Department of Cytogenetics. St. Vincent's Hospital. 41 Victoria Parade. Fitzroy. Victoria 3065 (Australia).
Downloaded by: Nagoya University 133.6.82.173 - 1/11/2019 10:36:51 PM
Fig. 1. (a) Diagram show ing the grain distribution on chro mosome 8 in 43 metaphases, (b) Partial metaphase showing a grain located at the site of hy bridization on replication Gbanded chromosomes.
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SP-40.40 localized to chromosome 8p 12 —»p21
Adams MD. Kelley JM. Gocayne JD. Dubniek M. Polymeropoulos MH, Xiao H. Merril CR. Wu A. Olde B. Moreno R. Kerlavage AR, McCombie WR. Venter JC: Complementary DNA sequencing: ex pressed sequence tags and human genome project. Science 252:1651-1656 (1991). Choo KH. Brown RM. Earle E: In situ hybridization of chromosomes, in Matthew CG (ed): Protocols in Human Molecular Genetics. Vol 7. pp 233-254 (Humana Press, New Jersey. 1991). de Silva HV. Stuart WD. Park YB. Mao SJT, Gil CM. Wettcrau JR. Busch SJ. Harmony JAK: Purifica tion and characterization of apolipoprotein J. J biol Chem 265:14292-14297 (1990). Dictzsch E. Hong J. Leslie DE. Martin L, Rolland J. Benson E. Toh BH. McCluskey J: T cell receptor and immunoglobulin gene rearrangement analysis as a laboratory aid in the diagnosis ofhum an malig nant Ivmphoproliferative diseases. Aust NZ J Med 21:307-313(1991).
containing a labeled chromosome 8. This study assigned the SP40.40 gene to 8p 12—>p21. A diagrammatic presentation of the distribution of grains observed in 43 metaphases and a photo graph of a silver grain located at the specific site of hybridization are shown in Fig. 1. Wc thank Michelle Glew for technical assistance and Miss .loan Osbourne for typing the manuscript.
Duguid JR. Bohmont CW. Liu N. Tortelotte WW: Changes in brain expression shared by scrapie and Alzheimer disease. Proc natl Acad Sei. USA 86: 7260-7264(1989). Harper ME, Saunders BF: Localization of single copy DNA sequences on human G-banded human chro mosomes by in situ hybridization. Chromosoma 83:431-439(1981). Kirszbaum L, Sharpe J. Murphy BF. d'Apicc AJF. Classon B. Hudson P. Walker ID: Molecular cloningand characterization of the novel, human complementassociated protein SP-40.40: a link between the complement and reproductive systems. EMBO J 8: 711-718(1989). Murphy BF. Saunders JR. O'Bryan MK. Kirszbaum L, Walker ID. d'Apice AJF: SP-40.40 is an inhibitor of C5b-6 initiated hemolvsis. Int Immunol 1:551-554 (1989). O’Bry an MK. Baker HWG. Saunders JR. Kirszbaum L. Walker ID. Hudson P. Liu DY. d'Apice AJF. Mur
phy BF: Human seminal clusterin (SP-40.40): isola tion and characterization. J clin Invest 85:14771486(1990). Purrcllo M. Bettuzzi S. Di Pietro C. Mirabile E. Di Blasi M. Rimini R. Grzeschik H-H. Ingletti C. Corti A, Sichcl G: The gene for SP-40,40. human homolog of rat sulfatcd glycoprotein 2. rat clusterin. and rat tes tosterone-repressed prostate message 2. maps to chromosome 8. Genomics 10:15 1- 156 ( 1991 ). Tobe T. Minoshimo S. Yamase S. Choi N-H. Tomita M, Shimizu N. Assignment of a human serum glyco protein SP-40,40 gene (CLI) to chromosome 8. Cytogenel Cell Genet 5 7:19 3 - 195 ( 1991 ). Zabel HU. Naylor SL. Sakaguchi AY. Bell GI. Shows TB: High-resolution chromosomal localization of human genes for amylase, proopiomelanocortin, somatostatin and a DNA fragment (D3SI ) by in situ hybridization. Proc natl Acad Sci, USA 80:69326936(1983).
Downloaded by: Nagoya University 133.6.82.173 - 1/11/2019 10:36:51 PM
Southern analysis showed that the human probe crosshybridizes with the mouse and Chinese hamster genes, produc ing species-specific DNA fragments that differ in size. Presence tr absence of the human DNA fragments were 100% concor dant with the presence or absence of human chromosome 8 in the hybrids studied. G-banded chromosomes were visualized down the micro scope and grain locations were recorded in metaphase spreads
Cvtogenet Cell Genet 61:178-179 ( 1992)
Regional localization of the gene for clusterin (SP-40,40; gene symbol CLI to human chromosome 8pl2—>p21 E. D ietzsc h ,1 B.F. M u rp h y ,2 L. K irszb au m ,3 1.D. W alker,3 and O .M . G a rso n 4 1University of Melbourne Department of Medicine, St. Vincent's Hospital. Fitzroy, Victoria. 2University of Melbourne Department of Medicine and Renal Unit. St. Vincent's Hospital. Fitzroy. Victoria,3University of Melbourne Department of Veterinary Sciences. Parkville. Victoria, and 4 Department of Cytogenetics. St. Vincent's Hospital. Fitzroy, Victoria (Australia)
Abstract. The human clusterin (SP-40,40) gene, designated CLI (complement lysis inhibitor) by the Human Gene Nomen clature Committee, has previously been assigned to chromo
some 8. In situ hybridization allowed us to map the locus at 8p12—>p 2 1.
Clusterin is an apparently multifunctional glycoprotein present in the blood and semen of many species (O'Bryan et al., 1990). Human clusterin (SP-40.40) has been demonstrated to be a regulator of the terminal complement pathway (Murphy et al., 1989). associated with lipoproteins in blood plasma (de Silva et al., 1990), upregulated in the brains of patients with Alzheimer disease (Duguid et al., 1989), and present in very high concen trations in seminal plasma (O'Bryan et al., 1990). In this study, using a somatic cell hybrid mapping panel, we have confirmed the chromosomal location of SP-40,40 (Adams et al., 1991; Purrello et al.. 1991; Tobe et al., 1991). Further more. by means of in situ hybridization, we have regionally mapped this gene to 8pl2-»p21.
Southern transfer, and hybridization were performed as described in Dietzsch et al. (1991). Final washes were in 1 x SSC. 0.5 % SDS at 65 ° C. In situ hybridization. Human metaphase chromosome spreads were pre pared from PHA-stimulatcd peripheral blood lymphocyte cultures synchro nized with BrdU (Zabel et al.. 1983). The probe was labeled by random priming (Boehringer) using [3H]dATP. [3H]dCTP, and [3H]dTTP (Amersham) to a specific activity of 2.25 x 108 cpm/pg DNA. The probe was added to the hybridization solution containing 50% forntamide, 10% (wt/vol) dextran sulphate, 5 x SSPE (pH 7.4), SDS to a final concentration of 33 pg/ml, and salmon sperm DNA at a concentration 500 times that of the probe. The technique of in situ hybridization was a modification of the procedures of Harper and Saunders (1981) and Choo et al. (1990). RNase treatment of the chromosome preparations and denaturation of the chromosomal and probe DNAs were as described. Probe mixes of 5. I, and 0.3 ng DNA per slide were hybridized to metaphase chromosomes overnight at 42 °C. The chro mosome preparations were washed at 44 °C in four changes each of 50% formamide in 2 x SSC and 2 x SSC (all pH 7.0). followed by dehydrating in ethanol. The slides were dipped in RPN 41 emulsion (Amersham) diluted 1:1 with distilled HyO and exposed for 5-43 d at 4°C . Autoradiographs were developed for 5.5 min in Kodak D -19 (diluted 1:1 with distilled HjO). fixed (Agfa), transferred to 10% NaySOj for 25 min. and rinsed under run ning tap HjO. Chromosomes were replication G-bandcd according to Choo et al. (1990).
Probe. An PcoRl cDNA fragment encompassing nucleotides 206-869 from the sequence of the clone LK.107 (Kirszbaum et al.. 1989) was isolated from the recombinant plasmid pUCI 8 by agarose gel electrophoresis. Southern analysis. DMA isolated from a panel of 18 human x rodent somatic cell hybrid lines was obtained from thcCoriell Institute. Camden. NJ. The DNA was digested with £coRI (Boehringer). Agarose gel electrophoresis,
E.D. and O.M.G. were in receipt of a gram from the Anti-Cancer Council of Victoria. O M .G . and B.F.M. were members of the stall'of St. Vincent's Hospital. Received 16 March 1992: accepted 5 May 1992.
Reprint requests from Mrs. Erin Dietzsch. Department of Cytogenetics. St. Vincent's Hospital. 41 Victoria Parade. Fitzroy. Victoria 3065 (Australia).
Downloaded by: Nagoya University 133.6.82.173 - 1/11/2019 10:36:51 PM
Fig. 1. (a) Diagram show ing the grain distribution on chro mosome 8 in 43 metaphases, (b) Partial metaphase showing a grain located at the site of hy bridization on replication Gbanded chromosomes.
179
SP-40.40 localized to chromosome 8p 12 —»p21
Adams MD. Kelley JM. Gocayne JD. Dubniek M. Polymeropoulos MH, Xiao H. Merril CR. Wu A. Olde B. Moreno R. Kerlavage AR, McCombie WR. Venter JC: Complementary DNA sequencing: ex pressed sequence tags and human genome project. Science 252:1651-1656 (1991). Choo KH. Brown RM. Earle E: In situ hybridization of chromosomes, in Matthew CG (ed): Protocols in Human Molecular Genetics. Vol 7. pp 233-254 (Humana Press, New Jersey. 1991). de Silva HV. Stuart WD. Park YB. Mao SJT, Gil CM. Wettcrau JR. Busch SJ. Harmony JAK: Purifica tion and characterization of apolipoprotein J. J biol Chem 265:14292-14297 (1990). Dictzsch E. Hong J. Leslie DE. Martin L, Rolland J. Benson E. Toh BH. McCluskey J: T cell receptor and immunoglobulin gene rearrangement analysis as a laboratory aid in the diagnosis ofhum an malig nant Ivmphoproliferative diseases. Aust NZ J Med 21:307-313(1991).
containing a labeled chromosome 8. This study assigned the SP40.40 gene to 8p 12—>p21. A diagrammatic presentation of the distribution of grains observed in 43 metaphases and a photo graph of a silver grain located at the specific site of hybridization are shown in Fig. 1. Wc thank Michelle Glew for technical assistance and Miss .loan Osbourne for typing the manuscript.
Duguid JR. Bohmont CW. Liu N. Tortelotte WW: Changes in brain expression shared by scrapie and Alzheimer disease. Proc natl Acad Sei. USA 86: 7260-7264(1989). Harper ME, Saunders BF: Localization of single copy DNA sequences on human G-banded human chro mosomes by in situ hybridization. Chromosoma 83:431-439(1981). Kirszbaum L, Sharpe J. Murphy BF. d'Apicc AJF. Classon B. Hudson P. Walker ID: Molecular cloningand characterization of the novel, human complementassociated protein SP-40.40: a link between the complement and reproductive systems. EMBO J 8: 711-718(1989). Murphy BF. Saunders JR. O'Bryan MK. Kirszbaum L, Walker ID. d'Apice AJF: SP-40.40 is an inhibitor of C5b-6 initiated hemolvsis. Int Immunol 1:551-554 (1989). O’Bry an MK. Baker HWG. Saunders JR. Kirszbaum L. Walker ID. Hudson P. Liu DY. d'Apice AJF. Mur
phy BF: Human seminal clusterin (SP-40.40): isola tion and characterization. J clin Invest 85:14771486(1990). Purrcllo M. Bettuzzi S. Di Pietro C. Mirabile E. Di Blasi M. Rimini R. Grzeschik H-H. Ingletti C. Corti A, Sichcl G: The gene for SP-40,40. human homolog of rat sulfatcd glycoprotein 2. rat clusterin. and rat tes tosterone-repressed prostate message 2. maps to chromosome 8. Genomics 10:15 1- 156 ( 1991 ). Tobe T. Minoshimo S. Yamase S. Choi N-H. Tomita M, Shimizu N. Assignment of a human serum glyco protein SP-40,40 gene (CLI) to chromosome 8. Cytogenel Cell Genet 5 7:19 3 - 195 ( 1991 ). Zabel HU. Naylor SL. Sakaguchi AY. Bell GI. Shows TB: High-resolution chromosomal localization of human genes for amylase, proopiomelanocortin, somatostatin and a DNA fragment (D3SI ) by in situ hybridization. Proc natl Acad Sci, USA 80:69326936(1983).
Downloaded by: Nagoya University 133.6.82.173 - 1/11/2019 10:36:51 PM
Southern analysis showed that the human probe crosshybridizes with the mouse and Chinese hamster genes, produc ing species-specific DNA fragments that differ in size. Presence tr absence of the human DNA fragments were 100% concor dant with the presence or absence of human chromosome 8 in the hybrids studied. G-banded chromosomes were visualized down the micro scope and grain locations were recorded in metaphase spreads
