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Title Author(s) CHROMOSOMAL ASSIGNMENT OF HUMAN GENES FOR GASTRIN, THYROTROPIN (TSH)-β SUBUNIT AND C-erbB-2 BY CHROMOSOME SORTING COMBINED WITH VELOCITY SEDIMENTATION AND SOUTHERN HYBRIDIZATION Fukushige, Shinichi Citation Issue Date Text Version ETD URL http://hdl.handle.net/11094/36004 DOI Rights Osaka University Osaka University BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 477-483 Vol. 134, No. 2,1986 January 29,1986 CHROMOSOMAL ASSIGNMENT OF HUMAN GENES FOR GASTRIN, THYROTROPIN (TSH)-~ SUBUNIT AND C-erbB-2 BY CHROMOSOME SORTING COMBINED WITH VELOCITY SEDIMENTATION AND SOUTHERNHYBRIDIZATION Shinichi Fukushige, Tomoaki Murotsu and Kenichi Matsubara I n s t i t u t e for Molecular and C e l l u l a r Biology, Osaka U n i v e r s i t y , Yamada-oka, Suita 565, Japan Received November 15, 1985 Human genes for g a s t r i n , thyrotropin (THS)-B subunit and c-erbB-2 were assigned to s p e c i f i c chromosomes using a s i n g l e - l a s e r cell s o r t e r . For this purpose, condensed human chromosomes prepared from a k a r y o t y p i c a l l y normal lymphoblastoid cell l i n e were p r e l i m i n a r i l y fractionated by v e l o c i t y sedimentation, and then sorted using a fluorescence-activated cell s o r t e r . DNA was then extracted from the chromosomes, cleaved by r e s t r i c t i o n enzymes, and subjected to Southern hybridization using gene-specific radioactive probes. When the assignment of specific chromosomes was not possible due to chromosomal size overlapping, sorted chromosomes from cell lines carrying chromosomal translocation or from hybrid cells carrying known human chromosomes were used in addition. The results indicate that human genes for gastrin, TSH-~, and c-erbB-2 are located on chromosomes 1 7 , 1 and 17, r e s p e c t i v e l y . ® 1986A e a d e m i e P . . . . . Inc. Chromosomal gene mapping is becoming increasingly important in basic, as well either as applied research in biology and medicine. by in situ hybridization (I) or It is usually done by discordance analysis using somatic hybrid cell panels (2). Both techniques present some drawbacks, requires time to obtain a decisive since conclusion, in situ hybridization and the hybrid cell panel method is subject to inconsistent results mostly due to the u n s t a b i l i t y of human chromosomes. In contrast cell sorter to fractionate to these two techniques, analysis chromosomes (3 7) seems using a to be the most chromosomes can not be powerful method for rapid and correct gene assignments. However, with sorted out sorting into a single-laser individual sorter, components. We have overcome this chromosomes from cell Abbreviations: cell problem by lines that carry translocated chromosomes or TSH, t h y r o t r o p i n ; base pair. NaDodSO4 , sodium dodecylsulfate; bp, 0006-291X/86 $1.50 477 Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved. VOI. 134, No. 2, 1986 from human-rodent Southern blot BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS hybrid analysis cells using the that sorted carry known human chromosomes. chromosomes (3, 4), rather than b l o t t i n g d i r e c t l y the chromosomes (5 - 7) greatly improved the accuracy of the assignments. MATERIALS AND METHODS I. Cell lines and chromosome preparation Two human lymphoblastoid cell lines GMOI31: 46, XX and GM3197: 46, XX, t(17;22) (17qter-17p13::22qll-22qter;22pter-22qll::17p13-17pter) (8) were obtained from "The Human Genetic Mutant Cell Repository (Camden, NJ)". These cell lines were grown in RPMI I~40 medium supplemented with 10% fetal c a l f serum under 5% C09/95% a i r at 37 C. . . Human-mouse hybrid c e l l s TAIA and TA3A (9) were kindly provided by Dr. N. Schimizu at Keio U n i v e r s i t y . TAIA carries human chromosomes I , 4, 7, 13, 15, 18, 19 and TA3A carries human chromosomes 3, 13, 14. These hybrids were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal c a l f serum. The c e l l s were then incubated for 16 hrs in the presence of colcemid (O.2~g/ml). Chromosome suspensions were prepared according to the polyamine method of S i l l a r and Young (I0) with some modifications: The cells containing condensed chromosomes were harvested by c e n t r i f u g a t i o n , resuspended in 75mM KCI, and kept on ice for 30 min. They were then collected by c e n t r i f u g a t i o n for I0 min, washed with polyamine buffer ( I 0 ) , resuspended in the same buffer containing 0.1% d i g i t o n i n , followed by vigorous a g i t a t i o n for 1 min using a Vortex mixer to release chromosomes. 2. Velocity sedimentation and sorting The chromosome suspensions (50ul) from 5 x 105 mitotic c e l l s were layered over a 16 ml l i n e a r sucrose gradient (5 - 30%, W/V) in po~yamine b u f f e r , centrifuged using a swing bucket at 900 rpm for 30 min at 4 C, and then fractionated into sixteen 1 ml a l i q u o t s . To each f r a c t i o n , ethidium bromide (50ug/ml) was added and the stained chromosomes were sorted using a fluorescence-activated cell sorter CHROSS 1 (Japan Spectroscopic Co., LTD.) monitored by the MULTI 16 computer (Mitsubishi E l e c t r i c , Tokyo). For e x c i t a t i o n , a Spectra-Physics model 2025-05 laser at 488 nm/3OOmW was used. The flow rate of sorting was I000 2000 chromosomes/sec. The preliminary f r a c t i o n a t i o n by sucrose gradient sedimentation f a c i l i t a t e d rapid sorting. To obtain m~ningful signals in Southern blot analysis of chromosomal DNA, about 4 x I0 sorted chromosomes were used. 3. DNA extraction he sorted chromosomes were collected by c e n t r i f u g a t i o n at 5 mi . DNA was prepared by incubating the samples for 5 hrs extraction buffer (75mM NaCI, 24mM EDTA, lOmM Tris.HCl, pH 7.5) proteinase K (lOOug/ml), yeast c a r r i e r tRNA (lO~g/ml) and I% followed by phenol extraction and ethanol p r e c i p i t a t i o n ( I I ) . ~ 14 krp~ for at 37UC in containing NaDodSO4 , 4. DNA probes The following probes were used: human g a s t r i n , a 260 bp H i n d l l l H i n d l l l fragment from cDNA (12); human TSH-B, a 210 bp EcoRV-Rsal fragment from genomic DNA (13); human c-er___bbB-2, a 440 bp Kpnl-Xbal fragment from genomic DNA (14). 5. Southern hybridization The DNA from sorted chromosome was digested with EcoRl ( g a s t r i n , TSHB) or Pvull (c-er___bbB-2), placed in 2mm slots in a 0.7% agarose gel and 478 Vol. 1 3 4 , No. 2, 1 9 8 6 B I O C H E M I C A L A N D B I O P H Y S I C A L RESEARCH C O M M U N I C A T I O N S elect~phoresed. B l o t t i n g onto n i t r o c e l l u l o s e f i l t e r s 8and h y b r i d i z a t i o n with P-labeled DNA probe ( s p e c i f i c a c t i v i t y 2 - 5 x lO cpm/ug) were done as described by Southern (15). RESULTS AND DISCUSSION TO sort chromosomes, metaphase chromosomes in a cell which has a normal karyotype (46, XX) lysate of GMOl31 w e r e fractionated by velocity sedimentation through sucrose (7, 16), and each f r a c t i o n was then subjected to flow s o r t i n g . histograms. Fig. l shows r e l a t i o n s between sucrose fractions and By combining sedimentation with flow s o r t i n g , the sorting rate increased by a factor of 5 - lO. 1.0 Eight chromosome fractions (A to H: ii ~,i i i i~4 i i i6i 18-16 i i i i i ilo) see >z 0.5 S o o m (J o . c 10 5 Fraction :~ :: i12-9 i 15 Number i ! i i : . i ~ 8 - ! 6 1 $ ) ........... ) ........i I'~ <aX: 7,~. . . .....i i + 12-9 i i :: i i ! 71 J122~ i .......i........ . . . . . . . . . ..............i :: 15-13! 8:;J ........i -~ :: :: L ) 8i :: ..... i........ i..... i ....... i..._~,_.L i i i i ! i i i [ ! i ! :3 s i ................. i ........ ¸ Figure I . Sedimentation profile (Panel A) and flow histograms of human chromosomes prepared from the cell line GMOl31. Chromosomes were preliminarily separated into 7 fractions (5 to l l ) by velocity sedimentation through sucrose. Each fraction was mixed with ethidium bromide and analysed by a fluorescence-activated cell sorter, ordinate: relative number of chromosomes; abscissa: relative fluorescence intensity. Panel B is the histogram from an unfractionated sample. Panels 5 - II show histograms of the corresponding sucrose fractions. Numbers in the histograms represent the positions of human chromosomes. For example, sucrose fraction 5 is enriched with chromosomes l , 2 and 3 to 5. 479 Vol. 134, No. 2, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS GM0131 A B I ! H G i 12-9 F E D C B A T ORIGIN 23Kb 2, ,~-i~ 9.4 ~i ..... ::..... 6.5 4.3 H'-I't'I °° I" " GM3197 D C h g f • d ¢ b • -.4 t (17;22 t(17;22) "1' '1 Figure 2. Assignment of human gastrin gene to chromosome 17. (A) Flow histogram of normal human chromosomes from the cell line GMOI31. Numbers in the figure represent the positions of human chromosomes. Fractions A to H were separately obtained as described in the text. (B) DNA was prepared from the sorted chrom~.omes, digested by EcoRI, and subjected to Southern blot analysis using P-labeled human gastrin gene (12) as a probe. Lane T: total human lymphocyte DNA. Lanes A to H; DNA from each sorted chromosome fraction. The arrow indicates the position of the DNA fragment hybridizing to the probe. Positions of marker DNAs using Hindlll-digested phage DNA are shown on the l e f t . The gastrin gene is in fraction F that contains chromosomes 16, 17, 18. (C) Flow histogram of chromosomes from a human cell l i n e GM3197 that carries a reciprocal translocation t(17;22). Chromosomes were sorted into fractions a to h as shown at the bottom of the histogram. The arrow head indicates the position of the t(17;22)(17qter17p13::22qll-22qter) chromosome. (D) DNA was prepared, cleaved with EcoRl, and subjected to Southern blot analysis as in (B). The gastrin gene is in fraction e that contains chromosomes 13, 14, 15, t(17;22) as well as in fraction f that contains chromosomes 16, 17, 18. Therefore, chromosome 17 should carry the gastrin gene. Fig. 2A) were obtained in a two-day o p e r a t i o n and were used in one f i l t e r assay. DNA was e x t r a c t e d appropriate restriction h y b r i d i z a t i o n which gives from each enzymes, chromosome f r a c t i o n , and then unambiguous s i g n a l s , 480 analysed digested by as compared to with Southern spot blot Vol. 134, No. 2, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS analysis (5 - 7). The f i l t e r was hybridized to nick-translated human genes including g a s t r i n , TSH-B or c-erbB-2. The gastrin probe showed a positive signal in fraction corresponds to the pool of chromosomes 16, 17 and 18 (Fig. next step, similarly. chromosomes from This cell translocated another cell carries a reciprocal line, translocation fractions e and f demonstrate that the human gastrin the same way, a c e l l u l a r (Fig. In the sorted t(17;22) is f r a c t i o n e that contains chromosomes 13, 14 and 15 (Fig. in 2B). GM3197 were chromosome, (17qter-17p13::22qll-22qter) DNA showed signals F that and the collected 2C). in Analysis of 2D). These results clearly gene is located on chromosome 17. In oncogene, c-erbB-2 was mapped on chromosome 17 (data not shown). The human TSH-~ gene probe hybridized to fraction A containing sorted chromosomes from GMOI31 c e l l s . In the next step, This f r a c t i o n contains chromosomes 1 and 2. we used a contains human chromosome I , human-rodent but not 2. human chromosomes 1 and 2 (see Southern blot detected hybrid in cell analysis the (Fig. 3B). corresponding TA3A that Fig. does hybrid The fraction 3A) In this translocated case, positive contain that corresponds to obtained were detected line by signal was another 1 or 2. GM3876 that carries supported in signal from human chromosomes chromosomes t ( l ; 2 0 ) signals TAIA that no positive chromosome f r a c t i o n Independent studies on chromosomes from the cell reciprocally line showed a positive In contrast, not cell this conclusion. two f r a c t i o n s , one that contains normal human chromosomes 1 and 2, and another that contains the translocated shown). chromosomes (data not These results indicate that human TSH-B gene is located on chromosome I . At present, hybrid c e l l s . with a panel sorting most of In p r i n c i p l e , of hybrid the gene mapping studies are performed using chromosome sorting is unnecessary when working cells. However, technique is more powerful we found that the since in many hybrid cell chromosome lines, human chromosomes are unstable, r e s u l t i n g in frequent loss of the o n c e - i d e n t i f i e d 481 Vol. 134, No. 2, 1 9 8 6 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS T T A A 3 o~ .Q A E 0 R I G I N -- 1 A M T H G F E D C B A ~,~~mF:~ > 6 : 5 -- 4.3~ ~ E o 2.3-2.0-- Relative Fluorescence Figure 3. Assignment of human TSH-B gene to chromosome I . (A) Flow histogram of chromosomes from a human-mouse hybrid cell TAIA that carries human chromosome l , but not 2. TAIA carries, in addition, human chromosomes 4, 7, 13, 15, 18, 19. The chromosome fractions corresponding to the position for human chromosomes l and 2 (shown by a horizontal arrow) were po3e2!ed. (B) EcoRl analysis of the DNA from sorted chromosome samples using P-labeled human TSH-B gene (13) as a probe. Lanes A to H contain DNA samples prepared in the same way as those shown in Fig. 2. Human chromosomes l and 2 ( i f they are present) in TAIA and TA3A, as recovered from the fraction shown by an arrow in (A), were analysed s i m i l a r l y . Lanes T and M: total human DNA and total mouse DNA, respectively. An arrow indicates the position of the fragment which hybridizes to the human TSH-B gene probe. Thus, the TSH-B gene must be located on chromosomeI . chromosomes or in fact, frequent translocations to rodent chromosomes (6). we tested 7 hybrid cell Therefore, when hybrid corresponding lines, cells and obtained inconsistent r e s u l t s . are chromosomal f r a c t i o n In used, the using a cell purification sorter greatly of the helps to reduce the complexity of these problems. Recently, further development of sorting techniques using a dual-laser cell sorter has made i t chromosomal types (6). possible to Our technique, sort viz. chromosomes into combination of 21 unique preliminary assignment with chromosome sorting from translocated or hybrid cell lines, is another practical way to assign genes to chromosomes. ACKNOWLEDGEMENTS We thank Dr. N. Shimizu (Keio U n i v e r s i t y ) for providing us with human -mouse hybrid cell l i n e s , Dr. T. Yamamoto (Tokyo U n i v e r s i t y ) , Dr. K. Kato, and Dr. Y. Hayashizaki for providing us with human c-erbB-2, g a s t r i n , and TSH-~ gene probes, respectively. This work was supported in part by s c i e n t i f i c grants from the M i n i s t r y of Education, Science and Culture of Japan. 482 Vol. 134, No. 2, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13 14. 15. 16. Harper, M. E. and Saunders, G. F. (1981) Chromosoma 83, 4314439. Ruddle, F. 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