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Immunospecific isolation of a human chromatin fraction from mouse-human hybrid cells
Molecular I eatmmlogy, Vol. 17, pa. 275-280. 0 Pcqmon Press Ltd. 1980. Printed in Great Brimin
IMMUNOSPECIFIC ISOLATION OF A HUMAN CHROMATIN FRACTION FROM MOUSE-HUMAN HYBRID CELLS* YAEL G. ALEVY? and JULIAN B. FLEISC~MAN Department of Microbiology and immunology, Washington University School of Medicine, St. Louis, MO 63110, U.S.A. tReceived 27 December 1978: received ,for publication 16 May 1979)
Abstract-This paper describes an immunospecific isolation of chromatin derived from a single human chromosome (No. 7) in a mouse-human hybrid cell line. Antisera to mouse and to human chromatin were prepared in rabbits and cross-absorbed with heterologous chromatin to render them species-specific. The specificities of the antisera were verified by micro~omplement fixation assays. Chromatin from the hybrid cells was treated batchwise with an IgG fraction of antiserum specific for mouse chromatin coupled to sepharose. The immunoabsorbent removed more than 90% of the mouse chromatin, leaving a supernatant fraction considerably enriched for the human chromatin of chromosome 7. This chromatin fraction reacted only with antiserum specific for human chromatin in the micro-complement fixation assay, and it contained non-histone chromatin proteins characteristic of the human parent of the hybrid cells when examined by high resolution two-dimensional gel electrophoresis.
The structure and function of chromatin are now under intensive study in many laboratories. Among the major components of chromatin, the non-histone chromatin proteins (NHCP) have attracted considerable interest because of their diversity, uniqueness, and potential role in regulating gene expression (Ma~Gillivray, 19761. As many as 500 NHCP have been found in some cell lines, but progress in isolating and studying these proteins, or even restricted fractions of them, has been slow. In the present paper we report an immunospecific approach to isoiating a restricted fraction of chromatin and its associated NHCP from moue-human hybrid cells. Intact chromatin is highly immunogenic in heterospecific recipients, and species-specific antisera to chromatin are readily obtained (Bustin, 1976). The principal immunogenic components are probably NHCP, since the other chromatin components, histones and DNA, are antigenicaliy similar among vertebrate species and are usually weak immunogens. In the present work we have used an immunoabsorbent prepared from a speciesspecific antiserum to isolate the chromatin *Supported by grants Nos. ROI CA20094 and P30 CA16217 from the U.S. Public Health Service. iThis work was conducted during the tenure of post doctoral fellowship No. F32 Af05178 from the U.S. Public Health Service. Present address: Division of Allergy and Immunology, St. Louis University School of Medicine, St. Louis MO 63104, U.S.A. 275
fraction co~esponding to human chromosome 7 from an interspecific (mouse-human) hybrid cell line. This approach may be generally applicable since other interspecific hybrid cell lines contain only one or a few copies of single human chromosomes from one parent (Davidson, 1974).
MATERIALS AND METHODS
Cell
lines
HeLa and mouse L-929 cells were maintained in spinner. cultures in Eagle’s minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS). Cells were harvested by centrifugation and used immediately or frozen at - 20°C. Hybrids of mouse macrophages and SV-40 transformed human fibroblasts (LN-SV) were kindly provided by Dr. C. M. Croce, of the Wistar Institute, Philadelphia. The hybrid clone used (No. 53-87-(l) Cl.211 Schlesinger et al., 1976) contains 2 copies of human chromosome 7, and an approximately diploid number of mouse chromosomes (Strand et al., 1976 and Fig. 1). Thirty-three chromosome spreads of the hybrid cells were counted using the Giemsa-1 1 method to distinguish mouse and human chromosomes (Bobrow & Cross, 1974). The hybrid celIs had 43.3 F 5.2 mouse chromosomes and 2.0 + 0.3 copies of human chromosome 7. The hybrid cells and the human parental line (LN-SV) were
276
YAEL G. ALEVY and JULIAN B. FLEISC~MAN
Fig. 1. Chromosome spread from 53-874 11 clone 21 mouse-human hybrid cells. The two copies of the metacentric human chromosome 7 are indicated by arrows.
maintained in T flasks or in roller bottles in MEM supplemented with 10% FCS. Mouse macrophages were obtained from peritoneal exudate cells of BALB/c mice after injection of thioglycolate medium (Stewart ef al., 197.5). Preparation of chromatin
Nuclei and chromatin were prepared from the above cell lines as described by Zardi et al. (1974). The final chromatin pellet was resuspended in 0.01 M Tris-HCl buffer pH 8, and centrifuged to removed homogenized, residual sucrose. Chromatin preparations were assayed for DNA by the diphenylamine method of Burton (1956) using calf thymus DNA as a standard, and for protein by the procedure of Lowry et af. (195 1) using bovine serum albumin as a standard. Protein to DNA ratios in chromatin ranged from 1.4: 1 to 2.6: 1 depending on the cell line. Preparation and assay of antisera
Rabbits were injected with HeLa, L-929 or LN-SV chromatin containing a total of 2-3 mg of protein in Freunds complete adjuvant, in two foot pads and at two subcutaneous sites. The immunization was repeated for each rabbit four times at seven-day intervals. The rabbits were bled IO days after the final injection. Antisera were assayed by microcomplement fixation (Champion et al., 1974). Chromatin
suspensions were sonicated for 10-20 set and assayed at concentrations between 1 and 20 Lrgof protein per ml. Preparafio~ of ~~~unou~sorbenfs
About 10 mg of chromatin in 0.5 M NaCl and 0.1 ,“MNaHCO, (pH 9) were coupled to CNBractivated sepharose 4B for 2 hr at room temperature and overnight at 4°C. IgG fractions of anti-chromatin antisera were prepared by precipitation with 50% saturated (NH,), SO, in the cold. After dialysis, the IgG was similarly coupled using 30 mg of IgG fraction per gram of Sepharose. Analysis of the supernatant fraction that revealed mixture of the reaction approximately 70-80% of the original IgG was bound to the Sepharose. Analysis of radiolabeiled non&stone proteins
RadioactiveIy labelled NHCP were prepared from cells grown 3-5 days in minimum essential medium (MEM) containing 5 ,&i of 3H-~leucine per ml (specific activity 58 Ciim-mole; Amersham) and 13.1 mg/l unlabelled L-leucine. Nuclei and chromatin were prepared from labelied cells as described above. Labelled NHCP were analyzed by two-dimensional gel electrophoresis (O’Farrell, 1975) of S 1 nucleasedigested chromatin (Peterson & McConkey, 1976). The labelled proteins were visualized by fluorography (Laskey & Mills, 1975).
277
Isolation of Chromatin from Hybrid Cells
n
s =
60-
i z
40-
4 2
20-
2
I-A CHROMATIN
(,,g Protein)
RESULTS
Species specificity of anti-chromatin antisera
Figure 2 shows the reaction of rabbit antiserum to mouse L-929 chromatin and its cross-reactivity with HeLa chromatin. To remove the antibodies reacting with human chromatin, the antiserum was absorbed with HeLa chromatin coupled to sepharose. The specificity of the antiserum was tested after each of two successive absorptions. Figure 2 indicates that the twice absorbed antiserum to L-929 chromatin was specific for L-929 chromatin within the range of antigen concentrations studied. However, at high antigen con-
-
CHROMATIN
20-
Fig. 2. Complement fixation reactions showing specificity of antiserum to L-929 (mouse) chromatin. Closed symbols: unabsorbed antiserum. Open symbols: antiserum absorbed with HeLa (human) chromatin. (0, 0) Are reactions with L-929 chromatin; (A, A) are reactions with HeLa chromatin. Antisera were assayed at l/l000 dilution.
80-
10
2
A
IO
2
12
(pg Protein)
Fig. 4. Complement fixation reactions showing specificity of antiserum to LN-SV (human) chromatin. Closed symbols: unabsorbed antiserum. Open symbols: antiserum absorbed with L-929 (mouse) chromatin. (0, 0) Are reactions with LN-SV chromatin; (A, A) are reactions with hybrid cell chromatin; (m, 0) are reactions with mouse macrophage chromatin. Antiserum was assayed at l/500 dilution.
centrations (60 pg HeLa chromatin protein/ml) even the absorbed serum showed some residual complement-fixing activity (approximately 10%) with HeLa chromatin. The species-specificity of the absorbed antiserum to L-929 chromatin was further demonstrated with other chromatin preparations (Fig. 3). The absorbed serum fixed complement with mouse macrophage chromatin but not with LN-SV human fibroblast chromatin. Thus the absorbed rabbit anti L-929 antisera appeared to be species-specific and could be used as a reagent to identify mouse chromatin but not human chromatin. Ideally, an antiserum to mouse macrophage chromatin should have been used for the experiment described below. However, it was technically impossible to grow enough macrophages to obtain the chromatin required for immunization and preparation of an immunoabsorbent. Antisera specific for human chromatin were prepared by immunizing rabbits with LN-SV chromatin. A typical antiserum predictably cross-reacted with mouse macrophage and with hybrid chromatin (Fig. 4). In order to render the Table 1. Recoveries of hybrid chromatin after successive immunoabsorptions with anti-mouse chromatin
CHROMATIN
(pg Protetn)
Fig. 3. Complement fixation reactions showing species specificity of antiserum to L-929 (mouse) chromatin, absorbed with HeLa (human) chromatin. 0, reaction with mouse macrophage chromatin; .& reaction with hybrid cell chromatin; 0, reaction with LN-SV chromatin. Antiserum was assayed at l/l000 dilution.
Absorption No. Unabsorbed
1 2 3
DNA (mg) 5.1 (1009;) 1.6 ( 314.J 0.7 ( 14,) 0.4 ( 80,)
Protein (mg) 7.0 (loo”/;) 2.2 ( 31” ) 1.2 ( 17
3H countsimin X 10-6 67.0 (IOO”,) 25.0 ( 37”,) 10.5 ( 16”J 4.1 ( 6”“)
YAEL G. ALEVY and JULIAN B. FLEISCHMAN
278
immunoabsorbent (Table 1). This figure approximates the amount of human chromatin in the hybrid cells: i.e. two out of 45 chromosomes (about .5’),). Tab-dimensional gel e~ectrophores~s oj’ nonhistone proteins from the enriched human chromatin fraction
2
CHROMATI
N (ug Ptatem)
10
Fig. 5. Compiement fixation reactions of hybrid cell chromatin treated three times with anti-mouse chromatinsepharose. (0) Reaction with antiserum (diluted l/500) specific for human chromatin; (0) reaction with antiserum (diluted l/1000) specific for mouse chromatin.
antiserum to LN-SV chromatin specific for human chromatin, it was absorbed with L-929
chromatin coupled to Sepharose. Figure 4 shows that the antiserum to LN-SV chromatin absorbed in this way was specific for LN-SV chromatin. The absorbed antiserum still reacted with hybrid cell chromatin which contains human chromatin derived from chromosome 7. The absorbed rabbit antiserum to LN-SV chromatin can thus be used as a specific reagent for human LN-SV chromatin. Separation of the human chromatin component corresponding to human chromosome 7
This fraction was isolated from 7 mg (protein content) of radiolabeled hybrid chromatin after removing the mouse chromatin by successive batchwise absorptions with an ‘anti-mouse chromatin immunoabsor~nt. The immunoabsorbent was prepared by coupling an IgG anti-L-929 (mouse) fraction of specific chromatin antiserum to Sepharose. After each absorption the residual chromatin was tested for its complement fixing reactivity with the specific anti-mouse or anti-human chromatin antisera described above. The results of these tests are shown in Table 1 and Fig. 5. Two successive immunoabsorptions with anti-mouse chromatin did not entirely remove the mouse chromatin. However, the third absorption effectively removed the residual immunologically reactive mouse chromatin leaving a supernatant fraction that reacted signi~cantly with only antiserum to human (LN-SV) chromatin (Fig. 5). The fraction obtained after three absorptions was 6-9% of the original protein and DNA treated with the
We wished to confirm that the immunoabsorbed fraction of the radiolabeled hybrid cell chromatin contained chromatin proteins characteristic of the human (LN-SV) parental line. The NHCP of the immunoabsorbed chromatin fraction were examined by twodimensionai gel electrophoresis and fluorography (O’Farrell, 1975) following digestion with Sl nuclease to remove DNA (Peterson & McConkey, 1976). Histones remained in the preparation, but because of their high isoelectric points, they were not resolved by the 2-D gel electrophoresis. The NHCP prepared from the enriched human component of hybrid cells [Fig. 6(B)] are compared with those of the human LN-SV parental cells [Fig. 6(A)] and also with those of the pre-absorption hybrid cell chromatin (Fig, 6(C); the latter were less satisfactorily resolved by this method). Nine non-histone proteins (arrows) are identical in the immunospecificaliy enriched human component of hybrid cells and in the human LN-SV parental line [Figs. 6(A) and (B)], and five of these proteins are also faintly visible in the hybrid pre-absorption chromatin [Fig. 6(C)]. Proteins in Fig. 6(B) not corresponding to those in the LN-SV parental cell line may be residual mouse NHCP remaining in the preparation. The gel patterns in Fig. 6 were readily reproducible; at least two gels of each preparation were run, which yielded essentially identical patterns. The NHCP indicated by were superimposable when arrows the autoradiograms were aligned along their bottom and ~ght-hand edges and viewed by transmitted light. We were unable to compare the patterns in Fig. 6 with those of mouse macrophages, since the slow growth of the macrophages in culture did not yield enough cells to prepare their NHCP. However, Fig. 6 was compared with a similar profile of mouse myeloma (MPCI 1) NHCP (gei not shown); six of the nine proteins indicated in Figs. 6(A) and 6(B) were undetectable in the mouse myeloma cells, and are thus believed to be of human (LN-SV) origin.
Isolation of Chromatin from Hybrid Ceils
279
Fig. 6. Two~imensionai gel efectrophoretic @‘Farrell. 19751 fluorograms of non-histone chromatin .proteins from LN-SV cells (A); from the immunos~c~ficaliy isolated human chromatin fraction of hybrid cells (B); and from the original ~pre-absorption} hybrid cell chromatin (CI. Arrows indicate the proteins which migrate at identical positions in the three preparations. Proteins identified in the fluorograms, but not readily visible in the printed figure, are circled. The horizontal (first) dimension is isoelectric focusing, with a final gradient from pH 4 (right) to pH 7 (left). The vertical (second) dimension is electrophoresis in SDS-poiyacrylamide gel, from top to bottom. DISCUSSION
Our experiments indicate that mouse and human chromatin in an interspecific hybrid line can be immunospecificaliy separated. The human chromatin component that we have isolated in this way is derived from a single human chromosome (No. 7). The method is potentially useful since many other interspecific hybrid cell lines are selected for retention of individual chromosomes of one parental species. Isolation of such restricted chromatin fractions would facilitate studies of complex chromatin functions, such as the role of NHCP in the regulation of gene expression in transcriptionally active chromatin. Although our experiments thus far are on an analytical scale, it seems reasonable that preparative quantities of a desired chromatin fraction could be obtained. The serologica and two-dimensional gel M.I.M.U. 17 ?---I
analyses of the immunoabsorbed hybrid chromatin (Figs. 5 and 6) demonstrate that it is considerably enriched for the human component, but that it is probably not pure. Additional immunoabso~tions on larger initial quantities of hybrid chromatin may be required to remove all the mouse chromatin. The NHCP spots in Fig. 6(B) not identifiable in the LN-SV parental line probably are residual mouse macrophage NHCP contaminating the immunoabsorbed preparation. However, we cannot unambiguously assign them to mouse macrophages since we were unable to obtain sufficient mouse macrophages to prepare the NHCP for a comparative two-dimensional gel. An alternate explanation is that the enriched human chromatin fraction representing chromosome 7 permits detection of some NHCP whose concentrations in total LN-SV chromatin are too low for detection.
280
Y.AEL G. ALEVY
and JULIAN
The NHCP having the same molecular weight but differing only slightly in their effective pIs could represent charge isomers of single proteins. generated by post-translational modification or by chemical changes during purification. Thus eight of the spots [Figs. 6(A) and (B)j could actually correspond to a minimum of three proteins. Structural gene assignments for individual NHCP have not been made in most cases. An interesting corollary of the present work is that the human NHCP which we find associated with chromosome 7 are probably coded for by that chromosome since the rest of the human genome is absent in these hybrids (Strand P? ai., 19761. Acknowledgemenrs-We are grateful to Ms. Donna Thurmond for her technical assistance, to Ms. Judith Connett for performing the two-dimensional electrophoreses, and to Mr. Mitchell Scott for the chromosome counts.
REFERENCES Bobrow M. & Cross J. (1974) Differential staining of human and mouse chromosomes in interspecific cell hybrids. Narure. Lond. 251. 77-79. Burton K. (1956) A study of the conditions and mechamsm of the diphenylamine reaction for the coiorimetric estimation of deoxyribonucleic acid. Biochrm. J. 62, 315-323.
Bustin
M.
(1976)
Chromatin
structure
and
specificity
B. FLEISCHMA~
revealed
by tmmunologtcal
techmques.
FEBS
LL’II. 70.
l-10. Champion
A. B., Prager
E. M.. Wachter D. & Wilson A. C. fixation. In Biochemrcal urlti lrnrnunoiogrcal Taronom,r of .4nimals (Edited by Wrtght C. A.) pp. 397-416. Academic Press, London. Davidson R. L. (1974) Gene expression in somatic cell hybrids. A. Rev. Gener. 8, 195-218. Laskey R. A. & Mills A. D. (1975) Quantitat;ve film detection of 3H and “C in polyacrylamide gels by fluorography.
( 1974) Microcomplement
Ew. J. Biochrm. 56, 335-341. Lowry 0. H.. Rosebrough N. J.. Farr A. L. Sr Randall R. J. ( 19.51) Protein measurement with the folin ohenol reagent. J. brol. Chem. 193, 265-275. MacGillivray A. J. (1976) Non-histone nuclear proteins as gene regulators? Biochem. Sot. Truns. 4, 976-975. O’Farrell P. H. (1975) High resolution two-dimensionaf electrophoresis of protems. J. biol. Chem. 250,4007-&12 1. Peterson J. L. & McConkey E. H. (1976) Uon-histone chromosomal proteins from HeLa ceils. A survey by high resolution, two-dimensional electrophorests. J. b~ol. C/rem. 251, 548-554. Schlesinger S., Lagwinska E., Stewart C. C. & Croce C. M (1976) Lactic dehydrogenase virus replicates m somattc cell hybrids of mouse peritoneal macrophages and SV40transformed human fibroblasts. &-oio~v 74, 535-539. Stewart C. C., Lin H. & Adles C. (1975
Zardi L., Lin J.-C.. Petersen R. 0. & Baserga R. (lY?.%) Specificity of antibodies to nonhistone chromosomal proteins of cultured fibroblasrs. In Cortrrol uf Proiiferarion in Ammal Cells (Edited by Clarkson B. & Baserga R.) pp. 729-741. Cold Spring Harbor. New York.
IMMUNOSPECIFIC ISOLATION OF A HUMAN CHROMATIN FRACTION FROM MOUSE-HUMAN HYBRID CELLS* YAEL G. ALEVY? and JULIAN B. FLEISC~MAN Department of Microbiology and immunology, Washington University School of Medicine, St. Louis, MO 63110, U.S.A. tReceived 27 December 1978: received ,for publication 16 May 1979)
Abstract-This paper describes an immunospecific isolation of chromatin derived from a single human chromosome (No. 7) in a mouse-human hybrid cell line. Antisera to mouse and to human chromatin were prepared in rabbits and cross-absorbed with heterologous chromatin to render them species-specific. The specificities of the antisera were verified by micro~omplement fixation assays. Chromatin from the hybrid cells was treated batchwise with an IgG fraction of antiserum specific for mouse chromatin coupled to sepharose. The immunoabsorbent removed more than 90% of the mouse chromatin, leaving a supernatant fraction considerably enriched for the human chromatin of chromosome 7. This chromatin fraction reacted only with antiserum specific for human chromatin in the micro-complement fixation assay, and it contained non-histone chromatin proteins characteristic of the human parent of the hybrid cells when examined by high resolution two-dimensional gel electrophoresis.
The structure and function of chromatin are now under intensive study in many laboratories. Among the major components of chromatin, the non-histone chromatin proteins (NHCP) have attracted considerable interest because of their diversity, uniqueness, and potential role in regulating gene expression (Ma~Gillivray, 19761. As many as 500 NHCP have been found in some cell lines, but progress in isolating and studying these proteins, or even restricted fractions of them, has been slow. In the present paper we report an immunospecific approach to isoiating a restricted fraction of chromatin and its associated NHCP from moue-human hybrid cells. Intact chromatin is highly immunogenic in heterospecific recipients, and species-specific antisera to chromatin are readily obtained (Bustin, 1976). The principal immunogenic components are probably NHCP, since the other chromatin components, histones and DNA, are antigenicaliy similar among vertebrate species and are usually weak immunogens. In the present work we have used an immunoabsorbent prepared from a speciesspecific antiserum to isolate the chromatin *Supported by grants Nos. ROI CA20094 and P30 CA16217 from the U.S. Public Health Service. iThis work was conducted during the tenure of post doctoral fellowship No. F32 Af05178 from the U.S. Public Health Service. Present address: Division of Allergy and Immunology, St. Louis University School of Medicine, St. Louis MO 63104, U.S.A. 275
fraction co~esponding to human chromosome 7 from an interspecific (mouse-human) hybrid cell line. This approach may be generally applicable since other interspecific hybrid cell lines contain only one or a few copies of single human chromosomes from one parent (Davidson, 1974).
MATERIALS AND METHODS
Cell
lines
HeLa and mouse L-929 cells were maintained in spinner. cultures in Eagle’s minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS). Cells were harvested by centrifugation and used immediately or frozen at - 20°C. Hybrids of mouse macrophages and SV-40 transformed human fibroblasts (LN-SV) were kindly provided by Dr. C. M. Croce, of the Wistar Institute, Philadelphia. The hybrid clone used (No. 53-87-(l) Cl.211 Schlesinger et al., 1976) contains 2 copies of human chromosome 7, and an approximately diploid number of mouse chromosomes (Strand et al., 1976 and Fig. 1). Thirty-three chromosome spreads of the hybrid cells were counted using the Giemsa-1 1 method to distinguish mouse and human chromosomes (Bobrow & Cross, 1974). The hybrid celIs had 43.3 F 5.2 mouse chromosomes and 2.0 + 0.3 copies of human chromosome 7. The hybrid cells and the human parental line (LN-SV) were
276
YAEL G. ALEVY and JULIAN B. FLEISC~MAN
Fig. 1. Chromosome spread from 53-874 11 clone 21 mouse-human hybrid cells. The two copies of the metacentric human chromosome 7 are indicated by arrows.
maintained in T flasks or in roller bottles in MEM supplemented with 10% FCS. Mouse macrophages were obtained from peritoneal exudate cells of BALB/c mice after injection of thioglycolate medium (Stewart ef al., 197.5). Preparation of chromatin
Nuclei and chromatin were prepared from the above cell lines as described by Zardi et al. (1974). The final chromatin pellet was resuspended in 0.01 M Tris-HCl buffer pH 8, and centrifuged to removed homogenized, residual sucrose. Chromatin preparations were assayed for DNA by the diphenylamine method of Burton (1956) using calf thymus DNA as a standard, and for protein by the procedure of Lowry et af. (195 1) using bovine serum albumin as a standard. Protein to DNA ratios in chromatin ranged from 1.4: 1 to 2.6: 1 depending on the cell line. Preparation and assay of antisera
Rabbits were injected with HeLa, L-929 or LN-SV chromatin containing a total of 2-3 mg of protein in Freunds complete adjuvant, in two foot pads and at two subcutaneous sites. The immunization was repeated for each rabbit four times at seven-day intervals. The rabbits were bled IO days after the final injection. Antisera were assayed by microcomplement fixation (Champion et al., 1974). Chromatin
suspensions were sonicated for 10-20 set and assayed at concentrations between 1 and 20 Lrgof protein per ml. Preparafio~ of ~~~unou~sorbenfs
About 10 mg of chromatin in 0.5 M NaCl and 0.1 ,“MNaHCO, (pH 9) were coupled to CNBractivated sepharose 4B for 2 hr at room temperature and overnight at 4°C. IgG fractions of anti-chromatin antisera were prepared by precipitation with 50% saturated (NH,), SO, in the cold. After dialysis, the IgG was similarly coupled using 30 mg of IgG fraction per gram of Sepharose. Analysis of the supernatant fraction that revealed mixture of the reaction approximately 70-80% of the original IgG was bound to the Sepharose. Analysis of radiolabeiled non&stone proteins
RadioactiveIy labelled NHCP were prepared from cells grown 3-5 days in minimum essential medium (MEM) containing 5 ,&i of 3H-~leucine per ml (specific activity 58 Ciim-mole; Amersham) and 13.1 mg/l unlabelled L-leucine. Nuclei and chromatin were prepared from labelied cells as described above. Labelled NHCP were analyzed by two-dimensional gel electrophoresis (O’Farrell, 1975) of S 1 nucleasedigested chromatin (Peterson & McConkey, 1976). The labelled proteins were visualized by fluorography (Laskey & Mills, 1975).
277
Isolation of Chromatin from Hybrid Cells
n
s =
60-
i z
40-
4 2
20-
2
I-A CHROMATIN
(,,g Protein)
RESULTS
Species specificity of anti-chromatin antisera
Figure 2 shows the reaction of rabbit antiserum to mouse L-929 chromatin and its cross-reactivity with HeLa chromatin. To remove the antibodies reacting with human chromatin, the antiserum was absorbed with HeLa chromatin coupled to sepharose. The specificity of the antiserum was tested after each of two successive absorptions. Figure 2 indicates that the twice absorbed antiserum to L-929 chromatin was specific for L-929 chromatin within the range of antigen concentrations studied. However, at high antigen con-
-
CHROMATIN
20-
Fig. 2. Complement fixation reactions showing specificity of antiserum to L-929 (mouse) chromatin. Closed symbols: unabsorbed antiserum. Open symbols: antiserum absorbed with HeLa (human) chromatin. (0, 0) Are reactions with L-929 chromatin; (A, A) are reactions with HeLa chromatin. Antisera were assayed at l/l000 dilution.
80-
10
2
A
IO
2
12
(pg Protein)
Fig. 4. Complement fixation reactions showing specificity of antiserum to LN-SV (human) chromatin. Closed symbols: unabsorbed antiserum. Open symbols: antiserum absorbed with L-929 (mouse) chromatin. (0, 0) Are reactions with LN-SV chromatin; (A, A) are reactions with hybrid cell chromatin; (m, 0) are reactions with mouse macrophage chromatin. Antiserum was assayed at l/500 dilution.
centrations (60 pg HeLa chromatin protein/ml) even the absorbed serum showed some residual complement-fixing activity (approximately 10%) with HeLa chromatin. The species-specificity of the absorbed antiserum to L-929 chromatin was further demonstrated with other chromatin preparations (Fig. 3). The absorbed serum fixed complement with mouse macrophage chromatin but not with LN-SV human fibroblast chromatin. Thus the absorbed rabbit anti L-929 antisera appeared to be species-specific and could be used as a reagent to identify mouse chromatin but not human chromatin. Ideally, an antiserum to mouse macrophage chromatin should have been used for the experiment described below. However, it was technically impossible to grow enough macrophages to obtain the chromatin required for immunization and preparation of an immunoabsorbent. Antisera specific for human chromatin were prepared by immunizing rabbits with LN-SV chromatin. A typical antiserum predictably cross-reacted with mouse macrophage and with hybrid chromatin (Fig. 4). In order to render the Table 1. Recoveries of hybrid chromatin after successive immunoabsorptions with anti-mouse chromatin
CHROMATIN
(pg Protetn)
Fig. 3. Complement fixation reactions showing species specificity of antiserum to L-929 (mouse) chromatin, absorbed with HeLa (human) chromatin. 0, reaction with mouse macrophage chromatin; .& reaction with hybrid cell chromatin; 0, reaction with LN-SV chromatin. Antiserum was assayed at l/l000 dilution.
Absorption No. Unabsorbed
1 2 3
DNA (mg) 5.1 (1009;) 1.6 ( 314.J 0.7 ( 14,) 0.4 ( 80,)
Protein (mg) 7.0 (loo”/;) 2.2 ( 31” ) 1.2 ( 17
3H countsimin X 10-6 67.0 (IOO”,) 25.0 ( 37”,) 10.5 ( 16”J 4.1 ( 6”“)
YAEL G. ALEVY and JULIAN B. FLEISCHMAN
278
immunoabsorbent (Table 1). This figure approximates the amount of human chromatin in the hybrid cells: i.e. two out of 45 chromosomes (about .5’),). Tab-dimensional gel e~ectrophores~s oj’ nonhistone proteins from the enriched human chromatin fraction
2
CHROMATI
N (ug Ptatem)
10
Fig. 5. Compiement fixation reactions of hybrid cell chromatin treated three times with anti-mouse chromatinsepharose. (0) Reaction with antiserum (diluted l/500) specific for human chromatin; (0) reaction with antiserum (diluted l/1000) specific for mouse chromatin.
antiserum to LN-SV chromatin specific for human chromatin, it was absorbed with L-929
chromatin coupled to Sepharose. Figure 4 shows that the antiserum to LN-SV chromatin absorbed in this way was specific for LN-SV chromatin. The absorbed antiserum still reacted with hybrid cell chromatin which contains human chromatin derived from chromosome 7. The absorbed rabbit antiserum to LN-SV chromatin can thus be used as a specific reagent for human LN-SV chromatin. Separation of the human chromatin component corresponding to human chromosome 7
This fraction was isolated from 7 mg (protein content) of radiolabeled hybrid chromatin after removing the mouse chromatin by successive batchwise absorptions with an ‘anti-mouse chromatin immunoabsor~nt. The immunoabsorbent was prepared by coupling an IgG anti-L-929 (mouse) fraction of specific chromatin antiserum to Sepharose. After each absorption the residual chromatin was tested for its complement fixing reactivity with the specific anti-mouse or anti-human chromatin antisera described above. The results of these tests are shown in Table 1 and Fig. 5. Two successive immunoabsorptions with anti-mouse chromatin did not entirely remove the mouse chromatin. However, the third absorption effectively removed the residual immunologically reactive mouse chromatin leaving a supernatant fraction that reacted signi~cantly with only antiserum to human (LN-SV) chromatin (Fig. 5). The fraction obtained after three absorptions was 6-9% of the original protein and DNA treated with the
We wished to confirm that the immunoabsorbed fraction of the radiolabeled hybrid cell chromatin contained chromatin proteins characteristic of the human (LN-SV) parental line. The NHCP of the immunoabsorbed chromatin fraction were examined by twodimensionai gel electrophoresis and fluorography (O’Farrell, 1975) following digestion with Sl nuclease to remove DNA (Peterson & McConkey, 1976). Histones remained in the preparation, but because of their high isoelectric points, they were not resolved by the 2-D gel electrophoresis. The NHCP prepared from the enriched human component of hybrid cells [Fig. 6(B)] are compared with those of the human LN-SV parental cells [Fig. 6(A)] and also with those of the pre-absorption hybrid cell chromatin (Fig, 6(C); the latter were less satisfactorily resolved by this method). Nine non-histone proteins (arrows) are identical in the immunospecificaliy enriched human component of hybrid cells and in the human LN-SV parental line [Figs. 6(A) and (B)], and five of these proteins are also faintly visible in the hybrid pre-absorption chromatin [Fig. 6(C)]. Proteins in Fig. 6(B) not corresponding to those in the LN-SV parental cell line may be residual mouse NHCP remaining in the preparation. The gel patterns in Fig. 6 were readily reproducible; at least two gels of each preparation were run, which yielded essentially identical patterns. The NHCP indicated by were superimposable when arrows the autoradiograms were aligned along their bottom and ~ght-hand edges and viewed by transmitted light. We were unable to compare the patterns in Fig. 6 with those of mouse macrophages, since the slow growth of the macrophages in culture did not yield enough cells to prepare their NHCP. However, Fig. 6 was compared with a similar profile of mouse myeloma (MPCI 1) NHCP (gei not shown); six of the nine proteins indicated in Figs. 6(A) and 6(B) were undetectable in the mouse myeloma cells, and are thus believed to be of human (LN-SV) origin.
Isolation of Chromatin from Hybrid Ceils
279
Fig. 6. Two~imensionai gel efectrophoretic @‘Farrell. 19751 fluorograms of non-histone chromatin .proteins from LN-SV cells (A); from the immunos~c~ficaliy isolated human chromatin fraction of hybrid cells (B); and from the original ~pre-absorption} hybrid cell chromatin (CI. Arrows indicate the proteins which migrate at identical positions in the three preparations. Proteins identified in the fluorograms, but not readily visible in the printed figure, are circled. The horizontal (first) dimension is isoelectric focusing, with a final gradient from pH 4 (right) to pH 7 (left). The vertical (second) dimension is electrophoresis in SDS-poiyacrylamide gel, from top to bottom. DISCUSSION
Our experiments indicate that mouse and human chromatin in an interspecific hybrid line can be immunospecificaliy separated. The human chromatin component that we have isolated in this way is derived from a single human chromosome (No. 7). The method is potentially useful since many other interspecific hybrid cell lines are selected for retention of individual chromosomes of one parental species. Isolation of such restricted chromatin fractions would facilitate studies of complex chromatin functions, such as the role of NHCP in the regulation of gene expression in transcriptionally active chromatin. Although our experiments thus far are on an analytical scale, it seems reasonable that preparative quantities of a desired chromatin fraction could be obtained. The serologica and two-dimensional gel M.I.M.U. 17 ?---I
analyses of the immunoabsorbed hybrid chromatin (Figs. 5 and 6) demonstrate that it is considerably enriched for the human component, but that it is probably not pure. Additional immunoabso~tions on larger initial quantities of hybrid chromatin may be required to remove all the mouse chromatin. The NHCP spots in Fig. 6(B) not identifiable in the LN-SV parental line probably are residual mouse macrophage NHCP contaminating the immunoabsorbed preparation. However, we cannot unambiguously assign them to mouse macrophages since we were unable to obtain sufficient mouse macrophages to prepare the NHCP for a comparative two-dimensional gel. An alternate explanation is that the enriched human chromatin fraction representing chromosome 7 permits detection of some NHCP whose concentrations in total LN-SV chromatin are too low for detection.
280
Y.AEL G. ALEVY
and JULIAN
The NHCP having the same molecular weight but differing only slightly in their effective pIs could represent charge isomers of single proteins. generated by post-translational modification or by chemical changes during purification. Thus eight of the spots [Figs. 6(A) and (B)j could actually correspond to a minimum of three proteins. Structural gene assignments for individual NHCP have not been made in most cases. An interesting corollary of the present work is that the human NHCP which we find associated with chromosome 7 are probably coded for by that chromosome since the rest of the human genome is absent in these hybrids (Strand P? ai., 19761. Acknowledgemenrs-We are grateful to Ms. Donna Thurmond for her technical assistance, to Ms. Judith Connett for performing the two-dimensional electrophoreses, and to Mr. Mitchell Scott for the chromosome counts.
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