EXPERIMENTAL
CELL RESEARCH
189,145-147
(1990)
SHORT NOTE Lamins A and C Are Not Expressed at Early Stages of Human Lymphocyte Differentiation ~LLE GUILLY,*,~ JEAN-PIERRE KOLB,?
MARIE-N•
FRANC•
IS GODEAU,$
AND JEAN-CLAUDE
FRANQOISE COURVALIN*
GOSTI,*
*Centre de G&&que Molhdnire, CNRS, 91190 Gif-sur- yvette, France; tlnstitut Curie, INSERM U 196,26 rue d’Ulm, F-75231 Paris Ceder 05, France; and $Institut Pasteur, INSERM U 277,25 rue du Dr Roux, 75724 Paris Cedez 15, France
Lamins are major proteins of the nuclear envelope that are members of the intermediate filament protein family. In vertebrates, nuclei from differentiated tissues usually contain both lamins of the A and B subtypes, while embryonic tissues contain the B-type lamin only. We have examined the composition of the nuclear lamina in human B and T lymphocytes representative of distinct stages of lymphoid differentiation. We show here that, in both cell lineages, while lamin B is constitutively expressed at all stages of differentiation, Atype lamin expression is restricted to later developmen0 1990 Academic Press. Inc. tal stages.
INTRODUCTION
The nuclear lamina is a protein meshwork situated between the inner nuclear membrane and the heterochromatin. In higher eucaryotes, this lamina is composed of two major types of polypeptides: the acidic B-type lamins and the neutral A-type lamins (lamins A and C in mammals) (for review see Ref. [l]). Both types of lamins have primary and secondary structural similarities to intermediate filament proteins [2-41. In vertebrates, lamins of both A and B types are present in the nuclear envelope of most somatic cells [5,6]. However, we have found a human T lymphoblastic cell line lacking lamins A and C [7]. In the present study, we have systematically analyzed for their lamin content human B and T lymphoid cells at different stages of differentiation. MATERIALS
AND
mic cells display a cortical thymocyte phenotype and a partial deletion of the short arm of chromosome 2. RPMI-6666 line is an EBV-transformed lymphoblastoid B cell line and U266 line is an IgE-producing human myeloma. Leukemic cells from two patients with B cell precursor acute lymphoblastic leukemia were characterized as CD 10 (CALLA, common acute lymphoblastic leukemia antigen)-positive cells by immunological marker analysis (by indirect immunofluorescence and flow cytometry). Peripheral blood lymphocytes were obtained from healthy donors as residues from platelet preparations and B cells were purified according to a technique previously described [9]. Thymocytes were taken from 3-week-old Wistar rat thymus. Cell fractionation procedure. Cell ghosts were prepared from each cell type as previously described [7]. A nuclear lamina-enriched fraction was then obtained according to Gerace et al. [lo] RNA analyses. Total RNA from HeLa, KE 37, and RPM1 6666 cells was extracted by the LiCl-urea procedure according to Auffray and Rougeon [ll]. Poly(A)+ RNAs were isolated and hybridized to a lamin A cDNA clone and glyceraldehyde 3-phosphate dehydrogenase cDNA clone as previously described [ 71. SDS-polyacrylamide gel ekctrophoresis and protein transfer to nitrocelluhe. SDS-polyacrylamide gel electrophoresis was performed on 10% polyacrylamide slab gels according to Laemmli [12]. Proteins were transferred to nitrocellulose by electrophoresis using the semi-dry method [13]. Immobilized proteins were probed with either a 1:500 dilution of an anti-lamin A + C serum (LS-1) or a 1:200 dilution of an anti-lamin B serum (F). These sera have been previously characterized [14, 151. Finally, blots were incubated with ‘261-protein A and reactive bands identified by autoradiography.
RESULTS AND DISCUSSION We have examined the composition of the nuclear lamina in several different human B and T lymphoid cells, typical of distinct differentiation stages, assuming that tumor cells are representative of their nontransformed counterparts [ 161. The early stage of differentiation was represented by pre-B leukemia cells, isolated from two patients, and the T lymphoblastic KE 37 cell line, which is of the cortical thymic type, respectively. As human thymocytes were not available, we included rat thymocytes as representatives of this group. The intermediate stage of differentiation was represented by B and T lymphocytes prepared from peripheral blood lymphocytes of healthy subjects. The fully differentiated stage in the B cell lineage was represented by the human
METHODS
Cells and ceU Zincs. Human established cell limes were grown in RPM1 1640 medium, containing 10% (v/v) fetal calf serum. The T lymphoblast RE 37 cell line was described previously [a]. These leuke-
1 To whom correspondence and reprint requests should be addressed at CEA, DPS/SPE, BP 6,92265 Fontenay-aux-Roses Cedex, France. 145
0014~4S27/90
$3.00
Copyright Q 1990 by Academic F’rsss, Inc. All rights of reproduction in any form reserved.
146
SHORT NOTE
firming our previous results [ 71, whereas two mRNAs of the expected size were present in poly(A)+ RNA isolated from lymphoblastoid cells. As a control, a RNA species that hybridized to a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe was detected in both cell lines. Concomitant expression of lamins of A- and B-type is generally found in vertebrate somatic nuclei [5,6]. Few Plasm. Pre.B PBL exceptions have been reported so far, all of them consistFIG. 1. Immunoblot analysis of lamins. Nuclear envelopes from ing of the absence of lamins A and C: early chicken emlymphoid T cells (upper row) and B cells (lower row) were probed with bryos [ 171, mouse embryonal F9 teratocarcinoma cells either lamin B-specific antibodies (left lanes) or lamiis A + C antibod[5, 18, 191, human T lymphoblasts [ 71, mouse and rat ies (right lanes). Cells used were rat thymocytes (R. Th.), KE 37 lymphoblastic cell line (KE 37), peripheral blood T and B lymphocytes lymphoid cells [20,21], human promyelocytic leukemia (PBL), pre-B acute lymphoblastic leukemias (pre-B), and lymphoHL60 cells [20, 221, and possibly mouse embryos 123, blastoid RPM14366 (Plasm.). In the figure, cells at the same stage of 241. The present study allows us to add rat thymocytes differentiation are aligned vertically. Note in rat thymocytes, KE 3’7 and human pre-B lymphoblasts to this list. Furthercells, and pie-B-ALL the absence of a signal for lamins A and C. more, our results show that there is a correlation between the stage of differentiation of the cells and their lymphoblastoid RPMI-3366 and U266 plasmocytoma lamin A and C complement, as previously reported in cell lines. amphibians for lamins L1 and Lri [25,26]. An apparent Cellular fractions enriched in nuclear lamins were exception to this general pattern is the recent report of prepared from these different cells using a procedure Ig-secreting mouse myeloma cells that lack lamins A and that minimizes proteolysis, and protein samples corre- c [22]. sponding to an identical number of cells were separated So far, there is no example of any somatic cells that by SDS-PAGE and then transferred to nitrocellulose. do not contain lamin B. This particular lamin is the tarLamin B and lamins A and C were identified on these get of extensive cell cycle-dependent post-translational immunoblots by using two autoimmune antisera which modifications, including phosphorylation, methylation, have been previously characterized [14, 151. Figure 1 and isoprenylation (for review see Ref. [l]). It is closely shows the results of these experiments. The lamin B associated with the inner nuclear membrane by interaccontent was nearly equivalent in the five human cell tion with an integral membrane protein [27]. During types, while the amount of lamins A and C varied to a lamina depolymerization at mitosis, lamin B remains aslarge extent. Lamins A and C were undetected in rat thy- sociated with membrane vesicles, whereas the A-lamins mocytes, human T lymphoblasts, and pre-B cells. In become soluble [lo]. It has been suggested that lamin B contrast, in B and T lymphocytes, lamins A and C were could be a component of the nuclear envelope essential present in nearly equivalent amounts in both cell types. for nuclear vesicle targeting during mitosis [lo]. It may Furthermore, an intense signal for lamins A and C was 1234 567 detected in the RPMI-3366 lymphoblastoid cell line and the U266 plasmocytoma cell line (data not shown). -3Kb Since these results were obtained with immunological -2dKb techniques, the observed variations in signal intensity could reflect qualitative rather than quantitative differences. However, we have previously shown that data obtained with these antibodies were in agreement with those obtained using Coomassie staining, proteolytic peptide analysis, and Northern blotting [ 71. Moreover, 8 9 10 11 12 13 titration experiments have shown that binding of autoFIG. 2. Northern blot analysis of mRNA from HeLa cells, KB antibodies to lamins is linear over a wide range of protein concentrations [5]. To further ascertain these observed 37 T lymphoblasts, and RPM143666 lymphoblastoid cells. Total RNA HeLa cells (lane l), KE 37 cells (lane 2), RPM14666 cells (lane variations, Northern blot analysis was performed with a from 5), poly(A)+ RNA from KE 37 cells (lane 3), RPM16666 cells (lane 6) 1.3-kb fragment from the 3’ end of a cDNA coding for and poly(A)- RNA from KE 37 cells (lane 4), RPM14366 cells (lane human lamin A as a probe [3]. This analysis was per- 7) was fractionated on a 1% agarose gel, transferred t.0 nitrocellulose formed on two cell lines exhibiting the two extreme pat- filters and hybridized with a human lamin A-specific probe. As a control of the RNA preparation, total RNA from KE 37 cells (lane 8) and terns of lamin A and C expression, i.e., T lymphoblasts RPM16666 cells (lane ll), poly(A)+ RNA from KE 37 cells (lane 9) (KE 37) and lymphoblastoid cells (RPMI-3366). Figure and RPM16666 cells (lane 12), and poly(A)- RNA from KE 37 cells 2 shows that no RNAs for lamins A and C were detected (lane 10) and RPM14366 cells (lane 13) was hybridized with a rat in the poly(A)+ RNA extracted from KE 37 cells, con- GAPDH probe. R.Th.
KE37
PBL
SHORT NOTE
be because of this critical function that lamin B is expressed constitutively in all somatic cells such as the lymphoid cells at all stages of differentiation used in the present study. Since isolated lamin B self-associates only weakly in vitro [28], it remains to be explained how a functional lamina can be assembled without lamins A and C. Because of the existence of cells that express only lamin B, it should be postulated that lamin B is able to form homopolymers in Co. We thank Dr. F. McKeon for the gift of anti-lamin A + C serum and lamin probes, Dr. F. Danon for the gift of anti-lamin B serum, Dr. J-M. Blanchard for the gift of GAPDH probe, Dr. F. Sigaux for providing and characterizing the pre-B ALL cells, Drs. J. Morizet and A. Bernard for the gift of anti-T mAb, and Drs. B. Guiard and Moreau for precious technical advice. We thank also Drs. C. Klotz, B. Maro, and G. Kerryer for their constant interest, Dr. J. Beisson for material and moral support, and Dr. H. Worman for critical reading of the manuscript. This research was supported by Grant ARC 6334. REFERENCES 1. Nigg, E. A. (1989) Curr. Opinion Cell Biol. 1,435-440. 2. Fisher, D. Z., Chaudhary, N., and Blobel, G. (1986) Proc. Natl. Acad. Sci. USA 83,6450-6454. 3. McKeon, F. D., Kirschner, M. W., and Caput D. (1986) Nature (London) 319,463-468. 4. Krohne, G., Wolin, S. L., McKeon, F. D., Franke, W. W., and Kirschner, M. W. (1987) EMBO J. 6,3801-3808. 5. Worman, H. J., Laxaridis, I., and Georgatos, S. D. (1988) J. Bial. &em. 263,12,135-12,141. 6. Lehner, C. F., Kurer, V., Eppenberger, H. M., and Nigg, E. A. (1986) J. Bial. Chem. 261,13,293-13,301. 7. Guilly, M-N., Bensussan, A., Bourge, J-F., Bomens, M., and Courvalin, J-C. (1987) EMBO J. 6,3795-3799. Received December 11,1989 Revised version received March 12,199O
147
8. Mayer, L., Shu Man Fu, and Kunkel, H. G. (1982) J. Exp. Med. 166,1860-1865. 9. Petit-Koskas, E., Genot, E., Lawrence, D., and Kolb, J-P. (1988) Eur. J. Immunol. 18.111-115. 10. Gerace, L., Comeau, C., and Benson, M. (1984) J. Cell Sci. Suppl. 1,137-160. 11. Auffray, C., and Rougeon, F. (1980) Eur. J. B&hem. 107,303314. 12. Laemmli, U. K. (1970) Nature (Landon) 227,~685. 13. Kyhse-Andersen, J. (1984) J. Biochem. Biaphys. Methods 10, 203-209. 14. McKeon, F. D., Tuffanelli, D. L., Fukuyama, K., and Kirschner, M. W. (1983) Proc. Natl. Acad Sci. USA 80,4374-4378. 15. Guilly, M-N., Danon, F., Brouet, J-C., Bornens, M., and Courvalin, J-C. (1987) Eur. J. Cell Bial. 43.266-272. 16. Osborn, M., and Weber, K. (1986) Trends Bzixhem. Sci. 11,469472. 17. Lehner, C. F., Stick, R., Eppenberger, H. M., and Nigg, E. A. (1987) J. Cell Bial. 105,577-587. 18. Stewart, C., and Burke, B. (1987) CeU61,383-392. 19. Lebel, S., Lampron, C., Royal, A., and Raymond, Y. (1987) J. CeU Bial. 105,1099-1104. 20. Hornbeck, P., Huang, K-P., and Paul, W. E. (1988) J. BbL Chem. 86,2279-2283. 21. Kaufman, S. H. (1989) J. Bial. Chem. 264,13,946-13,955. 22. Paulin-Levasseur, M., Scherbarth, A. M., Traub, U., and Traub, P. (1988) Eur. J. Cell Biol. 47,121-131. 23. Houliston, E., Guilly, M-N., Courvahn, J-C., and Maro, B. (1988) Development 102,271-278. 24. Ribber, R-A, Weber, K., and Osbom, M. (1989) Development 105,365-378. 25. Stick, R., and Hausen, P. (1985) CeU41,191-200. 26. Benavente, R., Krohne, G., and Franke, W. W. (1985) Cell 41, 177-190. 27. Worman, H. J., Yuan, J., Blobel, G., and Georgatos, S. D. (1988) Prac. Natl. Acad Sci. USA 85,8531-8534. 28. Georgatos, S. D., Stoumaras, C., and Blobel, G. (1988) Proc. Natl. Acad. Sci. USA 86,4325-4329.
CELL RESEARCH
189,145-147
(1990)
SHORT NOTE Lamins A and C Are Not Expressed at Early Stages of Human Lymphocyte Differentiation ~LLE GUILLY,*,~ JEAN-PIERRE KOLB,?
MARIE-N•
FRANC•
IS GODEAU,$
AND JEAN-CLAUDE
FRANQOISE COURVALIN*
GOSTI,*
*Centre de G&&que Molhdnire, CNRS, 91190 Gif-sur- yvette, France; tlnstitut Curie, INSERM U 196,26 rue d’Ulm, F-75231 Paris Ceder 05, France; and $Institut Pasteur, INSERM U 277,25 rue du Dr Roux, 75724 Paris Cedez 15, France
Lamins are major proteins of the nuclear envelope that are members of the intermediate filament protein family. In vertebrates, nuclei from differentiated tissues usually contain both lamins of the A and B subtypes, while embryonic tissues contain the B-type lamin only. We have examined the composition of the nuclear lamina in human B and T lymphocytes representative of distinct stages of lymphoid differentiation. We show here that, in both cell lineages, while lamin B is constitutively expressed at all stages of differentiation, Atype lamin expression is restricted to later developmen0 1990 Academic Press. Inc. tal stages.
INTRODUCTION
The nuclear lamina is a protein meshwork situated between the inner nuclear membrane and the heterochromatin. In higher eucaryotes, this lamina is composed of two major types of polypeptides: the acidic B-type lamins and the neutral A-type lamins (lamins A and C in mammals) (for review see Ref. [l]). Both types of lamins have primary and secondary structural similarities to intermediate filament proteins [2-41. In vertebrates, lamins of both A and B types are present in the nuclear envelope of most somatic cells [5,6]. However, we have found a human T lymphoblastic cell line lacking lamins A and C [7]. In the present study, we have systematically analyzed for their lamin content human B and T lymphoid cells at different stages of differentiation. MATERIALS
AND
mic cells display a cortical thymocyte phenotype and a partial deletion of the short arm of chromosome 2. RPMI-6666 line is an EBV-transformed lymphoblastoid B cell line and U266 line is an IgE-producing human myeloma. Leukemic cells from two patients with B cell precursor acute lymphoblastic leukemia were characterized as CD 10 (CALLA, common acute lymphoblastic leukemia antigen)-positive cells by immunological marker analysis (by indirect immunofluorescence and flow cytometry). Peripheral blood lymphocytes were obtained from healthy donors as residues from platelet preparations and B cells were purified according to a technique previously described [9]. Thymocytes were taken from 3-week-old Wistar rat thymus. Cell fractionation procedure. Cell ghosts were prepared from each cell type as previously described [7]. A nuclear lamina-enriched fraction was then obtained according to Gerace et al. [lo] RNA analyses. Total RNA from HeLa, KE 37, and RPM1 6666 cells was extracted by the LiCl-urea procedure according to Auffray and Rougeon [ll]. Poly(A)+ RNAs were isolated and hybridized to a lamin A cDNA clone and glyceraldehyde 3-phosphate dehydrogenase cDNA clone as previously described [ 71. SDS-polyacrylamide gel ekctrophoresis and protein transfer to nitrocelluhe. SDS-polyacrylamide gel electrophoresis was performed on 10% polyacrylamide slab gels according to Laemmli [12]. Proteins were transferred to nitrocellulose by electrophoresis using the semi-dry method [13]. Immobilized proteins were probed with either a 1:500 dilution of an anti-lamin A + C serum (LS-1) or a 1:200 dilution of an anti-lamin B serum (F). These sera have been previously characterized [14, 151. Finally, blots were incubated with ‘261-protein A and reactive bands identified by autoradiography.
RESULTS AND DISCUSSION We have examined the composition of the nuclear lamina in several different human B and T lymphoid cells, typical of distinct differentiation stages, assuming that tumor cells are representative of their nontransformed counterparts [ 161. The early stage of differentiation was represented by pre-B leukemia cells, isolated from two patients, and the T lymphoblastic KE 37 cell line, which is of the cortical thymic type, respectively. As human thymocytes were not available, we included rat thymocytes as representatives of this group. The intermediate stage of differentiation was represented by B and T lymphocytes prepared from peripheral blood lymphocytes of healthy subjects. The fully differentiated stage in the B cell lineage was represented by the human
METHODS
Cells and ceU Zincs. Human established cell limes were grown in RPM1 1640 medium, containing 10% (v/v) fetal calf serum. The T lymphoblast RE 37 cell line was described previously [a]. These leuke-
1 To whom correspondence and reprint requests should be addressed at CEA, DPS/SPE, BP 6,92265 Fontenay-aux-Roses Cedex, France. 145
0014~4S27/90
$3.00
Copyright Q 1990 by Academic F’rsss, Inc. All rights of reproduction in any form reserved.
146
SHORT NOTE
firming our previous results [ 71, whereas two mRNAs of the expected size were present in poly(A)+ RNA isolated from lymphoblastoid cells. As a control, a RNA species that hybridized to a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe was detected in both cell lines. Concomitant expression of lamins of A- and B-type is generally found in vertebrate somatic nuclei [5,6]. Few Plasm. Pre.B PBL exceptions have been reported so far, all of them consistFIG. 1. Immunoblot analysis of lamins. Nuclear envelopes from ing of the absence of lamins A and C: early chicken emlymphoid T cells (upper row) and B cells (lower row) were probed with bryos [ 171, mouse embryonal F9 teratocarcinoma cells either lamin B-specific antibodies (left lanes) or lamiis A + C antibod[5, 18, 191, human T lymphoblasts [ 71, mouse and rat ies (right lanes). Cells used were rat thymocytes (R. Th.), KE 37 lymphoblastic cell line (KE 37), peripheral blood T and B lymphocytes lymphoid cells [20,21], human promyelocytic leukemia (PBL), pre-B acute lymphoblastic leukemias (pre-B), and lymphoHL60 cells [20, 221, and possibly mouse embryos 123, blastoid RPM14366 (Plasm.). In the figure, cells at the same stage of 241. The present study allows us to add rat thymocytes differentiation are aligned vertically. Note in rat thymocytes, KE 3’7 and human pre-B lymphoblasts to this list. Furthercells, and pie-B-ALL the absence of a signal for lamins A and C. more, our results show that there is a correlation between the stage of differentiation of the cells and their lymphoblastoid RPMI-3366 and U266 plasmocytoma lamin A and C complement, as previously reported in cell lines. amphibians for lamins L1 and Lri [25,26]. An apparent Cellular fractions enriched in nuclear lamins were exception to this general pattern is the recent report of prepared from these different cells using a procedure Ig-secreting mouse myeloma cells that lack lamins A and that minimizes proteolysis, and protein samples corre- c [22]. sponding to an identical number of cells were separated So far, there is no example of any somatic cells that by SDS-PAGE and then transferred to nitrocellulose. do not contain lamin B. This particular lamin is the tarLamin B and lamins A and C were identified on these get of extensive cell cycle-dependent post-translational immunoblots by using two autoimmune antisera which modifications, including phosphorylation, methylation, have been previously characterized [14, 151. Figure 1 and isoprenylation (for review see Ref. [l]). It is closely shows the results of these experiments. The lamin B associated with the inner nuclear membrane by interaccontent was nearly equivalent in the five human cell tion with an integral membrane protein [27]. During types, while the amount of lamins A and C varied to a lamina depolymerization at mitosis, lamin B remains aslarge extent. Lamins A and C were undetected in rat thy- sociated with membrane vesicles, whereas the A-lamins mocytes, human T lymphoblasts, and pre-B cells. In become soluble [lo]. It has been suggested that lamin B contrast, in B and T lymphocytes, lamins A and C were could be a component of the nuclear envelope essential present in nearly equivalent amounts in both cell types. for nuclear vesicle targeting during mitosis [lo]. It may Furthermore, an intense signal for lamins A and C was 1234 567 detected in the RPMI-3366 lymphoblastoid cell line and the U266 plasmocytoma cell line (data not shown). -3Kb Since these results were obtained with immunological -2dKb techniques, the observed variations in signal intensity could reflect qualitative rather than quantitative differences. However, we have previously shown that data obtained with these antibodies were in agreement with those obtained using Coomassie staining, proteolytic peptide analysis, and Northern blotting [ 71. Moreover, 8 9 10 11 12 13 titration experiments have shown that binding of autoFIG. 2. Northern blot analysis of mRNA from HeLa cells, KB antibodies to lamins is linear over a wide range of protein concentrations [5]. To further ascertain these observed 37 T lymphoblasts, and RPM143666 lymphoblastoid cells. Total RNA HeLa cells (lane l), KE 37 cells (lane 2), RPM14666 cells (lane variations, Northern blot analysis was performed with a from 5), poly(A)+ RNA from KE 37 cells (lane 3), RPM16666 cells (lane 6) 1.3-kb fragment from the 3’ end of a cDNA coding for and poly(A)- RNA from KE 37 cells (lane 4), RPM14366 cells (lane human lamin A as a probe [3]. This analysis was per- 7) was fractionated on a 1% agarose gel, transferred t.0 nitrocellulose formed on two cell lines exhibiting the two extreme pat- filters and hybridized with a human lamin A-specific probe. As a control of the RNA preparation, total RNA from KE 37 cells (lane 8) and terns of lamin A and C expression, i.e., T lymphoblasts RPM16666 cells (lane ll), poly(A)+ RNA from KE 37 cells (lane 9) (KE 37) and lymphoblastoid cells (RPMI-3366). Figure and RPM16666 cells (lane 12), and poly(A)- RNA from KE 37 cells 2 shows that no RNAs for lamins A and C were detected (lane 10) and RPM14366 cells (lane 13) was hybridized with a rat in the poly(A)+ RNA extracted from KE 37 cells, con- GAPDH probe. R.Th.
KE37
PBL
SHORT NOTE
be because of this critical function that lamin B is expressed constitutively in all somatic cells such as the lymphoid cells at all stages of differentiation used in the present study. Since isolated lamin B self-associates only weakly in vitro [28], it remains to be explained how a functional lamina can be assembled without lamins A and C. Because of the existence of cells that express only lamin B, it should be postulated that lamin B is able to form homopolymers in Co. We thank Dr. F. McKeon for the gift of anti-lamin A + C serum and lamin probes, Dr. F. Danon for the gift of anti-lamin B serum, Dr. J-M. Blanchard for the gift of GAPDH probe, Dr. F. Sigaux for providing and characterizing the pre-B ALL cells, Drs. J. Morizet and A. Bernard for the gift of anti-T mAb, and Drs. B. Guiard and Moreau for precious technical advice. We thank also Drs. C. Klotz, B. Maro, and G. Kerryer for their constant interest, Dr. J. Beisson for material and moral support, and Dr. H. Worman for critical reading of the manuscript. This research was supported by Grant ARC 6334. REFERENCES 1. Nigg, E. A. (1989) Curr. Opinion Cell Biol. 1,435-440. 2. Fisher, D. Z., Chaudhary, N., and Blobel, G. (1986) Proc. Natl. Acad. Sci. USA 83,6450-6454. 3. McKeon, F. D., Kirschner, M. W., and Caput D. (1986) Nature (London) 319,463-468. 4. Krohne, G., Wolin, S. L., McKeon, F. D., Franke, W. W., and Kirschner, M. W. (1987) EMBO J. 6,3801-3808. 5. Worman, H. J., Laxaridis, I., and Georgatos, S. D. (1988) J. Bial. &em. 263,12,135-12,141. 6. Lehner, C. F., Kurer, V., Eppenberger, H. M., and Nigg, E. A. (1986) J. Bial. Chem. 261,13,293-13,301. 7. Guilly, M-N., Bensussan, A., Bourge, J-F., Bomens, M., and Courvalin, J-C. (1987) EMBO J. 6,3795-3799. Received December 11,1989 Revised version received March 12,199O
147
8. Mayer, L., Shu Man Fu, and Kunkel, H. G. (1982) J. Exp. Med. 166,1860-1865. 9. Petit-Koskas, E., Genot, E., Lawrence, D., and Kolb, J-P. (1988) Eur. J. Immunol. 18.111-115. 10. Gerace, L., Comeau, C., and Benson, M. (1984) J. Cell Sci. Suppl. 1,137-160. 11. Auffray, C., and Rougeon, F. (1980) Eur. J. B&hem. 107,303314. 12. Laemmli, U. K. (1970) Nature (Landon) 227,~685. 13. Kyhse-Andersen, J. (1984) J. Biochem. Biaphys. Methods 10, 203-209. 14. McKeon, F. D., Tuffanelli, D. L., Fukuyama, K., and Kirschner, M. W. (1983) Proc. Natl. Acad Sci. USA 80,4374-4378. 15. Guilly, M-N., Danon, F., Brouet, J-C., Bornens, M., and Courvalin, J-C. (1987) Eur. J. Cell Bial. 43.266-272. 16. Osborn, M., and Weber, K. (1986) Trends Bzixhem. Sci. 11,469472. 17. Lehner, C. F., Stick, R., Eppenberger, H. M., and Nigg, E. A. (1987) J. Cell Bial. 105,577-587. 18. Stewart, C., and Burke, B. (1987) CeU61,383-392. 19. Lebel, S., Lampron, C., Royal, A., and Raymond, Y. (1987) J. CeU Bial. 105,1099-1104. 20. Hornbeck, P., Huang, K-P., and Paul, W. E. (1988) J. BbL Chem. 86,2279-2283. 21. Kaufman, S. H. (1989) J. Bial. Chem. 264,13,946-13,955. 22. Paulin-Levasseur, M., Scherbarth, A. M., Traub, U., and Traub, P. (1988) Eur. J. Cell Biol. 47,121-131. 23. Houliston, E., Guilly, M-N., Courvahn, J-C., and Maro, B. (1988) Development 102,271-278. 24. Ribber, R-A, Weber, K., and Osbom, M. (1989) Development 105,365-378. 25. Stick, R., and Hausen, P. (1985) CeU41,191-200. 26. Benavente, R., Krohne, G., and Franke, W. W. (1985) Cell 41, 177-190. 27. Worman, H. J., Yuan, J., Blobel, G., and Georgatos, S. D. (1988) Prac. Natl. Acad Sci. USA 85,8531-8534. 28. Georgatos, S. D., Stoumaras, C., and Blobel, G. (1988) Proc. Natl. Acad. Sci. USA 86,4325-4329.
