RICHARD M. M. van den HEUVEL1, GUILLAUME J. J. M. van EYS1*, FRANS C. S. RAMAEKERS2, ... and HANS BLOEMENDAL1 J. 'Department of ...
Intermediate filament formation after transfection with modified hamster vimentin and desmin genes RICHARD M. M. van den HEUVEL1, GUILLAUME J. J. M. van EYS 1 *, FRANS C. S. RAMAEKERS2, WIM J. QUAX'f, WILMA T. M. VREE EGBERTS1, GERT SCHAART2, H. THEO M. CUYPERS' and HANS BLOEMENDAL1 J 'Department of Biochemistiy and zDepartmenl of Pathology, University of Sijmegen, Geerl Ginoteplein Xoord 21, 6525 /•.'/ Sijmegen, The Xetherlands * Present address: Laboratory of Tropical Hygiene (I WO), Royal Tropical Institute, Amsterdam, The Netherlands | Present address: Department of Microbiology, Gist Brocades, Delft, The Netherlands X Author for correspondence
Summary Previously we cloned and characterized the hamster intermediate filament genes coding for vimentin and desmin. It was demonstrated that the cloned desmin gene was expressed after gene transfer and that the newly synthesized protein assembles into intermediate filaments. Here we present data on the transfection of modified vimentin and desmin genes onto simian virus 40transformed hamster lens cells and HeLa cells. Modifications included: (1) removal of exons encoding the desmin COOH-terminal domain; (2) exchange of exons encoding the COOH-terminal domain of vimentin and desmin; and (3) deletion of part of exon I of desmin, coding for the NH2-terminal amino acids 4-148. In transient transfection assays it was shown that the modifications in the COOH region had no detectable effects on the filament forming potential of the encoded proteins as demonstrated with
desmin antibodies in the indirect immunofluorescence test. On the other hand, deletion of a considerable part of the first exon of the desmin gene results in a lack of bona fide intermediate filament formation. Immunoblotting with desmin antibodies of cell populations enriched for the transfected modified genes showed that the presence of the modified genes results in the synthesis of the corresponding proteins with the expected molecular weights. From our results we conclude that in vivo: (1) the presence of the COOH terminus is not essential for filament formation; (2) that an exchange of COOH-terminal parts of vimentin and desmin does not prevent assembly into intermediate filaments; and (3) that removal of the NH2 terminus of desmin affects intermediate filament formation.
Introduction
cytokeratins, while neurofilament proteins are found in neurones. Glial cells contain IF composed of the glial fibnllary acidic protein, and desmin is found in muscle cells. Vimentin is the IF protein in cells of mesenchymal origin and is also found in many cultured cells (for a review, see Traub, 1983). Recently, the lamins also have been described as being members of the IF protein family (Franke, 1987). The structures of the proteins that constitute the IF have been studied extensively, but major problems
Intermediate filaments (IF) have been characterized as a unique type of cytoskeletal structure in eukaryotic cells. Immunological, biochemical and sequence data have permitted the distinction of five different classes of IF constituents. The expression of these different classes is regulated in a tissue-specific manner, which parallels the differentiation of tissues (Franke et al. 1982; Osborn & Weber, 1982). Epithelial cells contain Journal of Cell Science 88, 475-482 (1987) Printed in Great Britain © The Company of Biologists Limited 1987
Key words: vimentin gene, desmin gene, hamster lens cells, HeLa cells.
475
concerning regulation of gene expression, IF organization and, in particular, function are still unsolved. Amino acid sequence data have demonstrated a common structure for all IF proteins (Geisler et al. 1982; Steinert et al. 1985). They all share an alpha-helical central domain (or rod) of about 311-314 amino acids, which has a highly conserved secondary structure and is flanked by end-domains of variable size and chemical characteristics. The alpha-helical domain is thought to be involved in coiled-coil interactions between two identical or different IF molecules and to play an essential role in filament formation (Frazerei al. 1986; Weber & Geisler, 1984). In vitro reassociation experiments have given conflicting and incomplete results concerning the contribution of NH 2 - and COOHterminal regions in filament formation (Geisler et al. 1982; Kaufmann et al. 1985; Lu & Johnson, 1983; Traub & Vorgias, 1983). Moreover, these experiments do not give information about the role of the different IF protein domains in interactions of filaments with other cellular components. Introduction of IF mRNAs (Franke et al. 1984; Kreis et al. 1983) or IF genes (Quax et al. 1985) into cells offers opportunities to study the function of the IF domains in filament formation in vivo and their subsequent interaction with cellular structures. In the present report we describe a system in which the role of different domains of IF proteins in filament formation can be studied within the living cell. For this purpose modified vimentin and desmin genes were constructed and transfected onto hamster lens cells. The choice of such a homologous cell line has the advantage that potential species barriers do not have to be considered in the interpretation of the results. Effects of these modifications on IF formation, cell shape and cell division were studied. Materials and methods Construction of modified IF genes Isolation and characterization of the vimentin (pVViml) and desmin (pDDesl) genes have been described extensively (Quax et al. 1983, 1984, 1985; Quax-Jeuken et al. 1983). In general, the procedures suggested by Maniatis et al. (1982) were followed for construction of plasmids and preparation of plasmid DNA. All IF genes were ligated into the pUC19 plasmid. The plasmid designated pVVim2 contains the promoter region of the vimentin gene. A schematic representation of this hybrid gene is shown in Fig. 1. In order to construct this plasmid, the£ta/wHI-.Bg7II fragment (position 0 up to +9-2 on the physical map) of vimentin (Quax et al. 1984) was ligated into the BamHl site of pUC19. The EcoR\-Fsp\ fragment (position +4 up to +7-4 on the physical map) of the desmin gene (Quax et al. 1985) was ligated into the EcoRl-Smal site of pUC19. The vimentin BamHl-EcoRl fragment and the desmin £coRI-///wdIII fragment obtained from these plasmids were recloned into 476
R. M. M. van den Heuvel et al.
the Baw;HI-///«dIII site of pUC19, resulting in pVVim2. The coding sequences of this construct are exons 1-6 of the vimentin gene and exons 7-9 of the desmin gene. The protein encoded by this construct contains amino acids 1-408 of vimentin followed by amino acids 414-468 of desmin (Fig. 2). The pDDes2 construct contains the desmin promoter region. In order to construct this hybrid gene (Fig. 1), the EcoRl-Bglll fragment (position —3-3 up to + 4 8 on the physical map) of desmin (Quax et al. 1985) was combined with the BglW fragment (position + 9 2 up to +13-7 on the physical map) of the vimentin gene (Quax et al. 1983) in the EcoRl-BamHl site of pUC19. The coding sequences of pDDes2 comprise exons 1-6 of the desmin and exons 7-9 of the vimentin gene. This construct encoded a protein that contains amino acids 1-413 of desmin and amino acids 409-465 of vimentin (Fig. 2). The pDDes3 construct contains the regulatory sequences and exons 1-6 of the desmin gene. Exons 7-9 were replaced by exon 9 of vimentin, which also contained the polyadenylation signal. The frameshift at the beginning of the vimentin exon 9 resulted in a protein composed of amino acids 1-413 of desmin (Fig. 2). In order to construct this plasmid, the Hindi fragment (position +11 0 up to +14-3 on the physical map) of vimentin (Quax et al. 1983) was cloned in the Sinai site of pUC19. The plasmid was linearized with EcoRl and the EcoRl fragment (position - 3 - 4 up to +4-0 on the physical map) of desmin (Quax et al. 1985) was cloned in this site. The pDDes4 construct contains the desmin gene with a 435 bp (base-pair) deletion in exon 1 encoding the NH2 terminus of desmin. This was achieved by replacing the BamHl fragment (position 0 up to +0-8 on the physical map) of the desmin gene (Quax et al. 1985) by a 450 bp deleted BamHI fragment. This deleted fragment was made by cloning the BamHl fragment in pUC19 and subsequent removal of the sequences between the Stu\ and Sma] sites (position +0-2 up to +0-6 on the map). The protein encoded by this plasmid lacks amino acids 4-148, while amino acid 3 changes from alanine to glycine (Fig. 2). Table 1 summarizes the most relevant data of the constructs, while Fig. 2 depicts a schematic representation of the expected protein products resulting from these constructs after transfection. Transfection and immunochemical procedures Simian virus 40 (SV40)-transformed hamster lens cells (Bloemendal et al. 1980) and HeLa cells were transfected by calcium phosphate precipitation with the constructs described above and assayed 48 h after transfection with a polyclonal rabbit desmin antiserum (polyDes). For doublelabel immunofluorescence studies a monoclonal antibody to vimentin (RV202) was applied in combination with polyDes. Methods of transfection, immunofluorescence and preparation of the antisera have been described (Broers et al. 1986; Quaxef al. 1985; Ramaekers et al. 1987). In addition, the hamster lens cells were cotransfected with pSV-neo in combination with each of the constructs described above (ratio 1:10) (Southern & Berg, 1982). Stable transformants
Desmin
Vimentin
COOH
NH 2
COOH
NH2
i
—
poly(A)
t I
m
CZ
Tt_
2
21
I M
2B3ZE1
12
Y
21
U S E IX
TATA-box H
BIHB
4HHFH
pDDesi
pVVimi
HE YVl SI 211
I
III222I
SIB
poly (A)
ligation-sile
TATA-box
I
pVVim2 5
TATA-box
TATA-box
ligation-site
2i
vi
a
a
poly(A)
' -HJ—
pDDes2
ligation-site poly(A)
pDDes3
TATA-box
poly(A)
ligation-site
pDDes4 i Fig. 1. Schematic representation of the relation between the structure of the vimentin and desmin genes and their respective protein products, as well as the structure of the modified genes pVVim2, pDDes2, pDDes3 and pDDes4. Black boxes represent exons derived from the desmin gene, whereas open boxes represent exons derived from the vimentin gene. Restriction sites are indicated as follows: B, Bam HI; Bl, Bglll; E, EcoRl; F, Fspl; H, Hindi; S, Smal; St, Still.
from these cotransfections, as selected for resistance to the antibiotic geneticin, were also assayed with the antiserum polyDes. The modified IF proteins encoded by the constructs in hamster lens cells were analysed by immunoblots of either cvtoskeletal preparations or total cell lvsates from enriched stably transformed cell populations. In the immunoblots the modified desmin and vimentin proteins were detected with the rabbit antiserum polyDes. For comparison, BHK-21 cell cvtoskeletal preparations were run parallel on the same immunoblot. Non-transfected hamster lens cells were used as controls and did not show a reaction with the desmin antiserum in the immunofluorescence or immunoblotting assays. Preparation of the cvtoskeletal extracts, gel electrophoresis and immunoblotting were performed essentially as described (Broers et al. 1986).
Results Transfer of the modified desmin and vimentin genes onto transformed hamster lens cells resulted in filament formation as summarized in Table 1. Transfection with the hamster desmin gene (pDDesi) was used as a control throughout these studies. As described before (Quax et al. 1985) transfection of pDDesi gave a number of cells that showed a filamentous staining pattern when tested with a polyclonal desmin antiserum (Fig. 3A). In order to estimate the importance of the non-helical protein domains in IF formation, four modified IF genes were constructed as described in Materials and methods and transfected onto hamster lens cells.
Filament formation by modified vimentin and desmin
477
Head
Rod
Tail
408
463
413
469
pVVim2
pDDcs2
1 L
pDDes3
I
pDDes4
1-2 D
413 3 Ala
468
149 1
I
1 Gly
Vimentin Desmin
Fig. 2. Schematic representation of proteins resulting from the modified intermediate filament gene constructs.
The gene sequences coding for the COOH-terminal domains of desmin and vimentin were exchanged in the constructs pVVim2 and pDDes2. Cells transfected with pVVim2 and assayed after 48 h by the indirect immunofluorescence technique with polyclonal antibodies directed against desmin showed typical IF patterns in approximately 1 or 2 % of the cells. From Fig. 3C,D it appears that the hybrid protein resulting from expression of pVVim2 (see Fig. 2) is generally able to assemble into a filamentous evtoskeletal network with normal IF features throughout the cytoplasm of the cell. Occasionally, intensely stained desmin-positive dots were observed within the cytoplasm of positive cells (Fig. 3E). Most of the positive cells occurred in small clusters of four to eight cells, suggesting that
the presence of the hybrid protein does not severely interfere with cell division. This could be further sustained by experiments using stably transfected cell lines, which showed a normal division pattern (unpublished data). Gross effects on cell shape, as judged by immunofluorescence, were not observed. The formation of filaments after introduction of the hybrid gene pDDes2, as assayed with the aid of a polyclonal antibody directed against desmin, occurred in a lower number of cells. Less than 0-5% of the transfected hamster lens cell population showed the typical IF staining pattern (Fig. 3B). The actual number of cells expressing pDDes2 may be higher, since we also observed cells with very low levels of fluorescence, which were not included in the counting of positive cells. However, intensely stained desmin-positive dots occurred more often in cells transfected with pDDes2 than those transfected with pVVim2. Transfection of hamster lens cells with pDDes3, the construct encoding a desmin polypeptide without the COOH-terminal domain (Fig. 2), resulted in IF with normal features (Fig. 3F,G). The fluorescence pattern was comparable to the one found in cells transfected with the constructs described previously. Different results were, however, obtained after transfection with pDDes4, the construct missing sequences encoding amino acid residues 4-148 of the NH2-terminal domain and about 15% of the alphahelical region (Fig. 2). Most cells positive with the desmin antibody polyDes showed a diffuse pattern of fluorescence (Fig. 3H). Since the results obtained with pDDes4 differed dramatically from those with the other constructs, the experiments were repeated in HeLa S3 cells. Transfection with pDDes4 onto these cells also resulted in a diffuse staining pattern with polyDes (Fig. 31). No cells with filamentous structures were observed, although transfection with pDDesl
Table 1. Code and characteristics of the different constnicts used in this study with their respective desminstaining reaction patterns after transfection onto hamster lens and HeLa cells Construct code pDDcsl pVVini2 pDDes2 pDDcs3
pDDes4 pUC19
Characteristics of the construct Hamster desmin gene Exons 7, 8, 9 of vimentin gene replaced by exons 7, 8, 9 from desmin gene Exons 7, 8, 9 of desmin gene replaced by exons 7. 8, 9 from vimentin gene Desmin gene in which exons 7, 8, 9 have been replaced by exon 9 of vimentin, creating a stop-codon at the beginning of exon 9 Desmin gene lacking nuclcotides 181—621 within exon If Vector
Amino acid sequences encoded for
Staining reaction with polvDcs*
Desmin, 1-468 Vimentin, 1-408 Desmin, 414-468 Desniin, 1—413 Vimentin, 409-465 Desmin, 1-413
+F ++F
Desmin, 1, 2; Glv: 149-468 None
+D
+F +F
* + / + + F, filamentous staining reaction observed in part of the cells after transfection and immunostaining with the desmin antiserum polyDes. + D , diffuse staining reaction observed in most of the polyDes positive cells. —, no staining reaction with polyDes. fQuaxef (it. (1985).
478
R. M. M. van den Heuvel et al.
Fig. 4. Double-label indirect immunofluorescence assay of HeLa cells, transfected with pVVim2, stained for DNA with Hoechst 33258 (A), and incubated with the monoclonal vinientin antibody RV202 (B) and the polyclonal antiserum polyDes (C). Bar indicates 20/lm.
resulted in the typical IF network (Quax et al. 1985). Preliminary results using the double-label immunofluorescence technique indicate that the hybrid proteins encoded by pVVim2 and pDDes2 colocalize with the
native vimentin, as far as can be deduced from the light-microscopic observations (see, e.g., Fig. 4). To enrich the population of cells expressing the modified genes, hamster lens cells were cotransfccted
Fig. 3. Indirect immunofluorescence assays for filament formation of the proteins encoded by constructs pDDesl (A), pDDes2 (B), pVVim2 (C,D,E), pDDes3 (F,G) and pDDes4 (H) after transfection onto hamster lens cells. I. He La S3 cells transfected with pDDes4. The cells were studied with a polyclonal rabbit antiserum to chicken gizzard desniin. Bars, 20,um.
Filament formation by modified vimentin and desmtn
479
M r x10~ — 57 D
— 53 •
S
m
—45 —37
a
b
c
with the plasmid pSV-neo and put under geneticin selection. In this way we increased the number of positive cells to about 15-25 %. The IF patterns in the neomycin-selected cells, expressing the constructs, were identical to the patterns of cells observed in the transient assay. Extracts were prepared from these cell pools and studied by immunoblotting using the polyclonal desmin antiserum (Fig. 5). For pDDesl-transfected cells the desmin protein could be detected and shown to comigrate with hamster desmin derived from BHK-21 cells. For pVVim2-transfected cells a protein band was demonstrated with an apparent molecular weight of approximately 57xlO 3 . This protein reacted with the polyclonal desmin antiserum, whereas no such product was found in non-transfected cells. The molecular weight of the pVVim2 product as calculated from the protein blot, deviates from the expected molecular weight based on sequence data. A similar phenomenon has also been described for hamster vimentin (Quax-Jeuken et al. 1983) comigrating with the pVVim2 product on SDS-polyacrylamide gels. For pDDes3- and pDDes4-transfected cells, protein bands with molecular weights of approximately 45 X103 and 37X103, respectively, were found to cross-react with the desmin antiserum. The calculated molecular weights based on the amino acid sequence are 47-Ox 103 and 37-5X103, respectively, in good agreement with the experimental values.
Discussion Assembly of IF has been investigated in test tube assays by studying reassociation and filament formation of dissociated and modified IF proteins (Geisler et al. 480
R. M. M. van den Heuvel et al.
g
3
Fig. 5. Immunoblotting of geneticin-selected hamster lens cells transfected with constructs pVVim2 (lane b), pDDesl (lane d), pDDes3 (lane e) and pDDes4 (lane f). The proteins encoded by these constructs were detected with a rabbit antiserum to chicken gizzard desmin (polyDes) and are indicated with arrows and their respective molecular weights. For comparison, lanes a and c contain immunoblots of a cytoskeletal preparation from BHK-21 cells, containing intact hamster desmin (D) and its breakdown products (bracket). Note that the antiserum does not react with any component in the vimentin region. Lane g shows the reaction of non-transfected hamster lens cells with polyDes, with an aspecific band indicated by an asterisk.
1982; Kaufmann et al. 1985; Lu & Johnson, 1983; Quinlan et al. 1986; Weber & Geisler, 1984). This approach has two disadvantages. First, reassociation /;/ vitro does not necessarily reflect filament formation /;/ vivo. In assays in vitro relevant factors may be absent, whereas aspecific reassociation cannot be excluded. Second, enzymic modification of the proteins is restricted, as compared to the modifications that can be achieved at the DNA level. In this paper we present a different approach to the study of IF formation. We took advantage of the availability of cloned desmin and vimentin genes (Quax et al. 1983, 1985), and investigated the possibility of using native and modified genes to study the contribution of different protein domains in filament formation. The results with a number of constructs demonstrate the potential of this method. Of the constructs used in this series of experiments only pDDes4, missing the sequences coding for the NH 2 -tenninal domain and a small part of the alpha-helix of desmin, did not generate bona fide IF. This observation confirms results of in-vitro reassociation studies (Geisler et al. 1982; Kaufmann et al. 1985; Traub & Vorgias, 1983; Weber & Geisler, 1984). In contrast, Lu & Johnson (1983) observed in in-vitro reassociation studies, that the large thrombic fragment (residues 97—463) of desmin was found to form IF structures indistinguishable from those of intact desmin. In view of this discrepancy future studies will have to elucidate whether the missing 15 % of the alpha-helical domain is responsible for the lack of IF formation. The diffuse fluorescence patterns seen in hamster as well as HeLa cells and the results of immunoblotting demonstrating the presence of the pDDes4 protein suggest a block on
filament formation only, but not on transcription of the plasmid or translation of the mRNA. On the other hand, removal of sequences of the desmin gene, encoding most of the COOH-terminal domain does not apparently interfere with the capability of the altered protein to form IF. Also, exchange of the last three exons (coding for the COOH domain) of desmin and vimentin has no effect on IF formation as far as can be concluded from indirect immunofluorescence staining. These data indicate that the COOH-terminal domain has only a minor function, if any, in the formation of IF in the cell. This conclusion is in agreement with most of the results of in-vitro reassociation assays (Geisler et al. 1982; Kaufmann et al. 1985; Traub & Vorgias, 1983; Weber & Geisler, 1984). At any rate our experiments show that transfection of altered IF genes can generate proteins that participate in a typical IF structure. From our results one cannot deduce whether the plasmid-encoded proteins form an independent IF network, stick to pre-existing filaments or associate with IF proteins encoded by the endogenous vimentin gene. However, our double-label studies do not support the first possibility. The results obtained with the pDDes4-transfected cells indicate that coiled-coil interactions per se are not sufficient for IF formation, although such an interaction may explain the fibrillar fragments observed in some of these cells. Preliminary results with transfection of cells devoid of a vimentin cytoskeleton indicate that our constructs are expressed in a similar way as that described here for hamster lens cells and HeLa cells. These data will be discussed in a future report. Future studies using immunoelectron microscopy may eventually define more accurately regions involved in IF formation and will permit investigations on the interaction of IF structures with IF-associated proteins. We thank Kick Verrijp for excellent technical assistance and Yvonne Stammes for secretarial help in the preparation of the manuscript. This study was supported by grants from the Netherlands Cancer Foundation (KWF) and the Netherlands Organization for the Advancement of Pure Research (ZWO).
analysis using a panel of monoclonal and polyclonal antibodies. J. Cell Sci. 83, 37-60. FRANKE, W. W. (1987). Nuclear lamins and cytoplasmic intermediate filament proteins: a growing multigene family. Cell 48, 3-4. FRANKE, W. W., SCHMID, E., MITTNACHT, S., GRUND, C.
& JORCANO, J. L. (1984). Integration of different keratins into the same filament system after microinjection of mRNA for epidermal keratins into kidney epithelial cells. Cell 36, 813-825. FRANKE, W. W., SCHMID, E., SCHILLER, D. L., WINTER, W., JARASCH, E. D., MOLL, R., DENK, H., JACKSON, B.
W. & ILLMENSEE, K. (1982). Differentiation-related patterns of expression of proteins of intermediate-size filaments in tissues and cultured cells. Cold Spring Harbor Symp. quant. Biol. 46, 431-454. FRAZER, R. D. B., MACRAE, T . P., PARRY, D. A. D. &
SUZUKI, E. (1986). Intermediate filaments in alpha.keratins. Proc. natn. Acad. Sci. U.S.A. 83, 1179-1183. GEISLER, N., KAUFMANN, E. & WEBER, K. (1982).
Protein-chemical characterization of three structurally distinct domains along the protofilament unit of desmin 10 nm filaments. Cell 30, 277-286. KAUFMANN, E., WEBER, K. & GEISLER, N. (1985).
Intermediate filament forming ability of desmin derivatives lacking either the amino-terminal 67 or the carboxy-terminal 27 residues. _7. molec. Biol. 185, 733-742. KREIS, T . E., GEIGER, B., SCHMID, E., JORCANO, J. &
FRANKE, W. (1983). De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin. Cell 32, 1125-1137. Lu, P. & JOHNSON, P. (1983). The N-terminal domain of desmin is not involved in intermediate filament formation: evidence from thrombic digestion studies. Int.jf. biol. Macromol. 5, 347-350. MANIATIS, T., FRITSCH, E. F. & SAMBROOK, J. (1982).
Molecular Cloning, A Laboratoiy Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. OSBORN, M. & WEBER, K. (1982). Intermediate filaments: Cell-type-specific markers in differentiation and pathology.- Cell 31, 303-306. QUAX, W . , VAN DEN BROEK, L . , VREE EGBERTS, W . , RAMAEKERS, F. & BLOEMENDAL, H. (1985).
Characterization of the hamster desmin gene: expression and formation of desmin filaments in nonmuscle cells after gene transfer. Cell 43, 327-338.
References
QUAX, W . , VAN DEN HEUVEL, R . , VREE EGBERTS, W . ,
BLOEMENDAL, H., LENSTRA, J. A., DODEMONT, H., RAMAEKERS, F. C. S., GROENEVELD, A. A., DUNIA, I. &
BENEDETTI, E. L. (1980). SV40-transformed hamster lens epithelial cells: A novel system for the isolation of cytoskeletal messenger RNAs and their translation products. Expl Eye Res. 31, 513-525.
QUAX-JEUKEN, Y. & BLOEMENDAL, H. (1984).
Intermediate filament cDNAs from BHK-21 cells: Demonstration of distinct genes for desmin and vimentin in all vertebrate classes. Proc. natn. Acad. Sci. U.S.A. 81, 5970-5974. QUAX, W., VREE EGBERTS, W., HENDRIKS, W., QUAX-
BROERS, J. L. V., KARNEY, D. N., KLEIN ROT, M.,
JEUKEN, Y. & BLOEMENDAL, H. (1983). The structure of
SCHAART, G., LANE, E. B., VOOUS, G. P. &
RAMAEKERS, F. C. S. (1986). Intermediate filament proteins in classic and variant types of small cell lung carcinoma cell lines: a biochemical and immunochemical
the vimentin gene. Cell 35, 215-223. QUAX-JEUKEN, Y., QUAX, W. & BLOEMENDAL, H. (1983).
Primary and secondary structure of hamster vimentin
Filament fonnation by modified vimentin and desmin
481
predicted from the nucleotide sequence. Pmc. natn. Acacl. Sci. U.S.A. 80, 3548-3SS2. QUINLAN, R. A., HATZFELD, M., FRANKE, W. W., LUSTIG,
STEINERT, P. M., STEVEN, A. C. & ROOP, D. R. (1985).
The molecular biology of intermediate filaments. Cell 42, 411-419.
A., SCHULTHESS, T. & ENGEL, J. (1986).
TRAUB, P. & VORGIAS, C. E. (1983). Involvement of the
Characterization of dimer subunits of intermediate filament proteins..7. molec. Biol. 192, 337-349.
N-terminal polypeptide of vimentin in the formation of intermediate filaments. J. Cell Sci. 63, 43-67. TRAUB, P. (1983). Intermediate Filaments, a Reviezv. Berlin, Heidelberg, New York, Tokyo: Springer-Verlag. WEBER, K. & GEISLER, N. (1984). Intermediate filaments, from wool alpha-keratins to neurofilaments: a structural overview. In Cancer Cells, The Transfonned Phenotvpe (ed. A. J. Levine, G. F. van de Woude, W. C. Topp & J. D. Watson), vol. 1, pp. 153-159. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
RAMAEKERS, F. C. S., HUIJSMANS, A., SCHAART, G.,
MOESKER, 0 . & Voous, G. P. (1987). In Application of Monoclonal Antibodies in Tumor Pathology (ed. D. J. Ruiter, G. J. Fleuren & S. O. Warnaar), pp. 65-85. Dordrecht, Boston, Lancaster: Martinus Nijhoff. SOUTHERN, P. J. & BERG, P. (1982). Transformation of
mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. molec. appl. Genet. 1, 327-341.
482
R. M. M. van den Henvel et al.
(Received 1 June 1987 -Accepted 20 July 1987)
