characterization of a second highly conserved b-type lamin present in cells previously thought to...

Chromosoma (1990) 99:379-390 C H R O M O S O M A © Springer-Verlag 1990

Characterization of a second highly conserved B-type lamin present in cells previously thought to contain only a single B-type lamin Thomas H. H~iger 1, Kurt Zatloukal 1, Irene Waizenegger 1, and Georg Krohne 1

1 Division of Membrane Biology and Biochemistry, Institute of Cell and Tumor Biology, German Cancer Research Center, im Neuenheimer Feld 280, D-6900 Heidelberg, Federal Republic of Germany z Division of Molecular Pathology, Institute of Pathology, University of Graz, Auenbruggerplatz 25, A-8036 Graz, Austria

Received June 13, 1990 Accepted June 15, 1990 by U. Scheer

Abstract. Previous analyses of the nuclear lamina of mammalian cells have revealed three major protein components (lamins A, B and C) that have been identified by protein sequence homology as members of the intermediate filament (IF) protein family. It has been claimed that mammalian cells contain either all three lamins or lamin B alone. Using monoclonal antibodies specific for B-type lamins and cDNA cloning we identified a second major mammalian B-type lamin (murine lamin B2) , thus showing that lamin composition in mammals is more complex than previously thought. Lamin Bz is coex- pressed with lamin B1 (formerly termed lamin B) in all somatic cells and mammalian species that we analysed, including a variety of cells currently believed to contain only a single lamin. This suggests that two B-type lamins are necessary to form a functional lamina in mammalian somatic ceils. By cDNA cloning we found that Xenopus laevis lamin LII is the amphibian homolog of mammalian lamin B 2. Lamin expression during embryogenesis of amphibians and mammals shows striking similarities. The first lamins expressed in the early embryo are the two B-type lamins, while A-type lamins are only detected much later in development. These findings indicate that the genomic differentiation into two B-type lamins occurred early in vertebrate evolution and has been main- tained in both their primary structure and pattern of expression.

Introduction

The nuclear lamina is a karyoskeletal structure closely apposed to the inner nuclear membrane of many, and probably all eukaryotic cells. Its components, the nuclear lamins (Mr between 60000 and 75000), are best characterized in vertebrate cells (for recent reviews see Gerace and Burke 1988; Nigg 1989), and display structural homology to the cytoplasmic intermediate filament (IF)

Offprint requests to: G. Krohne

proteins (McKeon et al. 1986; Fisher et al. 1986; Aebi et al. 1986; for review see Franke 1987). Lamins have been implicated to play a functional role in the formation of the nuclear envelope and in chromatin organization (Burke and Gerace 1986; Benavente and Krohne 1986; Holtz et al. 1989).

Based mainly on the analysis of lamins derived from rat liver nuclei and a few cultured cell types (Gerace et al. 1978; Gerace and Blobel 1980; Gerace et al. 1984) a relatively simple model of lamina composition and formation has been proposed: mammalian cells contain three major lamins termed A, B and C, usually present in similar amounts, which occur in a polymeric form in interphase cells and are disassembled during mitosis accompanied by their hyperphosphorylation. While lamin B remains membrane associated during mitosis, lamins A and C are transiently solubilized as discrete molecular entities (Gerace and Blobel 1980). The distinction of two categories of lamins, i.e. A- and B-type lamins, has also been supported by sequence analysis and can be extended to amphibian and avian species (Krohne et al. 1987; Wolin et al. 1987; H6ger et al. 1988; Peter et al. 1989; Vorburger et al. 1989a).

There are, however, recent experimental findings which cannot be readily accommodated in this model: (i) the sequence of lamin Lin of the amphibian species, Xenopus laevis, the only major lamin present in oocytes (Krohne et al. 1981), does not fall into one of these two subfamilies (Stick 1988). (ii) In chicken, two different B-type lamins have been identified by cDNA cloning (Peter et al. 1989; Vorburger et al. 1989a). (iii) Lamin B is not only phosphorylated but also carboxy-methyl- esterified in a cell-cycle dependent manner (Chelsky et al. 1987), a modification which might play a role in the reformation of the nuclear envelope in telophase (Chelsky et al. 1989). (iv) Lamins A and B are isopreny- lated (Beck et al. 1988; Wolda and Glomset 1988), probably by an all-trans geranylgeranyl group (Rilling et al. 1990; Farnsworth et al. 1990) at a hallmark cysteine near the carboxy-terminus, a region that is crucially involved in nuclear lamina assembly (Krohne et al. 1989; Holtz

380

et al. 1989; see also Vorburge r et al. 1989b). However , this mo ie ty is los t f rom lamin A by c a r b o x y - t e r m i n a l t r i m m i n g (Beck et al. 1988; see also Weber et al. 1989). (v) Moreove r , lamins A and C have no t been found in the l aminae o f a var ie ty o f cells and tissues ( K r o h n e et al. 1981 ; S tewar t and Burke 1987; Lebel et al. 1987; Gu i l ly et al. 1987), and the m o l a r ra t io o f the ind iv idua l l amin po lypep t ide s can differ g rea t ly in di f ferent cell types (Benavente et al. 1985; Lehner et al. 1987; Wor - m a n et al. 1988a).

The existence o f a f ou r th m a m m a l i a n lamin, l amin B2, has recent ly been shown by di rec t p ro t e in sequencing (Weber et al. 1990; for i m m u n o l o g i c a l inves t iga t ions see Lehner et al. 1986; K a u f m a n n 1989), suggest ing tha t the l amin fami ly o f p ro te ins in m a m m a l s m a y be as complex as in a m p h i b i a n s in which five di f ferent lamins h a d pre- v ious ly been ident i f ied (Benavente et al. 1985; Benavente and K r o h n e 1985; Stick a n d Hausen 1985; Wol in et al. 1987). The ini t ia l s tudies on a m p h i b i a n lamins had re- vea led var ious pa t t e rn s o f cel l - type specific express ion o f lamins ( K r o h n e e t a l . 1981; Benavente et al. 1985; K r o h n e and Benavente 1986), a concep t tha t was la ter ex tended to m a m m a l s (Stewar t and Burke 1987; Lebel et al. 1987) and bi rds (Lehner et al. 1987).

Us ing m o n o c l o n a l an t ibod ie s which recognize exclusively B- type lamins in somat i c cells o f a m p h i b i a n s and m a m m a l s , we have ident i f ied a fou r th m a j o r m a m m a l i a n lamin. Its a m i n o acid sequence as deduced f rom a mur - ine c D N A clone shows s t r ik ing h o m o l o g y to l amin Bz o f chicken. M o s t in teres t ingly , it is expressed in several cell types inc lud ing mouse e m b r y o n a l cells which have so far been t hough t to con ta in only one B-type lamin. In add i t ion , we have c loned and sequenced the o r tho lo - gous a m p h i b i a n lamin , i.e. the p o l y p e p t i d e lamin Ln o f X. laevis, which a l lows us to ex tend the obse rva t ion o f the w idesp read and c o n c o m i t a n t occur rence o f bo th B- type lamins to a wide range o f cells and species.

Materials and methods

Preparation of nuclear pore complex-lamina fractions. Nuclear pore complex-lamina fractions from murine, rat and bovine liver were isolated essentially as previously described (Krohne and Franke 1983). Cytoskeletal fractions enriched in nuclear pore complex- laminae from cultured cells were obtained according to Benavente et al. (1985). Fractionation of mouse liver nuclear envelopes and lamins with 4 M guanidinium-HCl was performed as described by Senior and Gerace (1988).

Cell lines, tissues and embryos. The following murine cell lines were studied: mesenchymal cells (line 3T3, American Type Culture Col- lection, ATCC, no. CCL 92), myeloma cells (Ag 8.653), teratocarcinoma cells (F9), and embryonal stem cells (ES, line D3 ; Doetsch- man et al. 1985). In addition we examined Chinese hamster ovary cells (CHO-KI, ATCC No. CCL 61), rat hepatoma cells (MHIC1, ATCC No. CCL 114), and X. laevis kidney epithelial cells (XLKE, line A6). Mouse tissues were dissected from adult mice and tissue pieces immediately frozen by immersion in isopentane cooled with liquid nitrogen to - 140 ° C. Mouse embryos were prepared as described (Jackson et al. 1980).

Monoclonal antibodies. Male Balb/c mice were immunized either with nuclear pore complex-lamina fractions from bovine liver or XLKE cells or with a cytoskeletal-karyoskeletal fraction from

mouse liver. Spleen cells of mice immunized with bovine liver or XLKE cell nuclear pore complex-lamina fractions were fused with Ag 8.653 cells and further processed (cf. Hazan et al. 1986). The hybridoma clones X223 and R29 (subclass IgGl) were used in the present study. In an independent experiment, spleen cells of mice immunized with routine liver cytoskeletons were fused with SP2/O-Ag-14 myeloma cells, and hybrid cell clones were selected in Dulbecco's modified Eagle medium (DMEM) containing 20% fetal calf serum, 1 gg/ml azaserine medium and 1.36 gg/ml hypo- xanthine. From this fusion, the hybrid clone GL-35 (subclass IgM) was obtained. Monoclonal antibody PKB 8 has been described previously (Krohne et al. 1984).

Gel electrophoresis and immunoblotting. Proteins of nuclear pore complex-lamina fractions were separated by one-dimensional SDS- polyacrylamide gel electrophoresis (SDS-PAGE, Laemmli 1970) and two-dimensional gel electrophoresis (isoelectric focusing: IEF; O'Farrell 1975; non equilibrium pH gradient electrophoresis: NEPHGE; O'Farrell et al. 1977).

For immunoblotting, proteins separated by gel electrophoresis were electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, FRG) essentially as described by Towbin et al. (1979) with the following modifications. The membranes were blocked with either 0.1% Tween 20 in PBS overnight or 1% bovine serum albumin in a buffer containing 10 mM Tris- HC1, pH 8.0, 150 mM NaC1 and 0.05% Tween 20 (TBST) for at least 3 h. Thereafter, the membranes were incubated for 2 h under continuous rotation with the following monoclonal antibodies: GL-35, X223, R29 and PKB 8. After washing three times for 5 10 min with PBS or TBST the secondary antibodies were applied. These were either a peroxidase-coupled rabbit antibody to mouse IgG (Dakopatts, Glostrup, Denmark), alkaline phosphatase-conjugated goat antibody to mouse IgG (Promega Biotec, Madison, Wisconsin, USA), or 12SI-labeled goat antibodies to mouse Ig (Amersham Buchler, Braunschweig, FRG).

Bound antibodies were visualized either by a color reaction with 0.04% 4-chloro-l-naphthol (Aldrich-Chemic, Steinheim, FRG) and 0.003% H202 (in TBS) for peroxidase-conjugated antibodies, or with 0.033% nitro-blue tetrazolium (Promega Biotec) and 0.015% 5-bromo-4-chloro-3-indolyl phosphate (Promega Bio- tec) in a buffer containing 100 mM Tris-HC1, pH 9.5, 100 mM NaC1 and 5 mM MgCI2, for alkaline phosphatase-conjugated antibodies. Alternatively, fluorography was used for the detection of azsI-labeled antibodies.

Immunolocalization. Immunofluorescence microscopy on cultured cells and tissue cryosections was performed as described in Jahn et al. (1987). For immunogold localization, 5 Ixm cryosections of mouse liver were fixed for 5 min in ice-cold acetone, then incubated with the appropriate dilution of the primary antibody (X223) for 2 h, washed three times for 5 min with PBS and incubated overnight with a 1 : 5 dilution of 5 nm gold-conjugated goat anti-mouse Ig (Janssen Biochemicals, Beerse, Belgium). Fixation and embed- ding procedures were carried out as described (Kartenbeck et al. 1984; Cowin et al. 1986).

Selection and characterization of cDNAs encoding murine lamin B 2 and X. laevis lamin LH. A cDNA library (2-ZAP ii, generously provided by Dr. Siegfried Ruppert, German Cancer Research Center, Heidelberg, FRG; for references see Sch61er et al. 1990) constructed from poly(A +) RNA from murine F9 cells was screened with the 1 : 1,000 diluted ascites fluid of two monoclonal lamin antibodies (X223 and R29). The secondary antibodies were x2sI-labeled goat antibodies to murine Ig. All other steps were performed according to the manufacturer's protocol (Stratagene, San Diego, Calif., USA). From approximately 106 non-amplified recombinants screened, I clone (MLB2-4A1) that gave a positive signal with both antibodies was chosen for further characterization. The inserted DNA was subcloned by the in vivo excision protocol (Stratagene) and subsequently analyzed by dideoxy sequencing according to Sanger et al. (1977). Both orientations were sequenced by constructing oligonucleotides hybridizing to different parts of

the cDNA. Portions of the DNA sequence were confirmed by the chemical method of Maxam and Gilbert (1980).

In order to obtain a clone coding for X. laevis lamin Ln, 6 x 105 recombinant plaques of a 2gtl0 library prepared from stage 17 embryos (a kind gift of Dr. D.A. Melton, Harvard University, Cambridge, Mass., USA) were screened with an EcoRI fragment of the murine lamin B2 clone comprising 1,654 bp at its 5' end and, in parallel, with an EcoRI-NruI fragment covering only 146 bp at the 5' end of the clone MLB2-4A1. Both were radioactively labeled according to Feinberg and Vogelstein (1983). These probes were used for hybridization to nitrocellulose-bound D N A (prepared according to Maniatis et al. 1982) overnight at 42°C in a buffer containing 50% deionized formamide, 5 x Denhardt's solution, 5 x SSC, 0.1% SDS and 100 gg/ml yeast tRNA. Filters

381

were washed three times with 2 x SSC, 0.1% SDS at room temper- ature for 15 min, three times with 0.1 x SSC, 0.1% SDS at 50 ° C for 15 min and then exposed overnight at - 7 0 ° C. Of the 41 plaques hybridizing to the 1,654 bp probe, only 20 hybridized with the 146 bp fragment. Of 12 plaque purified phages 9 encoded lamin LI, and 3 (XLII-17-2, XLII-18-2, and XLII-19-2) displayed high sequence homology to chicken and murine lamin B2. Clones XLII- 17-2 and XLII-19-2 were completely sequenced, and the latter was characterized by a hybridization selection experiment (Ricciardi et al. 1979) essentially as described (Jorcano et al. 1984). The hybrid-selected m R N A was translated in vitro in a rabbit reticulocyte lysate system (Promega Biotec).

For RNA blot analyses, poly(A+)RNA from F9 cells and X. laevis ovaries was prepared as described (Kleinschmidt et al. 1986)

~ LA L Lu

AC~ ~ P G K

f

" ~ L A L=

Lit

g

Fig. l a -g . Immunological identification of lamin B 2 as a major component of the nuclear pore complex-lamina fraction of mouse liver and of its amphibian homolog, Xenopus laevis lamin LII. Pro- teins were separated by non equilibrium pH gradient electrophoresis, NEPHGE in the first dimension and by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. a Coomassie blue stained proteins of the murine nuclear pore complex-lamina fraction. Proteins separated in parallel gels were transferred to nitrocellulose and incubated with monoclonal antibodies X223 b, R29 c and PKB 8 d. 125I-labeled antibodies were used

as secondary antibodies. X223 and R29 exclusively recognize lamin B2, and PKB 8 recognizes lamins A, B1 and C. e Coomassie blue stained proteins of X. laevis nuclear pore complex-lamina fraction prepared from XLKE cells. Skeletal muscle actin (Ac) and phosphoglycerokinase (PGK) were co-electrophoresed as reference proteins. Proteins were transferred to nitrocellulose and incubated with X223 (f) and PKB 8 (g). As a secondary antibody, alkaline phosphatase-conjugated antibody was used. X223 reacts only with lamin Lib and PKB 8 reacts with lamins LI, Ln and LA

382

and hybridized to radioactively labeled EcoRI fragments of clones MLB2-4A1 and XLII-19-2, respectively. Size reference RNAs (Bethesda Research Laboratories, Bethesda, Md., USA were used for co-electrophoresis in an adjacent lane.

Results

Monoclonal antibodies reacting with a major murme lamin

During our previous characterization of a c D N A clone encoding murine lamin B (H6ger et al. 1988) we noticed a fourth major polypeptide enriched in the nuclear pore complex-lamina fraction migrating on two-dimensional gel electrophoresis at a similar position to lamin B (see Fig. 1 a). Employing monoclonal antibodies which exclusively recognize either this fourth component (X223 and R29; Fig. I b, c) or this component and lamin B (GL-35; data not shown), or exclude this polypeptide but recognize lamins A, B and C (PKB 8; Fig. I d) we concluded that this polypeptide might be an as yet unidentified, genuine mammal ian lamin which we termed lamin B2 following the nomenclature developed for chicken lamins (Lehner et al. 1986). Consequently, we had to spec- ify the mammal ian polypeptide called " lamin B" as " la - min B1"

Interestingly, monoclonal antibodies X223 (Fig. 1 f) and R29 (data not shown) react only with lamin LH in X L K E cells and not with lamins LI or A (Fig. 1 f) indicating the existence of epitopes common to m a m m a - lian lamin B 2 and amphibian lamin LII.

Biochemical characterization and localization of murine lamin B2

Further biochemical characterization of lamin B2 showed that this polypeptide occurred in large amounts similar to mouse lamins A, B1 and C. During nuclear subfractionation using high-salt buffers, and DNAse, RNAse and detergent t reatment lamin B2 is clearly enriched in the residual fraction (Fig. 1 a). When isolated mouse liver nuclear envelopes were treated under largely denaturing conditions with 4 M guanidinium-HC1 (cf. Senior and Gerace 1988) and subsequently centrifuged, lamin B 2 remained in the supernatant as did lamins A, B1 and C (data not shown). This indicates that lamin B2 is a peripheral, not an integral membrane protein.

Using specific antibodies in immunofluorescence and electron microscopy lamin B 2 w a s localized in interphase cells at the periphery of the nucleus (Fig. 2b, c), closely apposed to the inner nuclear membrane (Fig. 2d). In mitosis, however, it appeared in a punctate pat tern throughout the cytoplasm (Fig. 2b), possibly owing to an association with vesicular forms as proposed for B- type lamins (Gerace and Blobel 1980; Stick et al. 1988).

Widespread occurrence of lamin B2 & mammalian cells and tissues

As monoclonal antibodies X223 and R29 react exclusively with lamin B2 in the mouse we were able to study

Fig. 2a-d. Immunolocalization of lamin B2 in murine cells and tissues, a-e Indirect immunofluorescence microscopy on cultured mouse 3T3 cells a and b and liver sections e after incubation with X223. In interphase cells, lamin B2 is localized exclusively at the nuclear periphery b and e, whereas in mitotic cells (see telophase shown in a and b) it shows a punctate distribution throughout the cytoplasm, d Electron microscopical localization of lamin B2 on a frozen mouse liver section after incubation with X223 and secondary antibody conjugated to 5 nm colloidal gold particles. The nuclear periphery of an hepatocyte is shown. Gold particles are found exclusively subjacent to the inner nuclear membrane and not on pore complexes (denoted by arrows) or on the outer nuclear membrane. N nucleoplasm; C cytoplasm. Bars represent 10 gm a, b, 20 ~tm e and 0.1 gm d

Table 1. Detection of lamin B2 in murine tissues by immunofluores- c e n c e

Cell type Reaction

Oocytes + Granulosa cells + Interstitial cells of testis + Sertoli cells + Spermatocytes Spermatids Spermatozoa Neurons (brain and spinal cord) + Intestinal cells + Smooth muscle cells + Skeletal muscle cells + Hepatocytes + Lymphocytes + Embryonal cells (day 6 of gestation)

Ectoplacental cone + Primitive ectoderm +

Uterine epithelial cells +

S /~• ~i!!i~iiiiiiiiiiii~ ¸

S

B2

b

B1

C

Fig. 3a--e. Murine F9 cells lack A-type lamins but contain two B-type lamins, a Ponceau S stained pore complex-lamina proteins of F9 cells separated by two-dimensional gel electrophoresis (isoelectric focusing (IEF) in the first, 10% SDS-PAGE in the second dimension) and transferred to nitrocellulose. Lamins B1 and B2 are indicated, b, e Immunoblots of the same fractions as in a after incubation with X223 b and PKB 8 c). In e only lamin B~ shows a positive reaction and A-type lamins are absent. As a secondary antibody, alkaline phosphatase-conjugated antibody was used

383

Table 2. Detection of lamin B; in various cells by immunoblotting

Cell type Reaction

Murine Ag 8.653 cells + Murine F9 cells + Murine ES (line D3) cells + Murine 3T3 cells + Murine hepatocytes + Rat MHIC1 cells + Rat hepatocytes + Hamster CHO cells + Bovine hepatocytes + Human HeLa cells + Human HT29 cells +

N E P H G E

S LI ,~ D Lll S --

a Ac PGK

Fig. 4a, b. Identification of the polypeptide encoded by cDNA clone XLII-19-2 as Xenopus laevis lamin Ln by a hybridization release, in vitro translation experiment and subsequent two-dimensional co-electrophoresis with nuclear pore complex-lamina proteins of XLKE cells, a Coomassie blue stained gel; b fluorography of the gel shown in a. The in vitro translated polypeptide com- igrates exactly with lamin L~I (b arrowL For further details see legend to Fig. I e

its expression directly by immunof luorescence microscopy. L a m i n B2 was found in all mur ine cells a nd tissues examined with the exception of certain spermatogenic stages such as spermatocytes, spermatids and spermatozoa (see Table 1). A mos t unexpected f inding was its presence in a variety of cells, e.g. F9 (Fig. 3 a, b) and Ag 8.653 cells, previously characterized to con ta in only one B-type lamin, i.e. l amin B~ (Stewart and Burke 1987; Lebel et al. 1987; R 6be r et al. 1989). In the early mouse embryo (day 6 of gestation), nuclei of all cell types in- c luding the primit ive ec toderm and the ectoplacenta l cone reacted posit ively with m o n o c l o n a l an t ibodies spe-

384

1 AATAGTACAATCAGTTTGGCGGGAATATCTAACCGCAAT ATG Met

85 TCT CCT GCC CGG GGA ACA TCT ACC CCT CTT TCA Ser P r o A l a A r g G [ y Th r 8e r T h r P ro Leu Set

180 CAT CTC AAT GAC CGA CTG GCC GTC TAT ATA GAC H i s Leu A~n Asp A r g Leu A [ a V a t T y r l i e Asp

235 AAG ATT TCC GAA AAA GAG GAAJGTT ACC ACA CGG Lys l i e Ser G l u L y s G t u JG [u Va [ T h r T h r A rH

310 GAT GCT CGG AAA GTT TTG GAT GAA ACA GCC AGA Asp A t a A rH Lys V a [ Leu Asp G l u Th r A l a Ar H

385 GAT CTT GAC GAG CTT AAC AAA AAC TAC A.AA AAA Asp Leu A=p G{u Leu ASh Lys Asn T y r Lys Lys

480 TTG GAA GCT CTC TTT CAC CGA AGT GAA GCA GAA Leu G t u A l a Leu Phe H i s Ar 9 $e r G tu A t a G t u

635 GTA GCA GAT CTT CGT GCC CAG CTT TCC AAG ACT Va [ A l a Asp Leu Ar 9 A l a G i n Leu Ser Lys Th r

610 ACC TTG ATG AGA GTT GAC TTT GA.zk AAT CGC ATG Thr Leu Met Ar H Va [ Asp Phe GIU A~n Ar 8 Met

685 GAG GAG GAG TCC CGT GA.A ACA AGG ~ G CGT CAT G l u G l u G [ u J S e r Ar 6 G [ u T h r A rH Lys Ar 8 H i s

76O TAT GAA TOT A.&A TTG [G GCA CAA GCA TTG GAT GAA T y r G l u Ser Lys L e u L A t . G i n A t a Leu Asp G t u

835 GAA CTA GAA CAA ACC TAT CA.& GCT AAG CTT GAC G l u Leu G [ u G i n T h r T y r G i n A t a Lys Leu Asp

910 ACT GCC CTA GAA GAA TTA ACT GAG CGA CGC ATG T h r A t a Leu G tu G t u Leu Th r G t u A rH ArH Met

985 CAG GCA AAT GCT GCA GAG GAA CGT ATA CGT GAG G i n A l a Asn A l a A~a G{u G tu A r g l i e A r H G [ u

1060 CTT TTG GAC TCC AAA GAA AGA GAG ATG GCT GAA Leu Leu Asp Ser Lys G t u Ar H G [ u Met A~a G l u

1135 TTG CTT GAT GTC AAA CTT GCA CTT GAT TTG GAG Leu Leu Asp V a t Lys Leu A l a Leu ASp Leu G tu

1210 TTA AAG CTT] TCC CCA AGC CCA GAA TCA CGA GTC Leu Lys LeuJ Ser P ro Ser P r o G [ u Set A t 8 Va [

1285 ACC TCT AGG AGC AAA AGA AGG CGT GTT GAA GAG T h r Ser A rH Ser Lys A rH ArH ArH V a [ G l u GIU

1360 CAT TCC CTC GGT TCA AGC CGC ATC ACT GCT AGT

H i s Ser Leu G l y Ser Ser A r g l i e Thr A~a Set

1435 ACT CGA TTT CAC TTG TCC CAG CAG GCT TCG GCC Thr A r g Phe H i s Leu Ser G i n G i n A [ a Ser A [ a

1510 TAT GTC CAC CTG AAA A.A.T AAC TOT GAT AAG GAC T y r V a [ H i s Leu Lys A~n Asn Ser A~p Lys Asp

4565 GAG GAG GAA ATA GTT TAC AAA TTC ACG CCT AAA GlU G l u G i u l i e V a t T y r Lys Phe Th r P r o Lys

1680 GAT GCT GGG GTG GCT CAC AGC CCC CCT Asp A t a G l y V a t A t a H i s Ser P r o P ro

1735 ATT AGA ACA TAC TTG GTT AAC ACT GAA t i e Ar H Tnr T y r Leu V a l Ash T h r G l u

1810 GTA GAG GAG GAA GAG GAC GAA GAC GCA V a t G t u G l u G t u G t u Aep G l u Asp A l a

1685 ACA TCC AGA GGC TGC TCT GTC ATG TAA Thr Set A r g G l y Cy~ Set V a [ Met ~

GCT ACT ACT ACA CCA AGC CGG TCG ACC CGG TCG TCT ATG CAG A t a Th r Th r Th r P ro 8e r A r g Ser T h r A r 9 Ser 6e r Met G i n

CCA ACT CGC ATA TCT AGG CTT CAG GAG AAG GAGFGAG TTG AGG P r o Th r A r g l i e 8er A r £ Leu G i n G [ u Lys G l u E Gtu Leu A t 9

CGC GTG CGT GCG CTG GAG CTG GAA AAT GAC CGG CTG ATG GTG A r 8 V a t A r g A l a Leu G l u Leu G l u Asn Asp A r 9 Leu Met Va t

GAG GTT AGT GGG ATC AAG AAC TTG EAT GA~k TCA GAG CTT GCT G t u V a [ Ser G [ y l i e Lys Ash Leu ~ y r GtU Ser G l u Leu A [ a

AGA GCC CGG TTG CAA ATA GAG CTG GGA ~kAA TTT CGA TCA G l u Ar 9 A [ a A r g Leu G i n l i e G [ u Leu G [ y Lys Phe Ar 9 Set

A A A G A T GCT GAC CTG TCT ACG GCA CAG GGG CGT ATT A.AA GAC Lys Asp A [ a Asp Leu 8e r Th r A [ a G{n G t y A r 9 l i e Ly~ Asp

CTT GGA ACT GCA CTC GGT GAG AAG CGA AGT CTT GAG GCA GAG Leu G [ y Th r A [ a Leu G t y G l u Lys Ar 9 Ser Leu G t u A t a G tu

GAA GAT GCC CAT CGT GTT GCT AAA AAA CAG CTG GAA AAG GAG G [ u Asp A t a H i s Ar 9 V a l A t a Lys Lys G i n Leu G t u Ly~ G tu

CAG AGT TTG CAG GAG GAG ATG GAC TTC AGA AAG AAT ATT TAC G i n Ser Leu G i n G i u G t u Met Asp Phe A r g Lys Asn I t e Ty r

GA~, CGC CGT ATA GTA GAA GTG GAC AGG GGC CAT CAT TAT GAC G[u Ar 8 A r 9 l i e V a [ G l u V a l Asp Ar 8 G l y H i s H i s T y r Asp

CTC CGA ~ CA& CAT GAT GAG CA.& GTG AAG ATG TAC ,&AG GAA Leu A r g Lys G i n H i s Asp G l u G t n V a [ Lys Met T y r Ly~ G tu

AAC ATC AAA CGT TCT TCT GAC CAC AAT GAT AAA GCT GCC AAC Asn l i e Lys A r g Set Ser Asp H i s Asn Asp Lys A l a A18 Asn

AGA ATT GAA ACA CTT GGT TAC CAA CTG TCA GGC CTG CAG AAA A r g I r e G [ u Thr Leu G t y T y r G i n Leu Ser G t y Leu G i n Lys

TTA GAA GAG TTG CTA AGC AGT GAC CGT GAC AAG TAT CGT AAA Leu G t u G l u Leu Leu Ser £e r Asp A r 9 Asp Lys T y r Ar 8 Lys

ATG AGG GAG CAG ATG CAG CAG CAA CTA AAT GAG TAT CA,& GAA Met Ar 9 Asp G i n Met G~n G i n G i n Leu Ash G t u Ty r G i n G tu

ATA AAT GCT TAC CGC AAG CTG CTG GAA GGA GAA GAG GAA AGG I~e Asn A t a Ty r Ar 9 Lys Leu Leu G [ u G~y G [ u G [u GIU A r 9

ACT GTC TCC AGG GCG ACT TCC AGT AGC TCT TCT GCT ACC CGT Thr V a l Ser A r 9 A [ a Th r Set Set Set Ser Ser A [ a Thr A r 9

GAA TAT GAG GAA GGT GGG GCA AGT ACA GGC TTC GGT GCT GGC G l u T y r G l u G i u G l y G l y A [ a Set Th r G t y Phe G [ y A [ a G t y

GAG GGT TCT AGC CGC ACT ATT ACT AGT GGC CAG AGC AGT ACT G[u G l y Ser q3er Ar 9 Th r l i e Thr Ser G l y G i n Set 8er Th r

ACA GGA AGT ATC AGT ATT GAG GAG ATT GAT TTG GAA GGA AAA Thr G t y 6er I r e 8er l i e GIU GtU l i e Asp Leu G [ u G t y Lys

CAG TCC CTT GGT AAC TGG AGG CTG AAG AGA A M ATT GGA GAG G i n Set Leu G I y ASh T r p A r 8 Leu Lys A r 8 Lys l i e G t y G tu

TAT GTA CTT AAA GCA GGG CAG TCT GTT AAG ATA TAC AGT GCT T y r V a [ Leu Lys A l a G l y G i n Set V a l Ly~ l i e T y r 6er A l a

TCC ATT CTA GTG TGG AAG AAC CAG AGT TCC TGG GGT ACA GGC AGC AAC Set l i e Leu V a l T r p Lys Asn G i n Ser 6er T r p G t y Thr G [ y 8er Asn

GAG GAG GAA GTA GCT GTG AGA ACA GTT ACA AAA TCT GTC TTA AGA AAT G l u G t u G l u Va t A l a V a l A r g T h r V a t Thr Ly$ 8e f Va t Leu Ar 9 Asn

GAC TTT GGT GAG GAA GAT CTC TTT CAC CAG CAG GGT GAT CCA AGA ACA Asp Phe G l y G l u G [u Asp Leu Phe H i ~ G i n G i n G l y Asp P ro Ar 9 Thr

ACAACATTCATAACATCCTCCTTTTTTTTTTTACTTACTCCCAGAGCCACTGATATCTATTTT

1975 TATATG~A`AGTTCAGGTTATGT~TT~TGTATTTTATGCCTTTCATTTGTTAAG~GTG~AGTTA~a~kGGAAGTGA~&GAGTTACCTAATGCCATA~AATAACT

2074 TGCAAGG•TTTTGTTTTTTTCAGTTTT•A•TTGTAGTGGTTG•AAAAACGTACTG•TATGTTTTTAAGTATTTGTGTTCTAATATGGCA•AATTTTA••

2173 TATTCATAG••AGACAATGGTTTTAATGATTAC•AA•CATGTG•CTGCATGA•&T•ATGTTGAGCATAAAATATGAGGTGGCTGTTTGTAA•A•TAATATT

2272 TTGATTTGACTTTTGTCCAGAATTGGATAAAAATTGTCTGGTTAGAAGTCCATA•TAAAGATGATATACAGATGTAACTCGTAGGAATTAAACCATGTT

28T1 AATTGTTTAGGAAGTTTTAGAATA•AATTG•TATTGCAGAATGTGATCACTATTTCAGTAGTACTGAAGTCTCTTTCACACCTCAAATTAGCAACTATC

24?0 TAGAT~TTAAAGTTATCCTT~TGCTTCTGGTGTAGT~TTCTATATGTGcG~AG~TGGCA~TTCCTAAGACTGACTAGATTTTACACTTTTTTTGTTA

2569 ATAGAAACAAAGGTTGATTTAGC•TGTC•TTGATTTATGTTTAGGCACATGTTGCTATAGGTGT•CCAACAACTATTGTCATTGCTCAGGCAGTTCCCA

2668 CTATGTGCATAATTAC~TCATATAG~ATTGAA~GTTGT~AGGAGCCA~TTTAGAATATGTAGATTTATTTGTTTCTTAGACTTAGcCACTACATAATAA

2787 TTGTTGGTACATTTATTGAG~AGGCAGTATTT~AATTATGAATGGGTTG~TAcTTTAATTGCAAGGAAGCTGCAA~GATTGAGAGGAAATA~TA

2866 TAGCCTTTA~TATGAcTATGAAGATTTT~ATTCATC~AGGTCATAGTATATCT~GTATAGGTCAATCT'a~CAA~TGGACTTG~TGAGTAATCAATG

2985 AAGACGTTT•ACTACTCATCCGAG•AGTTTCTTCAGTTGATTTTAAGTAGGGGATATATTTCCCCTGTAGTCCA•TGCCATTCTTTTTTTTTTTTC•a`AC

3064 TAGAATAA•AATTTGTGTTTTTTTTCCTAGTTG•AATTGA•A•cAATA•TTTTATATAGCAGA•TAATGGTTTCAAGTAcATTTTCATTTTTTTTTTGT

3163 TTTTTTTTTATTT~A~ATATGTGGGTAATGTATTTTTTAATAACAGCT~A~AT~TGCACATGAAAGAA~AGAGGGATGAAGGGAAGC

Fig. 5. Nucleotide sequence of the cDNA clone MLB2-4A1 coding for murine lamin B2. Predicted e-helical domains are indicated by brackets. The putative nuclear migration signal is marked by a broken line. The highly conserved tetrapeptide CxxM is underlined. The probable polyadenylation signal is doubly underlined

385

1 ATTCGGCAACCGAGGTGATTCTCCCGCCGCCGCCACCTCCGCC ATG GCG TCT Met A~a Set

98 TCG CCC ACG CGT CTG TCT CGG CTG CAG GAG AAG GAG FGAG CTC Ser Pro Thr Ar9 Leu Ser A r9 Leu G in G tu Lys G [ u L G[u Leu

161 GAC CGT GTC CGC GCG CTG GAG CTG GAG .&AT GAT AGG TTG CTG Asp A r9 Va t A r g A l a Leu G [u Leu Gtu Asn Asp A r9 Leu Leu

236 CGC GAG GTG AGT GGC ATC AAG ACC CTG rT TAG GAG TCA GAG CTG Arg G lu Va [ Set G [ y l i e Lys Thr Leu L Tyr GIu Set Gtu Leu

311CGT GAA CGT GCC CGG CTG CAG ATT GAA ATT GGA AAG GTG GAG A r 9 G tu A r 9 A~a A r g Leu G i n l i e Gtu l i e G l y Ly8 Vat G i n

386 AAG CGG GAA GGT GAG CTC ACA GTG GCC CAG GGG CGA GTG AAG Lys Ar 9 G lu G [ y GIU Leu Thr VBI A [ a G in G [ y A r g Va [ Lys

461 GAG CTG GCC ACA GCC GTC AGT GAC AAC GAG GGC CTG GAG ACA G[u LeU A l a Thr A l a Leu Ser Asp Ash G i u G l y Leu G [u Thr

536 GCA GAA GAT GGT CAT GCT GTG GCC AAG AAG GAG TTG GAG AAG A l a GlU ASp G l y H i s A t a Va [ A [ a Ly~ Lys G i n Leu GiU Lys

611 TGC CAG AGC CTG CAG GAG GAG CTG GCT TTC AGC AAG AGT GTG Cys G in Set Leu G i n G tu G lu Leu A i a Phe Set Lys Ser Vat

686 GAG GAG CGG CGC TTG GTG GAG GTG GAG AGC AGC CGG CAA CAG H i s G tu A r g A t 9 Leu Va[ G tu Va [ ASD Ser Ser Ar 9 G i n G i n

761 GAG CTG CGC AGT CAG CAC GAT GAG CAA GTG CGC CTG TAC CGG Asp Leu A r g Ser G i n H i s Asp G i u G in Va [ A rg Leu Ty r Ar 9

836 GAG AAC GCC AAQ CTG CTC TCG GAG GAG AAT GAG AAG GCA GCC Asp Asn A t a Lys Leu Leu Ser Asp G i n Asn Asp Lys A l a A l a

8 1 1 A T G CGC GTG GAG TCC CTC AGC TAC CAG CTT TTA GGC CTC CAA Met A r g V s l G [u Ser Leu Set Ty r G in Leu Leu G [ y Leu G i n

986 GAG CTG GAG GAG GCC TTG CGT GGG GAG CGT GAG AAG TTC CGC G[U Leu G i u GiU A l a Leu Ar 9 G t y G[U A r g As~ Lys Phe A r g

1061 GAG GTG CGG GAG CGA ATG GAG GAG GAG CTG GCA GAG TAG CAA Gtu Va [ A r g Asp A r g Met G~n G i n G i n Leu A t a G[u T y r G i n

1136 GAG ATA AGC GCC TAG CGC AAG CTG CTG GAG GGC GAG GAG GAG AGG GtU l i e Ser A [ a Ty r Ar 9 Lys Leu Leu GIU G t y G[U G[u G[U At 9

1 2 1 1 A T C ACC ATC TCT CGG GCC ACA TCG AGC AGC AGC AGC AGC GGG GTT l i e Th r l i e Ser Ar 9 A l e Thr Set Ser Ser Ser Ser Set G~y Va [

1286 CGC CGG CGG CTG GAG GAG ACC TCA GGC TCA CCC AGC AGG GCT TCC A 2 9 A ~ g A ~ g . . L ~ _ G I ~ Asp Thr Set G [ y Ser Pro Ser Ar 9 A l a Ser

1361 ACT GTG GCC ACG GGT GTT GTG AAC ATC GAC GAG GTG GAG CCA GAG Thr Vet A l a Th r G [ y Va [ Va[ ASh l i e Asp Gtu Va [ ASD Pro GIU

1436 GAG AAG GAG GAG TCT TTG GGG AAC TGG AGG ATC AAG AGA GAG GTT AsD Lys ASp G i n Set Leu G l y ASh T rp A rg l i e Lys A r g G i n Va[

1 5 1 1 A C A CCC AAG TAT GTC CTC CGG GCC GGC CAG ACT GTC ACG GTG TGG Thr Pro Lys Ty r Va [ Leu A r9 A t a G [ y G in Thr Vat Thr V a l T r p

1686 CCA TCA ACC CTT GTG TGG AAA AGC CAG ACC AAC TGG GGC CCT GGG Pro Ser Th r Leu Va t T rp Lys Set G i n Thr ASh T rp G l y P ro G l y

1661 GAC GGT GAG GAG GTG GCC GTG AAG GCT GCA AAG CAC TCA TCT GTC Asp G [ y G l u G[u Va [ A l a Va t Ly~ ALa A [ a Lys N i s Set Ser Va[

CTG CCG CCC CAC GCG GGG CCC GCC ACG CCG CTG Leu Pro Pro H i s A t a G [ y P ro A t a Thr Pro Leu

CGT GAA CTG AAC GAG CGC CTG GCA CAC TAG ATC Arg GIu Leu Ash ASp A r g Leu A [ a H i s Ty r l i e

CTC CGG ATC TCC GAG AAG GAG GA(]GTG ACC ACT Leu A r9 l i e Set G lu Lys G tu G t u J V a l Thr Thr

GCT GAG GCC CGA CGG GTA CTG GAT GAG ACG GCC A t a Asp A l a A rg A r9 Va[ Leu Asp G iu Thr A i a

GCT GAG CTA GAG GAG GCC AGG AAG AGT GCC AAG A l a GLu Leu GIU GIU A l a A rg Lys Ser A t a Lys

GAT CTG GAA TCA CTG TTT GAG CGG AGT GAG GCT A~p Leu GLU Set Leu Phe H i s Ar 9 Ser GlU ALa

GAG GTG GCA GAG CTT CGA GCA CAG CTG GCC AAG GIU Va [ A~a G [u Leu A t 9 A [ a G in Leu A [ a Lys

GAG ACG CTG ATG CGG GTT GAG CTG GAG AAC CGA G lu Thr LeU Met Ar 9 V a i Asp Leu G [u Asn A rg

TTT GAG GAG GAG]GTA CGG GAG ACC CGA CGG AGG Phe Gtu G [u G~uJva~ A r9 G[U Thr A t 9 A r g Arg

GAA TAT GAC TTC AAG ATG GcTFcAG Gcc CTG GAG G lu Tyr A~p Phe Lys Met A l a L Gin A l a Leu G lu

GTA GAA CTG GAG GAG ACC TAC CAG GCC AAG CTG v a [ G l u Leu GIU G in Th r T y r G i n A l a Lys Leu

CAT GCA GCC CGC GAG GAG CFC AAG GAG GCC CGC H i s A [~ A l a A rg G[U G[u Leu Lys G[U A [ a Ar 9

AAG CAG GCC AGT GCT GCA GAG AAC CAC ATC CAT Lys G in A [ a Ser A t a A [ a G tu Asn H i s l i e H i s

AAG ATG GTG GAG GCT AAG GAA GAG GAG ATG AGG Lys Met Leu Asp A [ a Ly8 G[u G i n G[U Met Thr

GAG CTG CTG GAG ATT AAG CTG QCC CTG GAC ATG G lu Leu Leu ASD I r e Lys Leu A [ a Leu Asp Met

CTG 1 AAG CTG TCT CCC AGC CCT TCA TGA GGC Leu Lys Leu Ser Pro Ser P ro Ser Set A r9

GGC ATG TCT GTG GGC GAG CGC GGG GGC AAG G l y Met Ser Va l G l y G~n Ar 9 G [ y G t y ~ Z ~

AGA GTG AGC AGC GGC TCC CGC CTG GCT CAG Arg v a [ Ser Ser G i y Ser A r9 Leu A [ a G in

GGC AGG TTC GTG CGC CTT AAG AAC TCT TCA G t y A rg Phe Vat A r 9 Leu Lys ASh Ser Set

CTG GAG GGT GAG GAG ATT GCC TAG AAG TTC Leu G tu G t y G lu Asp l i e A i a Ty r Lys Phe

GCA GCT GGC GCA GGG GCT ACC CAC AGT CCC A l a A l e G i y A [ a G~y A [ a Th r H i s Ser Pro

GAG AGC TTC CGC ACT GCC CTG GTC AGT GCC G[u Ser Phe A r 9 Thr A [& Leu Va t Ser A l a

GAG GGG AGG GAG AAC GGG GAA GAG GAG GAA G i n G l y A rg G iu Ash G [ y G[U Gtu G [u G[U

1736 GAG GAA GAG GCA GAA TTC GGT GAA GAG GAG CTC TTC CAC CAG GAG GGG GAC CCA AGG ACT ACC TCA AGG GGC TGC G[u G [u G[U A [ a G l u Phe G [ y G [u G[u Asp Leu Phe H i s G i n G in G [ y ASp Pro A rg Thr Th r Set A r9 G [ y Cy~s

1611 CGA CTG ATG TGA AACCTGCCCCACGGTCACCATGGTCCCCAGAGCCCCTAAAACTACTTTTTTATGGTCTGCTCTCACTAGTCCTTGGTCTGTTT A r g Leu Met ~ w

1906 CTCGATGACTGTTAAG•AACCATGAGAATGTGGGTGGGCTCTGCTGGGTTT•CGAGATGGAGGTG•CCTCTGCCCAC•GGCCTGACCTGGCCCATGCCG

2104 ATGA~A~TGGTGATTCAGTTGTTTTCTTTGAA~CTTTC.&AATCAAAT~AAATTCAG~AT~AAGAGTG~TGTGGGGC~CCATAG~TGT~AAGGAATG

2200 TGGTGGG•TCTTAGGACACAGGGCAGA•CTCCTCTCCTGGAGTTGATGG•ACCTGA•ACCAGGGATGGCCCAGCTTTGTGCTCTGAATCTGAAGGGTTT

2401 TCACA~ATGGGACGACG~CT~CCATCCT~AGACTGAAGGGGACAGGCAAACCGTGGGCGTTGTGCCTTCAGGAGCA~AGGCTGCTGGTTCCCAGCCAG

2500 TGACTGCTCA~ATGAGATGTGGCCACACTAGCTATG~TCCTGGCCTCTCCTGTATGAATACTGTGTGGCCCCTCTGTGCCCCACCTCGATGAAGGCCTG

2698 TTTTTTACAT•GGTCAGTAACGCAG•TCACTGGGATGATGCTCTGGGGGTTCAGGCCTGTCCGGAGCGGCTGGCAGCTTCCTTTACTGAGCTAGGGCGA

2797 GTTCTTTCTCTCTCCTGGCACAGCTGATGGTAGGAAGAGTCCAGGCCTAGTCTTC••C•AAGGCAAGAGCTCTTGGCCACAGGATGCCACCACTGCCAC

2896 CAACCTCCAGAAC•CACCAGGCCACCTCCTGCTTCTAGAGTACCTCCGTTTTCTTTGTGACCGGCGTTGGATCCTGGATAGTTATGGCTGGCTGCAGTC

2685 ACTGAC-C•TCTGCTGC•TT•CGACAGGGCAGGTGTACTCTGCACTGGAGCTAGCTTCCTTGTATTTGAGGAGCAGGTCTAGAGTAATCTGCAGGT••TC

3064 AGGGGT~T~TTTCTGGCCCTGGCTAGGT~AGATGGTTA~CGGGAGTGCGA~TGG~T~G~TGGGGACGCG~CGGCACTCGCGCACAGCT~TCAGCTCTAC

3193 AGATTGTTT~TTTTAT~JkGATGCATG~CA~a#~CGTGTGTTC~AC~TTTTCTTTTA~GC~TATGATTTGT~a`~ATATACATTTTACAACTGGJ~J~CTTTTGTA

Fig. 6. Combined nucleotide sequences of c D N A clones XLII-17-2 and XLII-19-2 coding for Xenopus laevis lamin Lu. Predicted e-helical domains are indicated by brackets. The highly conserved tetrapeptide CxxM is underlined. The putative nuclear migration signal is marked by a broken line

I ' ~ Coil 1A 1 ME--TPSQRRATRSGAQAS--STPLS•TRITRLQEKEDLQELNDRLAVYIDRVRSLETENHGLRLRITESEEVVSREvSG HLA

1 MATATPVQQQRAGSRASA--PATPL•PTRL•RLQEKE•LRELNDRLAVYIDKvRSLETENSALQLQVTEREEVRGRELTG MLBI

1 MASLPP .... HAG ....... PATPLSPTRLSRLQEKEELRELNDRLAHYIDRVRALELENDRLLLRISEKEEVTTREVSG MLB2

1 MSG-TPIRGTPGG ......... TPLSPTRISRLQEKEELRQLNDRLAVYIDRVRALELENDRLLVKISEKEEVTTREVSG CLB2

1 MATTTPSRSTRSSMQSPARGTSTPL~PTRI~RLQEKEELRHLNDRLAVYIDRvRALELENDRLMVKISEKEELTTREvSG XLII

i- ~ Coil l B 77 Z~A~;mLGD~K~DSV~QLELSXVP~E~LKAAN~ZEGDL~QAALKD;mA~LNS~LSTALSE~R HLA 79 LKALYETELADABRALDDTARERAKLQIELGKFKAEHDQLLLNYAKKESDLSGAQIKLREY~AALNSKDAALATALGDKK MLB1 53 •KTLYESELADARRvLDETARERARLQIEIGKVQAELEEARKSAKKREG•LTvAQGRVKDLESLFHRSEAELATALSDKR MLB2 71 IKNLYESELADARR•LDETAKERARLQ•EIGKLRAELEEFNKSYKKKDADLSVAQGRIKDLE•LFHRSEAELNT•LN•KR CLB2 81 ~L~SEUm;mRV~D~A~RARLQ~HLG~RSD~DZLm~Nn~KDADLST~QGRLKD~A~,~%~;~G~ALGE~ ×LI~

Coil 1 B 157 TLEGELHDLRGQVAKLEAALGEAKKQLQDEMLRRvDAENRL•TMKEELDFQKNIYSEELRETKRRHETRLVEIDNGKQRE HLA

159 SLEGDLEDLKDQIAQLEASLSAAKKQLADETLLKvDLENRCQ•LTEDLEFRKNMYEEEINETRRKHETRL•EvDSGRQIE MLBI

133 GLETE~AEI~AQLAKAEDGHAVAKKQLEKETLMRvDLENRCQ~LQEELAFSKSVFEEEVP`ETRRRH~RRLVEVDSSRQQE MLB2

151 SLEAEvADLRAQLAKAEDGHAvAKKQLEKETLMRvDLENRCQSLQEDLDFRKNVFEEEIRETRKRHEHRLVEvDTSRQQE CLB2

161 SLEAEvADLRAQLSKTEDAHRvAKKQLEKETLMRVDFENRMQSLQEEMDFRKNIYEEESRETRKRHERRIVEVDRGHHYD XLII

I -~ Coil 2 237 FE~RLADALQELRAQHED~VEQYKKELEKTYSAKLDNARQ~AERN~NL~GAAHGELQQ~RIRIDSLSAQLSQLQKQLAAK HLA

239 YEYKLAQALHEM•%EQHDAQvRLYKEELEQTYHAKLENARLSSEMNTSTVNSAREELMESRMRIESLSSQLSNLQKESRAC MLBI

213 YDFKMAQALEDLRSQ~DEQvRLYRVELEQTYQA~LDNAKLL~DQNDKAAHAAP`EELKEARMRV~SLSYQLLGLQKQASAA MLB2

231 YENKMAQALEDLRNQHDEQvKLYKMELEQTYQAKLENAILASDQNDKAAGAAREELKEARMRIESLSHQLSGLQKQASAT CLB2

241 YESKLAQALDELRKQ~D~QVKMYKEELEQTYQAKLDNIKRSsD~NPKAANlTALE~LTERRMRI~TLGYQLSGLQKQANAA XLII

Coil 2 317 EAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARM•QQLDEYQELLDIKLALDMEIHAYRKLLEGEEERLRL•PSPTSQ HLA

319 LERIQELEDMLAKERDNSRRMLSDRERE~EIRDQMQQQLSDYEQLLDVKLALDMEISAY~KLLEGEEERLKLSPSPSSR MLBI

293 ENHIHELEEALRGERDKFRKMLDAKEQEMTEVRDRMQQQLAEYQELLDIKLALDMEISAYRKLLEGEEERLKLSPSPS•R MLB2

311 EDRIRELKETMAGERDKFRKMLDAKEREMTEMRDQMQLQLTEYQELLDVKLALDMEISAYRKLLEGEEERLKLSPSPSSR CLB2

321 EERIRELEELLSSDRDKYRKLLDSKEREMAEMRDQMQQQLNEYQELLDVKLALDLEINAYRKLLEGEEERLKLSPSPESR XLII

397 RSRGRASSHSSQTQGGGSV-T---KKR/<LE ............................ ST ........ ESRSSF-SQHAR HLA

399 VTVSRASSSRS ...... VRTTRG-KRKRVDVE ...................... ESEASSSVSIS ........... HSAS MLBI

373 ITISRATSSSSSSGVGMSVGQRGGKRP.RLE-D ...... TSG ....... SPSRASRV--SSGSR ............ LAQTV MLB2

391 VTVSRATSSSSSSSTSLVRSSRG-KRRRIEAEELSGSGTSGIGT .... GS--IS---GSSSSSS ........ FQMSQQAS CLB2

401 VTV~R~T~SS~---~ATRTSRS--KR~RR--VE-EEYEEGGASTGFGAGHSLGS~RITASEG~RTITSGQSSTTRFHLSQQAS XLII

435 TSGR•AVEEVDEEGKFvRLRNKSNGDQSNGNWQIKRQNGDDPLLTYRFPPKFTLKAGQVVTIWAAGAGATHSPPTDLvWK HLA

438 ATGNVC•EEIDVDGKFIRLKNTSEQDQ•MGGWEMIRKIGDTS-VSYKYTSRYvLKAGQTVTVWAANAGVTASPPTDLIWK MLBI

424 ATGVVNIDEVDP•GRFvRLKNSSDKDQSLGNWRIKRQVLEGEDIAY•FTPKYvLRAGQTvTVWAAGAGATHSPPSTLVWK MLB2

452 ATGSISIEEIDL•GKYvQLKNNSEKDQSLGNWRLKRQIGDG•EIAYKFTPKYvLRAGQTvTIWGADAGVSHSPPSVLvWK CLB2

476 ATGSISIEEIDLEGKYvHLKNNSDKDQSLGNWRLKRKIGEEEEIVYKFTPKYvLKAGQSVKIYSADAGVAHSPPSILvWK XLII w : *

515 KAQNTWGCGNSLRTALINSTGEEvAMRKLVR-SvTVV---EDDEDEDGDGLLHHHHGSHCSSSGDPAEYNLRSRTVLCGTC HLA

517 KNQNSWGTGEDVKVILKNSQGEEVAQRSTVFKTTIPEEE-EEEEEEPIGVAVEEE .......................... MLBI

504 KSQTNWGPGESFRTALVSADGEEVAVKAAKHYSVQGRENGEEEEEEEAEFG-EED .......................... MLB2

532 KNQGSWGTGGNIRTYLVNSDGEEVAVR-TVTKSVVVREN-EEEEDE-ADFG-EED .......................... CLB2

556 KNQSSWGTGSNIRTYLVNTEEEEVAVR-TVTKSVL--ENVEEEEDEDADFG-EED .......................... XLII

600 GQPADKASASGSGAQVQGPISSGSSASSVTVTRSYRSVGGSGGGSFGDNLVTRSYLLGNSS--PRTQSPQNCSIM 664 HLA

571 ........................................................ RFHQQGAPRAW-NKSCAIM 588 MLBI

558 ........................................................ LFHQQGDPRTTS-RGCRLM 575 MLB2

583 ........................................................ LFNQQGDPRTTS-RGCLVM 600 CLB2

606 ........................................................ LFHQQGDPRTTS-RGCSVM 623 XLII

Table 3. Amino acid sequence comparison of the rod domains of different vertebrate lamins

% Identical amino acid residues (% homology)

Xenopus laevis, X. laevis Murine Murine Murine Human Chicken lamin L~ lamin LH lamin B1 lamin B 2 lamin C lamin A lamin B2

387

X. laevis, - 57.4 (73.9) 73.3 (85.2) 57.4 (73.3) 60.2 (75.9) 59.4 (75.1) 58.5 (74.5) lamin LI X. laevis, 57.4 (73.9) - 61.1 (78.8) 73.1 (84.8) 61.1 (66.7) 59.7 (77.0) 79.3 (87.7) lamin Ln Murine 73.4 (85.2) 61.1 (78.8) - 59.9 (76.2) 61.6 (77.9) 60.2 (77.0) 62.2 (78.4) lamin B1 Murine 57.4 (73.3) 73.1 (84.8) 59.9 (76.2) - 59.1 (74.2) 57.4 (73.7) 82.9 (91.0) lamin Bz Murine 60.2 (75.9) 61.1 (66.7) 61.6 (66.7) 59.1 (74.2) 97.2 (98.3) 59.7 (75.3) lamin C Human 59.4 (75.1) 59.7 (77.0) 60.2 (77.0) 57.4 (73.7) 97.2 (98.3) - 58.3 (74.8) lamin A Chicken 58.5 (74.5) 79.3 (87.7) 62.2 (78.4) 82.9 (91.0) 59.7 (75.3) 58.3 (74.8) - lamin B2

Sequences were taken from Krohne et al. (1987), Riedel and Werner (1989), McKeon et al. (1986), Fisher et al. (1986), and Vorburger et al. (1989a). Homology means identical amino acid residues plus conservative exchanges. The latter are defined as follows (one letter code) : T/S; D/E; H/K/R; M/I/L/V/A; Y/F

cific for lamin B2. It was also found to be a component of the mouse oocyte (Table 1).

In other mammalian species such as hamster, rat, cow and man, X223 and R29 reacted with lamins B1 a n d B z as demonstrated by immunoblotting (data not shown). Such experiments revealed the presence of lamin B 2 in all mammalian species studied (Table 2), although in varying amounts. In most cells and tissues analyzed it appeared as a major component of the nuclear pore complex-lamina fraction, except for rat hepatocytes in which it was only a minor component.

c D N A cloning o f murine lamin B2 and its amphibian counterpart, lamin LII

To examine whether lamin B2 was a genuine polypeptide and not a lamin B1 modification we cloned a lamin B 2

c D N A from an F9 cell expression library. One clone' (MLB2-4A1) reacting with both X223 and R29 was analyzed. When this c D N A clone (insert ~ 3.3 kb) was used in R N A blot analysis on poly(A) + R N A from F9 cells, a weak band at ~ 3.5 kb was detected (data not shown),

Fig. 7. Amino acid sequence comparison of human lamin A (HLA), murine lamin B1 (MLBI), murine lamin B2 (MLB2), chicken lamin B2 (CLB2) and Xenopus laevis lamin Ln (XLII). Sequences have been aligned to optimize similarities. Amino acid residues in boM indicate identity between the three B2-type lamins, and when shared by lamins HLA and MLB1. Three sequence motifs charac- teristic of B2-type lamins are indicated by brackets. Coils 1A, 1B and 2 of the rod domains are delimited by arrows. The highly conserved carboxy-termini and the putative nuclear migration signal are underlined. Three of the four absolutely conserved tryptophan residues in the tail region of all lamins are marked by asterisks. The position of a highly conserved fourth tryptophan (tail) and of a cysteine residue (coil 1 B) are indicated by a double point

indicating that this clone contained most of the lamin B2 mRNA.

Using this mouse lamin B2 c D N A we then screened a 2gtl0 library constructed from stage 17 embryos of X. laevis and isolated two highly homologous c D N A clones with insert lengths of ~ 3.1 and ~ 1.6 kb, respectively (XLII-17-2 and XLII-19-2). R N A blot analysis using poly(A) + R N A from X. laevis ovaries revealed a signal at ~ 3.5 kb. For further characterization of the two c D N A clones a hybrid selection translation experiment was performed with one of them (XLII-19-2). Hy- brid-selected m R N A was translated in vitro and the re- sulting polypeptide co-electrophoresed with authentic la- rains prepared from X L K E cells. As shown in Fig. 4, this polypeptide comigrated with lamin LII, thus proving that both clones encode this lamin.

Amino acid sequence features o f murine lamin B 2 and X. laevis lamin LH

The nucleotide sequences of clones MLB2-4A1, XLII - 17-2 and XLII-19-2 were determined (Figs. 5 and 6). Lamin B z clone MLB2-4A1 is 3,224 nucleotides long and contains an open reading frame (nucleotides 95- 1,820). The sequence of nucleotides 1-94 was obtained from a genomic clone. The colinear mouse lamin Bz clones have 1,510 nucleotides including a poly-adenyla- tion signal of 3' and 43 nucleotides of 5' untranslated region. They encode a protein with 592 amino acids and a total molecular weight of 66,994 daltons.

Using restriction mapping and sequencing we showed that the two X. laevis lamin L~I clones XLII-17-2 and XL-19-2 are colinear and encode a protein with 623 amino acids and a total molecular weight of 71,471 daltons. Clone XLII-17-2 contains 1,811 nucleotides of the translated region and 1,341 nucleotides of the 3' un-

388

translated region which lacks a polyadenylation signal. The J(. laevis lamin LH clone XLII-19-2 is 1,575 nucleotides long, with 39 nucleotides of 5' untranslated region and an open reading frame encoding 512 amino acids and thus lacks part of the coding sequence at the carboxy-terminus.

Inspection of the deduced amino acid sequences of murine lamin Bz and X. laevis lamin L n and analysis of predicted conformation revealed conservation of the typical molecular organization of all lamins : a short non e-helical amino-terminal "head" rich in basic and hydroxyamino acids is followed by a long, mostly e-helical part (" rod domain") containing two interspersed possibly helix-breaking linker regions. Within coil 1B of the rod domain (Figs. 5, 6, and 7) the cysteine residue char- acteristic of B-type lamins is present in murine lamin B2, but absent in X. laevis lamin LII (marked by a double point in Fig. 7). At its carboxy-termini a long " ta i l" region contains a set of interesting sequence motifs, most of them common to all other sequenced lamins such as a putative nuclear migration signal. Murine lamin B2 and 35. laevis lamin LII contain an "acidic box" and the carboxy-terminal tetrapeptide "CxxM" (underlined in Fig. 7) which occur at the same positions as in all other lamins sequenced so far. Interestingly, murine lamin B2 contains within the CxxM motif (x normally being an aliphatic amino acid) a basic amino acid. Mur- ine lamin B2 contains four conserved tryptophan residues (marked by asterisks or a double point in Fig. 7) whereas X. laevis lamin Ln lacks the second one which is replaced by a tyrosine residue (marked by a double point in Fig. 7).

Comparison of the amino acid sequences of the cen- tral rod domains demonstrates the high degree of homology between murine lamin B2 and X. laevis lamin L n (84.8% homologous amino acid residues), and to chicken lamin B2 (87.7% to X. laevis giI ; 91.0% to murine lamin B2 ; for a detailed comparison with other lamins see Table 3). This suggests that these three polypeptides represent an orthologous series of polypeptides.

We found three sequence features (marked by brackets in Fig. 7) common to lamin B2 of mouse, lamin Bz of chicken and lamin Ln ofX. laevis which allow discrim- ination between lamins B1 and B2. There is a conspicu- ous cluster of hydroxyamino acids in the tail preceding the putative nuclear migration signal, Two segments of the amino acid residues, Phe-His-Arg-Ser within coil 1B, and Asp-Lys-Ala-Ala within coil 2, are exclusively conserved within this group of lamins.

Discussion

The identification of a second mammalian B-type lamin and its amphibian counterpart reveals that not only the protein structure of lamins as such, but also their expression patterns are highly conserved in evolution. Our in- vestigation of lamin B2 expression in mouse in comparison with the previous published observations in X. laevis (Benavente etal. 1985; Benavente and Krohne 1985; Stick and Hausen 1985) and chicken (Lehner et al. 1987) shows the common presence of a period when the laminae of the cells are composed solely of two B-type lamins.

M~ (kDa)

80

60

40-

A A A L,v

o

c L',,, ° L ii

B~ B~ B~ B, o ~ O ~ D

Li

PGK Ac

I I I

7.4 6.4 5.4 pl

Fig. 8. Scheme showing the typical position of all characterized vertebrate lamins after two-dimensional gel electrophoresis (NEPHGE in the first, SDS-PAGE in the second dimension). Open squares Xenopus laevis lamins L1, Ln, A; filled squares X. laevis lamins LHI and Liv. Open circles mammalian lamins A, B1, B2, C. Open triangles avian lamins A, B1, B2. Reference proteins (PGK, phosphoglycerokinase; Ac, skeletal muscle actin) are indicated by asterisks. Mr, relative molecular mass; pI, isoelectric point

In all three species studied the synthesis of A-type lamins occurs much later (in X. laevis, swimming tadpole stage, Wolin et al. 1987; in mouse, embryo proper at day 12 of gestation, R6ber et al. 1989; in chicken, day 5 of breeding, Lehner et al. 1987) and is regulated in a tissue- specific way which is not yet understood. Our data su- persede the currently held view that mammalian cells lacking A-type lamins contain only a single B-type lamin (Stewart and Burke 1987; Lebel et al. 1987).

Based on the existing sequence information it is now possible to group the majority of the identified vertebrate lamins as follows (also see Fig, 8; for discussion of X. laevis lamins LII I and LIV see below): (I) A-type lamins comprise mammalian lamins A and C (McKeon et al. 1986; Fisher et al. 1986), and amphibian and avian lamin A (Wolin et al. 1987; Peter et al. 1989). Their most striking sequence feature is the presence of an oligohisti- dine stretch in their extended tail region. Biochemically, they differ from B-type lamins in that they become completely soluble during mitosis (Gerace and Blobel 1980) and their pI is nearly neutral. The occurrence of lamin C - a "truncated" form of lamin A probably arising as an alternatively spliced product of the lamin A gene - appears to be restricted to mammals.

(II) The situation of the more acidic B-type lamins is somewhat more complicated. Clearly, the demonstra- tion of the existence of a second mammalian B-type lamin necessitates a revison of the nomenclature: we choose to refer, from now on, to mammalian lamin B as lamin B1 and to the newly identified second B-type lamin as lamin B2. This nomenclature reflects the evolu- tionary and functional relationships between all B-type lamins, cDNA sequence analysis of X. laevis lamins L~ (Krohne etal. 1987) and LII (this paper), murine B1 (H6ger et al. 1988) and B2 (this paper), and chicken B1 and B2 (Vorburger et al. 1989a; Peter et al. 1989) and direct protein sequence data on mouse B-type lamins (Weber et al. t990) prove that there are two distinct sub- types of the B-type lamins, namely B1- and B2-type la-

389

mins. They share i m p o r t a n t b iochemica l fea tures such as thei r m e m b r a n e assoc ia t ion dur ing mi tos is (Gerace and Blobel 1980; Burke and Gerace 1986; Stick et al. 1988).

The f indings o f Che l sky et al. (1987) who have de- scr ibed a second ca rboxy-methy l -es t e r i f i ed p ro t e in in ad- d i t ion to l amin BI , and o f Beck et al. (1988) who have f o u n d an ana logous s i tua t ion in Chinese h a m s t e r ova ry cells in which pro te ins mod i f i ed by a i sop reno id moie ty were ana lyzed suggest t ha t this as yet un iden t i f i ed p ro - tein p r o b a b l y co r r e sponds to l amin B2. I t thus appe a r s tha t all B- type lamins unde rgo a c o m p l i c a t e d series o f p o s t - t r a n s l a t i o n a l modi f ica t ions .

C o m p a r i s o n o f the amino acid sequences shows tha t there are only subt le differences be tween B~- and B2-type lamins (see Resul ts ) ; this p rov ides no clue as to any func t iona l specia l i sa t ion. I t also r emains to be seen which o f the B- type lamins in terac ts wi th o the r p ro te ins such as v iment in ( G e o r g a t o s et al. 1987) and the lamin B recep to r ( W o r m a n et al. 1988 b) in v i t ro and in vivo.

G iven the fact t ha t in ve r tebra tes the express ion o f A- and B-type lamins is h ighly conserved in somat ic cells and dur ing embryogenes i s , this can also be expected for l amins expressed p r e d o m i n a n t l y or exclusively in ge rm cells. So far, however , lamins wi th these cha rac te r - istics have been ident i f ied only in X. laevis. These are expressed in oocytes ( lamin Lni; Benavente et al. 1985; Stick 1988) a n d late spe rma togen ic s tages ( lamin Lw; Benavente and K r o h n e 1985). N o lamins at all have been found specific for ch icken (Stick and Schwarz 1982; Lehne r e t a l . 1987) and m a m m a l i a n spe rmatogenes i s ( L o n g o et al. 1987; for c o n t r a d i c t o r y results conce rn ing m a m m a l s see Mau l et al. 1986 and Ie ra rd i et al. 1983). W i t h our m o n o c l o n a l an t ibod ies reac t ing in somat i c cells exclusively wi th B- type lamins we have o b t a i n e d s ta in ing o f the l amina in bov ine spe rma tocy te s and spe rmat ids (T. H 6 g e r and G. K r o h n e , unpub l i shed obse rva t ions ) suggest ing tha t pub l i shed negat ive results migh t s imply be the resul t o f lack o f specific an t ibod ies a n d shou ld be r e -examined with a d e q u a t e probes .

Acknowledgements. We would like to thank Jutta Mfiller for her help with the photographic work, Dr. M.T. Murray for carefully reading the manuscript and Dr. W.W. Franke (German Cancer Research Center) for stimulating discussions. Monoclonal antibodies were kindly provided by Drs. K. Weber and M. Osborn (PKBS; Max Planck Institute for Biophysical Chemistry, G6t- tingen, FRG) and Dr. H. Denk (GL35; Institute of Pathology, Graz, Austria). This work has been supported by a grant of the Deutsche Forschungsgemeinschaft to G.K. (grant Kr 758/2-3 and 758/4-1) and by the Fond zur F6rderung der wissenschaftlichen Forschung (grant P5803/P6611M) to K.Z. and Dr. G. Denk (Insti- tute of Pathology, University of Graz, Austria). The nucleotide sequence data reported will appear in the EMBL, Genbank and DDBJ Nucleotide Sequence Databases under the accession numbers X54098 for murine lamin B2 and X54099 for X. laevis lamin Ln. The accession number of murine lamin B1, corrected in three amino acid positions, is X16705.

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characterization of a second highly conserved b-type lamin present in cells previously thought to...

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