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(Received for publication, July 24,
1995; and in revised form, January 16, 1996) From the
Expression of the A-type lamins was studied in the lung
adenocarcinoma cell line GLC-A1. A-type lamins, consisting of lamin A
and C, are two products arising from the same gene by alternative
splicing. Northern blotting showed in GLC-A1 a relatively low
expression level of lamin C and an even lower expression level of lamin
A as compared to other adenocarcinoma cell lines. Immunofluorescence
studies revealed highly irregular nuclear inclusions of lamin A,
suggesting protein or gene expression abnormalities. Reverse
transcriptase-polymerase chain reaction-based cDNA analysis followed by
sequencing indicated the presence of an as yet unidentified alternative
splicing product of the lamin A/C gene. This product differs from lamin
A by the absence of the 5` part of exon 10 (90 nucleotides). Therefore
we propose to designate this product lamin A
Lamins are intermediate filament-type proteins which form the
major components of the nuclear lamina. Two main types of lamins are
known in mammals, i.e. A-type lamins and B-type lamins. The
B-type lamins, B1 and B2, are encoded by two distinct
genes(1) , while lamin B3 is a recently discovered alternative
splicing product of the lamin B2 gene in embryonic cells(2) .
At least one of the B-type lamins is ubiquitously expressed in
mammalian cells and their expression appears to be independent of the
state of cellular differentiation(3, 4) . The A-type
lamins, represented by lamins A and C, are products arising from one
gene by alternative splicing(5, 6, 7) .
A-type lamin expression appears to be related to the state of cellular
differentiation. In general, well differentiated cells express A-type
lamins, whereas undifferentiated cells synthesize low or undetectable
levels of A-type lamins(8, 9, 10) . In
addition it is shown that A-type lamins are not expressed in
proliferating cells of some adult tissues such as basal cells of the
skin (11) or certain lineages of the hematopoietic
system(12) . The recent unraveling of the complete human lamin
A/C gene (7) has provided a better insight into the mechanism
by which lamins A and C are generated from the same gene. The site for
alternative splicing has been demonstrated to be located within exon
10. While exon 1 through 9 of the lamin A and C mRNAs are identical,
lamin A mRNA further contains the 5` 90 bases of exon 10, followed by
exon 11 and 12. In contrast the lamin C messenger contains the complete
111-base sequence of exon 10, but not exon 11 and 12. In this report we
describe a third splicing product of the lamin A/C gene, identical to
lamin A with the exception of the absence of exon 10.
Figure 2:
Northern blotting of cell lines GLC-A1,
GLC-A2, NL-Ac1, and NCI-H125 (lanes 1-4), hybridized
with the lamin A probe (Panel A). Note the reduced expression
of A-type lamins in GLC-A1 (lane 1). Panel B, lamin
B1 probe; Panel C, GAPDH probe.
Figure 3:
Panel A, region of interest of the lamin A
cDNA. Numbering started from nucleotide 1. Upper numbers indicate restriction sites and exon boundaries of interest. Lower numbers correspond to first nucleotide recognized by
primers 1 through 4 (p1-p4). Numbers in boxes correspond to exon numbering according to Lin and Worman (7) . Panel B, agarose gel electrophoretic analyses of
PCR products resulting from primers 1 and 2 on cell line GLC-A1 (lane 1). Note the presence of two bands with sizes between
800 and 900 bp (double arrow). Excision and purification of
each band, followed by electrophoresis, resulted in two bands with
distinct molecular weights (lanes 2 and 3). Panel
C, restriction fragment length analysis of the normal lamin A (lanes 1 and 3) and lamin A
Figure 1:
Immunofluorescence of cell line GLC-A1 (Panels A and B) and GLC-A2 (C), using an
antibody to lamin A (Panels A and C) or lamin B2 (Panel B). Bar represents 20
µm.
Figure 4:
Analysis of PCR products for the presence
of lamin A and lamin A
Figure 5:
PCR-based analysis of genomic DNA of human
placenta, human leukocytes, and cell lines NL-Ac1 and GLC-A1. In the first lane for each sample, primers 3 and 6 were used, and in
the second lane primers 3 and 4. Arrows indicate the
level of the 1404-bp band (upper) and 649-bp fragment (lower arrow). Note that, in the 100-bp ladder markers (m), bands below 600 bp are barely
visible.
Figure 6:
Immunoblot detection of lamin A and lamin
A
In this report we describe the widespread occurrence of an as
yet unidentified splicing product of the lamin A/C gene that we
designate lamin A Hybridization with oligonucleotides hybridizing to either lamin
A A positive
identification of the protein encoded by the lamin A It is feasible that the presence of lamin
A
Volume 271,
Number 16,
Issue of April 19, 1996 pp. 9249-9253
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
10. Deletion of the 30
amino acids encoded by exon 10 was predicted to result in a shift in pI
of the protein from 7.4 to approximately 8.6, which was confirmed by
two-dimensional immunoblotting. mRNA analysis in a variety of cell
lines, normal colon tissue as well as carcinomas demonstrated the
presence of lamin A10 in all samples examined, suggesting its
presence in a variety of cell types.
Cell Lines
Adenocarcinoma cell lines NCI-H125,
NCI-H23(13, 14) , GLC-A1, GLC-A2(15) , and
NL-Ac1(16) , the neuroendocrine non-small cell lung carcinoma
cell line NCI-H460(17) , the large cell undifferentiated lung
carcinoma cell line LCLC-103H(18) , the breast carcinoma cell
line T47D(19) , and the (bladder) transitional carcinoma cell
line T24 (20) were grown in Roswell Park Memorial Institute
Medium 1640 (RPMI 1640, ICN Flow, Irvine, UK) containing 2 g/liter
NaHCO
, supplemented with 10% newborn calf serum (ICN Flow).
The epitheloid lung carcinoma cell line MR65 (21) was grown in
minimal essential medium in 10% newborn calf serum, while the
neuroblastoma cell line SK-N-SH (22) was grown in RPMI with 15%
fetal bovine serum. All cell lines were maintained in a humidified
incubator at 37 °C in 5% CO
.Human Tissue Specimens
Human lung adenocarcinoma
specimens and a specimen of normal colon were obtained after surgery
and snap frozen in liquid nitrogen. Samples were homogenized in
ice-cold guanidine isothiocyanate buffer (23) using an Omni
1001 mixer (Omni, Waterbury, CT) at 20,000 rpm for 15-60 s and
stored at -70 °C until use.Immunofluorescence
The procedure for
immunofluorescence microscopy has been described(24) . Primary
antibodies used include 133A2 (25) recognizing lamin A and LN43 (26) recognizing lamin B2. As a secondary antibody, fluorescein
isothiocyanate-labeled rabbit anti-mouse immunoglobulins (DAKO,
Glostrup, Denmark) was used.RNA Isolation and Northern Blotting Analysis
Total
RNA was isolated from cell lines and tissue specimens by the guanidine
isothiocyanate procedure(23, 27) . Twenty µg of
total RNA were size-fractionated on a formaldehyde agarose
gel(28) , blotted onto Hybond-N+ membrane (Amersham Life
Science, Little Chalfont, UK), and cross-linked with 120 J/cm in an UV Stratalinker 1800 (Stratagene, La Jolla, CA). Blots were
hybridized overnight at 65 °C in a hybridization mix containing 7%
SDS (Life Technologies, Inc.), 1% bovine serum albumin (Sigma), 2
mM EDTA, 0.5 M NaHPO
(pH
7.0), and 0.1 mg/ml sheared salmon sperm DNA (Boehringer Mannheim GmbH,
Mannheim, Germany), to which [
P]dATP-labeled
probe (see below) was added. After hybridization the blots were washed
with saline citrate buffer (SSC, 0.15 M NaCl and 0.015 M sodium citrate pH 7.0) containing 0.1% SDS. Stringency conditions
were optimized for each DNA probe. Autoradiography was performed with
RX Fuji medical x-ray films (Fuji) at -70 °C using
intensifying screens.Probes
The lamin A/C probe, kindly provided by Dr.
F McKeon (6) , and the lamin B1 probe, a kind gift of Dr.
Pollard(29) , were labeled with
[
-P]dATP (Amersham) by random priming (30) and purified over a Sephadex G50 fine (Pharmacia, Uppsala,
Sweden) column. Each hybridization was performed with 50-200 ng
of probe DNA. A GAPDH probe (31) was used as a control for the
amount mRNA loaded onto the gel.cDNA Synthesis and Polymerase Chain Reaction
Analysis
The reverse transcriptase (RT) (
)reaction
was performed with 25 µg of total RNA or 5 µg of
poly(A)
RNA. Samples were incubated for 1.5 h at 37
°C in a reaction mixture containing 10 µg of oligo(dT) primers
(Pharmacia), 1 mM of each dNTP (Pharmacia), 40 units of RNasin
(Promega, Madison, WI), 0.01 M dithiothreitol (Sigma), and 600
units of Moloney leukemia virus reverse transcriptase (Life
Technologies, Inc.) in a total volume of 50 µl. After completion of
the RT reaction 5 µl of this reaction mixture were used for PCR in
a total reaction volume of 50 µl, containing 50 mM KCl
(Merck), 10 mM Tris-HCl, pH 8.3 (Merck), 4 mM MgCl (Merck), 0.001% gelatin, 1 mM of each
dNTP (Pharmacia), 1.5 µg of primer 1 (sense,
5`-GCCTACCGCAAGCTCTTGGA-3`; Eurogentec, Seraing, Belgium),
corresponding to lamin A codon 375-381 (nucleotides
1123-1142, see Fig. 2A), and 1.5 µg of primer
2 (antisense, 5`-GGTGAGGAGGACGCAGGAAG-3`, lamin A noncoding region,
nucleotides 2030-2049). This mixture was heated to 95 °C for
5 min and cooled to 80 °C for 5 min, and thereafter 0.2 unit of Taq polymerase (Super Taq, HT Biotechnology Ltd.,
Cambridge, UK) was added. The mixture was overlaid with 60 µl of
mineral oil (Sigma), heated at 94 °C for 2 min, and carried through
30 cycles of denaturation (94 °C, 1 min), annealing (54 or 58
°C, 2 min), and elongation (72 °C, 2 min), followed by a final
extension step of 72 °C for 10 min. PCR products were used for
subcloning (see below) or for a second round of PCR. For this latter
purpose, samples were first run on 1.5% agarose gel. Bands at the
800-900-bp level, corresponding to the predicted size of products
of lamin A and not lamin C cDNA, were excised and (electro-)eluted.
Next, PCR was performed under identical conditions as described above,
with primers 3 (sense, 5`-AGCCTGCGTACGGCTCTCAT-3`, lamin A codon
525-531, nucleotides 1573-1592) and 4 (antisense,
5`-GCTCCTGAGCCGCTGGCAGA-3`, lamin A codons 599-605, nucleotides
1795-1814).
Oligonucleotide Hybridization
PCR products were
run on a 2% alkaline agarose gel(32) , blotted onto Hybond
N+ (Amersham) and hybridized with
[
-P]ATP end-labeled oligonucleotides.
5`-End labeling of 10 pmol of oligonucleotides was performed using the
USB T4 polynucleotide kinase system (U. S. Biochemical Corp.).
Oligonucleotides comprised primer 3 (see above), hybridizing to a
region within exon 9, and an oligonucleotide complementary to the last
11 bases of exon 9 and the first 11 bases of exon 11 (oligo 5,
5`-CAGTGGGAGCCTTCCCCAGTGG-3`), thus enabling a specific hybridization
to lamin A cDNA, in which exon 10 is lacking. Hybridization was
performed at 46 °C in 5 
SSPE buffer containing 0.3% SDS.
Washes were at 52 °C in the same buffer. The same blot was first
hybridized with primer 3, stripped, and rehybridized with oligo 5.Genomic DNA Analysis
DNA was isolated from human
leukocytes, human placenta and cell lines GLC-A1 and NL-Ac1 using
standard procedures(32) . Purified genomic DNA was subjected to
a single round of PCR using either primers 3 and 4 (see above) or
primer 3 and primer 6, 5`-GAAAAGATTTTTGGCACGG-3`, complementary to the
sequence in the untranslated region of exon 10. The use of primers 3
and 4 in PCR is supposed to produce a 1405-bp fragment for the lamin
A/C gene, while primers 3 and 6 will yield a 649-bp fragment for this
gene.Plasmid Subcloning, Restriction Analysis, and
Sequencing
Two products of 927 and 837 bp resulting from PCR
with primers 1 and 2 were subcloned into the pGEM-T vector (Promega)
and used to transform Escherichia coli TG1 cells by
electroporation. Cloned plasmids were analyzed by restriction fragment
analysis. The restriction digestions were performed with a combination
of the endonucleases NcoI and SstII, or SstI
and PstI (Promega). Relevant restriction sites are indicated
in Fig. 3A. Cloned fragments of the PCR reaction with
primers 1 and 2 were digested with PstI, and the fragment from
nucleotide 1496-1707 was subcloned in pUC 19. Subsequently these
fragments were sequenced using the dideoxynucleotide chain termination
method (33) with 1.5-2 µg of template plasmid using a
T7 sequencing kit (Pharmacia).
10 (lanes 2 and 4) using SstI and NcoI (lanes 1 and 2), or PstI and SstII (lanes 3 and 4). Note that after digestion with the first set of
restriction enzymes the fragment of 704 bp (lane 1) is
shortened in lamin A10 (lane 2, arrow), and
after digestion with the second set of restriction enzymes the expected
fragment of 211 bp is shortened by approximately 100 bp (compare lanes 3 and 4, arrow). These results
indicate a deletion between the PstI sites of exon 9 and 11. Panel D, structure of the three different mRNAs resulting from
alternative splicing of the lamin A/C gene. Panel E, schematic
diagram (adapted from Nigg(42) ) showing the impact of the
missing exon 10 in lamin A at the protein level. P,
phosphorylation site; NLS, nuclear localization signal; (E/D), triple repeat of Glu and Asp; H, a sequence of four histidines; CAAX,
motif for isoprenylation of lamin A.
Two-dimensional Gel Electrophoresis and
Immunoblotting
Cytoskeletal preparations of cell line GLC-A1
were prepared as described previously (24) and subjected to
nonequilibrium pH gel electrophoresis(34) . Electrophoresis was
performed for 1800 V-h using 2% Bio-Lyte 3-10 carrier ampholytes
(Bio-Rad). In the second dimension 10% SDS/polyacrylamide gels were
used according to Laemmli (35) . Proteins were immunoblotted
onto nitrocellulose and the presence of lamin A was detected by
antibody 133A2 (25) using enhanced chemiluminescence (ECL kit,
Amersham, Buckinghamshire, UK) as described elsewhere(24) .
Immunofluorescence
In cell line GLC-A1 an
abnormal localization of lamin A has been detected using
immunofluorescence microscopy (Fig. 1A; see also (24) ). Instead of a perinuclear localization, as seen for the
B-type lamins, nuclear aggregates of lamin A were seen in these cells,
when stained with an antibody directed to lamin A. In contrast, lamin
B2 appears to be organized in a perinuclear lamina (Fig. 1B) in the same cell line. Another adenocarcinoma
cell line GLC-A2 expressed a normal fluorescence staining pattern with
the lamin A antibody (Fig. 1C).
Northern Blotting
Northern blotting studies,
comparing lamin expression in several adenocarcinoma cell lines of the
lung, revealed that in the cell line GLC-A1 A-type lamin mRNA levels
were largely reduced as compared to the other cell lines (Fig. 2). Lamin C mRNA levels in GLC-A1 were much lower than in
the other cell lines and the lamin A messenger was barely detectable on
Northern blot. No obvious abnormalities in the predicted sizes of the
A-type lamin mRNAs were observed.Detection of a New Type of Lamin A/C mRNA
To
examine whether minor mRNA abnormalities could be detected, RT-PCR was
performed. Special emphasis was put in the region coding for the
carboxyl terminus since the structure of the lamin tail is crucial for
correct nuclear assembly. Therefore, we designed primers 1 and 2 (Fig. 3A), starting at nucleotide 1123 and terminating
55 bases into the 3`-untranslated region of lamin A. Gel
electrophoretic analysis of the PCR fragments of GLC-A1 showed two
bands of about the predicted 927 bp (Fig. 3B, double arrows). Only one band was found in a control PCR using
a lamin A cDNA clone (not shown). In addition, RT-PCR was performed
with primer 1 and primer 6, complementary to part of the
3`-untranslated region of lamin C mRNA, resulting in a single band
around 650 bp, as predicted from the known cDNA sequence (data not
shown). Subcloning of the two PCR products using primers 1 and 2 showed
that one band was of the correct size (927 nucleotides), whereas the
other product was approximately 100 bp shorter (Fig. 3B). Restriction fragment length analyses of the
latter product showed that fragments containing exons 9, 10, and 11
were reduced by approximately 100 bp (Fig. 3C, arrows). Sequencing of both the normal and the short products
revealed that in both products the nuclear localization signal and the
CAAX motif were present (not shown), but that the 90 bp of
exon 10 were missing in the short product. The predicted mRNA structure
of this shortened lamin A, which we suggest to designate lamin
A10, is outlined in Fig. 3D. To map which region
is missing from the normal lamin A, we have included a schematic
drawing indicating the most important characteristics of lamin A (Fig. 3E). The amino acid region encoded by exon 10 is
located in the carboxyl-terminal region, which forms the tail of the
protein. This region contains a stretch of amino acids rich in aspartic
acid and glutamic acid (Asp/Glu), followed by a sequence of four
consecutive histidines (His).Presence of Lamin A
To
determine whether lamin A10 in Other Cell Types10 was unique for a single cell line,
nested PCR was performed using primers 1 and 2, followed by PCR with
primers 3 and 4 (Fig. 3A) on cDNA of several lung
cancer cell lines, other carcinoma cell lines, and a neuroblastoma cell
line. In addition, cDNA derived from tissues of normal colon and
adenocarcinomas of the lung were examined (Fig. 4, A and B). PCR of plasmid lamin A cDNA yielded the expected
242-bp product of lamin A, whereas the lamin A10 plasmid cDNA
yielded a fragment of 152 bp (Fig. 4A, lanes 1 and 2). Analysis of cDNA from different sources on
ethidium bromide stained agarose gels (A and B, upper panels) showed in addition to the expression of the 242
bp a band of 152 bp of variable intensity in most samples. The identity
of the stained bands was confirmed by an alkaline agarose gel, and PCR
products were hybridized to the end-labeled primer 3 (Fig. 4, A and B, middle panels) or oligo 5 (Fig. 4, A and B, lower panels).
Hybridization to primer 3 showed that lamin A and lamin A10 occur
in all samples, except for the controls (Panel A, lanes
1, 2, and 7). However, the ratio of lamin
A/A10 expression showed a large variation among samples.
Hybridization with oligo 5 allowed the positive identification of lamin
A10 in all samples tested, except for the two controls in Panel A (lanes 2 and 7). No detectable
cross-hybridization with normal lamin A was observed.
10 cDNA. PCR using primers 1 and 2 was
followed by a PCR using primers 3 and 4 in all cases, except for cell
lines LCLC-103H and NCI-H125, which were subjected to a single round of
PCR reactions using primer 3 and 4. Panel A, lamin A10
cDNA (lane 1), normal lamin A cDNA (lane 2), GLC-A1 (lane 3), LCLC-103 (lane 4), NCI-H125 (lane
5), NCI-H23 (lane 6), normal lamin A cDNA (lane
7), GLC-A2 (lane 8), and NL-Ac1 (lane 9). Panel B, cDNA from cell line T24 (lane 1), T47D (lane 2), NCI-H460 (lane 3), MR65 (lane 4),
SK-N-SH (lane 5), normal colon (lane 6), and four
different adenocarcinomas of the lung (lanes 7-10). The upper panel shows ethidium bromide-stained agarose gels with
levels of the expected lamin A and lamin A10 bands denoted by an arrow. Middle panel, hybridization with P-end-labeled primer 3, hybridizing to both lamin A and
lamin A10 cDNA. Lower panel, hybridization with P-end-labeled oligonucleotide 5, specifically hybridizing
to lamin A10 cDNA only. m = 100-bp ladder
markers
Genomic DNA Analysis
To investigate the
possibility that lamin A10 is a transcript from an as yet
unidentified lamin A-like gene, PCR was performed on genomic DNA
samples from carcinoma cell lines and normal tissues. Fig. 5shows that DNA from placenta, leukocytes, and from cell
lines GLC-A1 and NL-Ac1 all show a single band at the predicted levels
of 649 and 1409 bp.
Two-dimensional Immunoblotting
Two-dimensional
immunoblotting of GLC-A1 using the antibody to lamin A showed the
presence of a lamin A doublet at the predicted position (approximately
70 kDa, pI 7.0, Fig. 6). In addition, however, a clear protein
spot recognized by this antibody was observed with a molecular mass of
around 65 kDa and a pI value that was shifted approximately 1 pH unit
to the basic site (arrow). No such protein spot was observed
in immunoblots of cell line NCI-H125 (not shown).
10 after two-dimensional nonequilibrium gel electrophoresis. Note
the presence of normal sized lamin A with the expected pI of around
7.0, while an additional spot with lower molecular weight and more
basic pI is also found (arrow).
10, since exon 10 is absent in this transcript.
In a previous report we have demonstrated the presence of an A-type
lamin protein, which forms intranuclear aggregates in cell line
GLC-A1(24) . In contrast to normal, perinuclear A-type lamins,
these intranuclear aggregates could be largely extracted by Triton
X-100, indicating that these aggregates are not assembled into the
nuclear matrix(36) . This abnormal nuclear localization of the
protein suggested a distortion in the mRNA region coding for the
carboxyl-terminal part of the protein, since this part is known to
govern targeting to the nucleus by the nuclear localization signal, and
the CAAX-motif, the isoprenylation site of (pre)lamin A, which
is essential for a proper incorporation into the nuclear
lamina(37, 38, 39) . The structure of this
region of the mRNA was examined by RT-PCR. Sequencing showed that the
correct sequence for both motifs was present in the cDNA examined.
However, gel analysis showed the presence of an additional shortened
PCR product. Restriction fragment analysis, followed by sequencing
showed that in the otherwise normal lamin A cDNA sequence exon 10 was
lacking. Therefore we designated this novel protein lamin A10. 10 cDNA alone or both lamin A10 and lamin A cDNA showed that
lamin A10 is expressed in a variety of tissues, since all samples
examined were positive. In addition, we found that the ratio of
expression levels of lamin A10 and lamin A varied significantly
between samples. Especially in the lung cancer cell lines the relative
concentration of the lamin A10 can be high. Similar differences in
the expression ratio were observed between lamin A and lamin C at both
protein and mRNA level(8, 24) . Which mechanism is
involved in regulating the relative expression of the three alternative
splicing products of the lamin A/C gene, remains to be elucidated.
Preliminary studies show that different cultures from the same cell
line can express different ratios of lamin A to lamin A10, which
might be explained by differences in cell density.10 mRNA is not
yet possible since no antibody specific for this product is available.
Therefore we cannot yet state that the abnormal lamin A expression
patterns as seen in GLC-A1 are represented by the lamin A10
protein. However, evidence that indeed lamin A10 mRNA is
translated into protein comes from one-dimensional (24) and
two-dimensional gel electrophoresis followed by immunoblotting studies.
Computer assisted calculation of the pI of the lamin A 10 protein
indicates a value of 8.58 as compared to a theoretical pI of 7.4 for
normal lamin A (PepStats, CAOS/CAMM, Nijmegen, The Netherlands).
Furthermore, a deletion of 30 amino acids should give rise to an
approximately 3.5-kDa smaller protein. In one-dimensional gel
electrophoresis a protein smaller than lamin A was detected with the
lamin A antibody 133A2(24, 25) . The additional
protein spot detected in two-dimensional immunoblotting is
significantly more basic (approximately 1 pI value), about 5 kDa
smaller than the normal lamin, and fulfils the predicted
electrophoretic characteristics of lamin A10. The possibility that
lamin A10 is a result of a translocation or deletion of lamina A/C
or is a transcript from a closely related as yet unknown gene has been
examined. A previous study suggests that such a gene might
exist(40) . Our PCR analysis within the region between exon 9
and 11 of genomic DNA provides no evidence for an additional gene.
Thus, a single gene is responsible for lamins A, A10, and C. The
possibility that lamin A10 is the result of a mutated lamin A/C
gene has been eliminated by our finding that lamin A10 occurred in
all samples examined.10 mRNA has been overlooked in previous studies because of its
relatively low abundance as compared to lamins A and C expression. A
cell line with a low expression of normal lamins A and C has enabled us
to identify this lamin A10 mRNA by RT-PCR. The same holds true for
protein analyses, in which lamin A10 is easily overlooked,
especially because of the relatively large pI shift. Furthermore, only
in cells expressing low levels of the normal A-type lamins an aberrant
lamin can induce visible effects on the structure of the lamina.
Apparently, in GLC-A1 the concentration of lamin A10 can reach
relatively high levels resulting in a distorted nuclear phenotype. The
lamin A10 protein may be localized in the nuclear inclusions seen
in this cell line(24) . This would be in agreement with
transfection studies (39) which showed that different types of
nuclear distortions can be induced with constructs of lamin A
containing carboxyl-terminal deletions either starting at codon 456
(within exon 7) or starting at codon 550 (within exon 10). An altered
localization of the lamin A10 protein is likely, since the
deletion of the first part of exon 10 results in loss of an acidic
domain (7 consecutive Glu or Asp residues) and a polyhistidine domain.
This is bound to have an impact on the interaction of lamin A10
with other nuclear components. Although suggested, it is not yet shown
that this particular highly charged region is involved in chromatin
binding(41) . If the protein extracted by Triton X-100 from
GLC-A1 indeed represents lamin A10, then it is possible that this
protein is not or only partially bound to the nuclear matrix and may be
involved in other intranuclear interactions.
)
We thank Dr. Y. Raymond (Montreal, Canada) and E. B.
Lane (Dundee, UK) for providing antibodies, Dr. F. McKeon (Boston, MA),
Dr. C. Glass (La Jolla, CA) for allowing the use of the lamin A and B1
probes, and Dr. G. Bepler (Durham, NC), Dr. L. de Leij (Groningen, The
Netherlands), and Dr. D. Carney (Dublin, Ireland) for providing cell
lines used in this study.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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T. Ralle, C. Grund, W. W. Franke, and R. Stick Intranuclear membrane structure formations by CaaX-containing nuclear proteins J. Cell Sci., December 1, 2004; 117(25): 6095 - 6104. [Abstract] [Full Text] [PDF]
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J. L.V. Broers, E. A.G. Peeters, H. J.H. Kuijpers, J. Endert, C. V.C. Bouten, C. W.J. Oomens, F. P.T. Baaijens, and F. C.S. Ramaekers Decreased mechanical stiffness in LMNA-/- cells is caused by defective nucleo-cytoskeletal integrity: implications for the development of laminopathies Hum. Mol. Genet., November 1, 2004; 13(21): 2567 - 2580. [Abstract] [Full Text] [PDF]
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M. S. Zastrow, S. Vlcek, and K. L. Wilson Proteins that bind A-type lamins: integrating isolated clues J. Cell Sci., March 1, 2004; 117(7): 979 - 987. [Abstract] [Full Text] [PDF]
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 C. Favreau, D. Higuet, J.-C. Courvalin, and B. Buendia Expression of a Mutant Lamin A That Causes Emery-Dreifuss Muscular Dystrophy Inhibits In Vitro Differentiation of C2C12 Myoblasts Mol. Cell. Biol., February 15, 2004; 24(4): 1481 - 1492. [Abstract] [Full Text] [PDF]
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C. Ostlund, G. Bonne, K. Schwartz, and H. J. Worman Properties of lamin A mutants found in Emery-Dreifuss muscular dystrophy, cardiomyopathy and Dunnigan-type partial lipodystrophy J. Cell Sci., March 14, 2002; 114(24): 4435 - 4445. [Abstract] [Full Text] [PDF]
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R. D. Goldman, Y. Gruenbaum, R. D. Moir, D. K. Shumaker, and T. P. Spann Nuclear lamins: building blocks of nuclear architecture Genes & Dev., March 1, 2002; 16(5): 533 - 547. [Full Text] [PDF]
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 C. J. Lelliott, L. Logie, C. P. Sewter, D. Berger, P. Jani, F. Blows, S. O'Rahilly, and A. Vidal-Puig Lamin Expression in Human Adipose Cells in Relation to Anatomical Site and Differentiation State J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 728 - 734. [Abstract] [Full Text] [PDF]
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A. Garg, M. Vinaitheerthan, P. T. Weatherall, and A. M. Bowcock Phenotypic Heterogeneity in Patients with Familial Partial Lipodystrophy (Dunnigan Variety) Related to the Site of Missense Mutations in Lamin A/C Gene J. Clin. Endocrinol. Metab., January 1, 2001; 86(1): 59 - 65. [Abstract] [Full Text]
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J. Broers, B. Machiels, G. van Eys, H. Kuijpers, E. Manders, R van Driel, and F. Ramaekers Dynamics of the nuclear lamina as monitored by GFP-tagged A-type lamins J. Cell Sci., January 10, 1999; 112(20): 3463 - 3475. [Abstract] [PDF]
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T. Mical and M. Monteiro The role of sequences unique to nuclear intermediate filaments in the targeting and assembly of human lamin B: evidence for lack of interaction of lamin B with its putative receptor J. Cell Sci., January 12, 1998; 111(23): 3471 - 3485. [Abstract] [PDF]
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G. Pugh, P. Coates, E. Lane, Y Raymond, and R. Quinlan Distinct nuclear assembly pathways for lamins A and C lead to their increase during quiescence in Swiss 3T3 cells J. Cell Sci., January 10, 1997; 110(19): 2483 - 2493. [Abstract] [PDF]
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