Neuron,
Vol. 5, 881-888,
December,
1990, Copyright
0 1990 by Cell Press
Specificity of Expression of the Muscle and Brain Dystrophin Gene Promoters in Muscle and Brain Cells Efrat Barnes: Dorit Zuk: Rabi Simantov,+ Uri Nudel: and David Yaffe* *Department of Cell Biology +Department of Molecular Genetics and Weizmann Institute of Science Rehovot 76100 Israel
Virology
Summary The gene that is defective in Duchenne and Becker muscular dystrophies is expressed in the muscle and brain. However, the 5’ ends of the 14 kb mRNA in these tissues are derived from two different exons, indicating the involvement of at least two promoters in the regulation of the cell-type and developmental specificities of expression of this gene. In the study presented here, we used the polymerase chain reaction and RNAase protection methods and various cell cultures to investigate the specificities of expression of these promoters. The results indicate a very stringent control of expression of the two promoters. In cloned rat myogenic cells, only the muscle-type dystrophin transcript was detected, and its presence was correlated with the formation of multinucleated fibers. In neuronal cell cultures, the braintype transcript was detected. However, glial cell cultures expressed the muscle transcript only. Some cell lines derived from brain cells expressed both isoforms. Introduction Duchenne muscular dystrophy (DMD) is an X-linked recessive disease that results in a progressive degeneration of the muscle and death in the second or third decade of life. In addition to the severe damage to the muscle, some 30% of the affected patients also suffer various degrees of mental retardation (reviewed in Moser, 1984). The gene that, when defective, is responsible for the disease spans over 2300 kb. Its 14 kb mRNA codes for a 3685 amino acid protein (dystrophin). The predicted amino acid sequence suggests that the protein has a rod shape (Koenig et al., 1988). Electron microscopic and immunofluorescence investigations have indicated that the protein is associated with the cell membrane (Hoffman et al., 1987; Arahata et al., 1988; Sugita et al., 1988; Watkins et al., 1988; Zubrycka-Gaarn et al., 1988.) Early studies suggested that the gene is expressed in muscle only; however, it was later shown that significant amounts of dystrophin mRNA are also found in the brain and to a lesser extent in several other nonmuscle tissues (Nude1 et al., 1988, 1989). It was also demonstrated that the 14 kb isoform of the DMD gene mRNA, which is expressed in the brain, is not identical to the muscle isoform. The Sends of the two mRNAs are derived from different exons, indicating
the involvement of at least two promoters in the control of the tissue and developmental specificities of expression of this gene (Nude1 et al., 1989; Yaffe et al., 1989; Feener et al., 1989). In the current study we have employed the polymerase chain reaction (PCR) and RNAase protection methods and various cell cultures to test more rigorously the cell-type specificities of expression of these promoters. The results indicate a very stringent control of expression of the two promoters. Only the muscle-type DMD transcript was detected in cloned myogenic cells, and it was developmentally regulated. The major DMD isoform detected in neuronal cultures was the brain isoform, whereas primary glial cell cultures expressed the muscle isoform.
Results Expression of the Dystrophin mRNA lsoforms in Muscle and Brain To extend the analysis of the dystrophin gene expression in the brain, we dissected rat brains into various regions. Total RNA was prepared from the cerebellum, medulla, midbrain, thalamus, hypothalamus, hippocampus, striatum, temporal cortex, and occipital cortex. RNA samples were tested with a probe derived from the plasmid pMD-1, using the RNAase protection assay. This probe hybridizes with both muscleand brain-type mRNAs. As demonstrated in Figure 1, all parts of the brain tested contained dystrophin mRNA, with some variation in quantity. When using the RNAase protection assay, probes complementary to the first exon of the muscle-type dystrophin mRNA did not detect dystrophin transcripts in brain RNA (Nude1 et al., 1989). Likewise brain-type dystrophin mRNA was not detected in muscle RNA by probes derived from the first exon of the brain-type DMD gene transcript. However, when we used the extremely sensitive PCR technique and primers diagnostic for the muscleand brain-type DMD first exons (described in Experimental Procedures), we also detected small amounts of the brain isoform in the muscle and of the muscle isoform in brain (e.g., Figures 4 and 5).
Expression of the Dystrophin mRNA lsoforms in Cultured Cells The detection of both dystrophin mRNA isoforms in RNA preparations from muscle and brain tissue could be due to both isoforms being synthesized by the same cells, or a result of expression of the two isoforms in different cell types found in the tissues from which the RNA was prepared. To investigate these possibilities further, we analyzed the expression of the two isoforms in cloned cell populations of muscle and brain origin.
NWMl a82
Cloned
cells: Multi nucleated fibers! Primer:
Sk.muscle I
-
L185 +
II
-
+
I
M 81 M B M 81 M B I
NuC.
Figure Clones
2. PCRAnalysis of Myogenic
of Dystrophin Cells
mRNA
lsoforms
in Isolated
Mononucleated myoblasts from primary skeletal muscle cultures (Sk. muscle) and cells of the myogenic subline Ll85 (L185) were cloned and induced to differentiate as described in Results and Experimental Procedures. Single myogenic clones consisting of only mononucleated cells f-J and clones containing multinucleated fibers (+) were isolated, lysed, and analyzed by the PCR technique using primers diagnostic from the muscle-type first exon (M) or brain-type first exon (B).
:
i (a
Figure 1. Expression the Brain
of the Dystrophin
Gene
in Various
Parts of
The indicated regions were dissected from rat brains, and total RNA was extracted as described in Experimental Procedures. The RNAase protection assay was performed on 20 pg RNA samples. The radiolabeled probe used in this assay was MD-l, which is complementary to nucleotides 2502-2588 of rat dystrophin mRNA (numbering is according to the human dystrophin cDNA; Koenig et al., 1988). The mouse skeletal muscle mRNA (M.Sk. Muscle) protects a shorter stretch of the rat-derived probe, most probably because of some divergence between mouse and rat sequences (Nude1 et al., 1988).
M yogenic Primary pared plating.
Cell Cultures rat
and
skeletal enriched Secondary
muscle cell for myogenic cultures were
cultures were precells by differential plated at cloning
density. Between 4 and 6 days after cloning, the medium in part of the cultures was changed to the fusion-inducing medium (group A); the other cultures were refed with fresh proliferation-supportive medium (group 6). Between 1 and 2 days later, single colonies in group B, containing several dozen typical myogenic cells, were marked and carefully inspected with an inverted phase-contrast microscope to ensure that all ce,lls were mononucleated. Conversely, in group A, colonies containing multinucleated fibers were marked. The colonies were isolated using glass cylinders and lysed for cDNA preparation and PCR analysis as described in Experimental Procedures. The PCR analysis, using primers diagnostic for the first exons of the muscleor brain-type dystrophin mRNAs, did not reveal the DMD gene transcript in extracts from undifferentiated colonies. A clear signal for the muscle-type dystrophin mRNA was obtained in extracts from colonies containing multinucleated fibers; primers for the brain-type dystrophin transcript did not produce a positive signal in this RNA (Figure 2). A similar experiment was done using colonies formed by an established myogenic cell line, L185. As with cloned primary culture cells, only the muscletype dystrophin mRNA was detected, and this finding was correlated with the presence of multinucleated fibers (Figure 2). Moreover, no brain-type dystrophin mRNA was detected by PCR analysis of RNA extracted from differentiated or undifferentiated L185 mass cultures. The muscle-type dystrophin transcript was detected main-
Specificity 883
of Expression
of Dystrophin
Promoters
A Multinucleated
t
fibers: Primer:
+ M
Figure 3. PCR Analysis genie Cell Cultures
B
II B
M
of Dystrophin
RN A: Muscle
l+ln M
B
mRNA
lsoforms
Primer: BM
in Myo-
ly in the differentiated cultures containing multinucleated fibers (Figure 3). The small amount of this mRNA found in the myoblast cultures may be a result of the existence of small numbers of myofibers in mass myoblast cultures.
mRNA:
Figure
4. PCR Analysis
Neur.
Must. Brain
‘MB”MB”M ..
of Dystrophin
-tar
mRNA
lsoform
Glia
‘MBI’M8”M’M
G4 B’
B
Total RNA was extracted from undifferentiated (-) and differentiated (+) mass cultures of L185 cells. (A) Southern blot analysis of the size-fractionated PCR product. The blot was overexposed to allow for the detection of even minor amounts of the brain isoform. (B) Ethidium bromide staining of the size-fractionated PCR product. Lanes labeled M and B represent reactions with primers diagnostic for muscleand brain-type dystrophin mRNAs, respectively.
Primer:
Brain
in Neurons
RNA was extracted from rat skeletal muscle (Must.), rat brain (Brain), and primary neuron cultures (Neur.). Lanes labeled M and B represent reactions with primers diagnostic for muscleand brain-type dystrophin mRNAs, respectively. The blot was overexposed to allow for the detection of even minor amounts of muscle-type dystrophin mRNA in neurons. The upper band in each lane is double-stranded DNA, and the lower band is single-stranded DNA.
Figure Cells
5. PCR Analysis
of Dystrophin
mRNA
lsoforms
in Clial
RNAwas prepared from muscle, brain glial cells, and an immortalized clone isolated from a glial culture (G4). Lanes labeled M and B represent reactions with primers diagnostic for the first exons of muscleand brain-type dystrophin mRNAs, respectively.
Thus, at this level of detection, products of the brain-type promoter are not found in myogenic cultures at all, whereas the products of the muscle-type promoter are detected in colonies containing multinucleated fibers. Neuronal and Clial Cell Cultures Primary rat neuronal and glial cultures were prepared as described in Experimental Procedures. RNA was extracted and analyzed by the PCR using the primers specific for the muscleor brain-type first exons. The brain dystrophin mRNA isoform but not the muscle isoform was reproducibly found in one RNA preparation from neuronal cell cultures (Figure 4). A second RNA preparation from neuronal cultures gave a very weak signal with the primer diagnostic for the muscle-type first exon, in addition to the major signal obtained with the brain-type primers (data not shown). The weak signal obtained with primers diagnostic for the muscle-type dystrophin mRNA in this RNA preparation is probably due to contamination of the primary neuronal cultures with small amount of glial cells (see below). In contrast to the results obtained with the neuronal cell cultures, the muscle-type dystrophin mRNA, but not the brain-type, was reproducibly detected in RNA preparations from glial cell cultures (Figure 5). The presence of the muscle-type dystrophin mRNA isoform in glial cells was also demonstrated by the RNAase protection method (Figure 6). In accordance with the PCR results, the brain-type dystrophin transcript was not detected in the same RNA preparations (data not shown). Comparison of the signal produced by RNAase protection of glial cell RNA and that of muscle RNA (at shorter exposures than that of Figure 6) indicates that the DMD mRNA sequences in glial cells are far less abundant than those in muscle cells.
-nt
- 148
-I03
65 nt
Brain Probe Muscle
Figure6 Brain-Type
RNAase Protection Assay Analysis Dystrophin mRNAs in Clial Cells
of
Muscle-
and
Growth of glial cell cultures, preparation of total RNA, and RNAase protection assay were performed as described in Experimental Procedures. The RNA probewas synthesized on plasmid MD-12 containing sequences from the first and second exons of the muscle-type dystrophin mRNA (nucleotides 221285 according to the numbering of the human dystrophin cDNA by Koenig et al. [1988]). The 65 nucleotide RNA fragment was protected by muscle-type dystrophin mRNA only.
Expression of the Muscle-Type in Clial Cell Lines
Dystrophin
mRNA
To test whether the positive signals in glial cell-cultures obtained with the primer and probe diagnostic for the muscle-type DMD gene first exon were not the result of the presence in the glial cell cultures of some other cell types, we examined the expression of the dystrophin mRNA in cloned glial cell lines. Cg is a glioma cell line established by Benda et al.
Figure 7. RNAase Protection Brain-Type Dystrophin mRNAs
mRNA(l48nt) mRNA(l03nt)
Assay Analysis of Musclein Cloned C6 Cells
and
C6 cells were plated at cloning density. Subclones were isolated and grown to confluency, and total RNA was extracted and analyzed by the RNAase protection assay using probe MD-105 as described in Experimental Procedures. C6-3 and C,-6 are representative of the six subclones assayed. The brain- and muscle-type dystrophin mRNAs protected 148 and 103 nucleotides of the probe, respectively.
(1968). Cells of this line accumulate the glial-specific protein SIOO (Pfeifer et al., 1970) and display an increase in the activity of the glial-specific membrane protein 2’,3’-cyclic nucleotide 3’ phosphohydrolase (Maltese and Volpe, 1979) when reaching high cell density. Preliminary RNAase protection analysis revealed the presence of both dystrophin mRNA isoforms in RNA extracts of these cells (data not shown). As this cell line has been maintained for many passages in cell culture, it is possible that the line consists of a het-
Specificity 885
Figure
of Expression
8. The
Response
of Dystrophin
Promoters
of C4 Ceils
to Dibutyric
CAMP
(A) Cultures of the cloned cell line G4, grown in 33% (B) G4 cells grown CAMP to the nutritional medium. The cells were stained with Ciemsa.
erogeneous cell population and that the two isoforms are produced by different cell types. Alternatively, the results may indicate that the same cell type can produce both isoforms. To examine this question, we plated Cb cells under cloning conditions and six single clones were isolated and analyzed by RNAase protection and the PCR. The results clearly showed that all recloned Cr, cells contained both dystrophin mRNA isoforms, although there was some variation in the relative amounts of the two isoforms in the different clones (Figure 7). This indicates that the activation of both promoters in the same cell lineage is not mutually exclusive. It is of interest to note that comparable levels of the dystrophin mRNA were detected in both confluent and proliferating cultures of C6 cells (data not shown). This indicates that the giant DMD gene can be transcribed in proliferating cell populations. To examine the question of dystrophin mRNA synthesis in glial cells, we attempted to clone cells from primary glial cell cultures. Primary glial cell cultures were infected with the retrovirus RVtsA58 containing the early region of SV40, encoding a temperaturesensitive large T antigen, and a gene conferring resistance to the neomycin derivative C418 Uat and Sharp, 1989). The infected cultures were grown in medium
at 39OC for 6 days,
1 day after
addition
of 1 mM
dibutyric
containing G418, and one of the G418-resistant clones was amplified. When grown at 33%, these cells proliferated as an immortalized cell line, without overt morphological differentiation, and stained positively with the antibody Ran2, typical of progenitors of type-l astrocytes (Miller et al., 1989). Transfer of the cultures to 39OC and subsequent addition of N6,0*‘-dibutyryladenosine 3’,5’-cyclic monophosphate (dibutyric cAMP) resulted in the rapid acquisition of a glia-like morphology (Figure 8). The precise nature of these cells is currently under investigation. In PCR analysis, RNA preparations from either undifferentiated or differentiated cells produced a strong signal with the probes diagnostic for the first exon of the muscle-type dystrophin mRNA. No signal was detected when probes diagnostic for the brain-type dystrophin mRNA were used (Figure 5, G4). The signal observed when RNA from differentiated cultures was used was reproducibly stronger than that seen with RNA from undifferentiated cultures (data not shown). Discussion The investigation presented here the activation of the two DMD gene here brain-type and muscle-type,
demonstrates that promoters, termed can be very strin-
gently controlled. Analysis of cloned myogenic cell populations with the extremely sensitive PCR technique showed that products of the brain-type promoter are undetectable in myogenic cells and that the expression of the muscle-type promoter mRNA product is detectable only in colonies containing multinucleated fibers. Conversely, no muscle-type dystrophin mRNA is detected in an RNA preparation from neuronal cultures depleted of proliferating cells. Glial cells are ontogenetically closely related to neuronal cells and develop from a common precursor cell (e.g., Turner and Cepko, 1987; Holt et al., 1988; Wetts and Fraser, 1988; Frederiksen et al., 1988; Ready et al., 1978). Yet surprisingly, the dystrophin mRNA detectable in primary cultures of these cells differs from the brain-type dystrophin mRNA and contains the muscle-type first exon. The observation that RNA of a cloned population of glial cells immortalized by SV40 large T antigen also reacts only with the muscle-typespecific primers indicates that the presence of the muscle-type diagnostic sequences in mRNA extracted from glial cell cultures is not due to a contaminant of muscle cells, but rather to a genuine product of the glial cell lineage. Thus, the reproducible signal obtained in the PCR of RNA prepared from brain tissue with primers specific for the muscle-type dystrophin mRNA may originate from glial cells. It was previously reported that when a brain cDNA library was screened for DMD cDNA clones, two out of the three isolated cDNA clones had the muscle-type 5’ first exon (Feener et al., 1989). In view of the current findings, it is conceivable that these cDNA sequences were formed on RNA templates originating from glial cells. It should be pointed out that the relative amount of the muscle-type dystrophin mRNA in primary glial cell cultures is much smaller than the relative amount of dystrophin mRNA in total brain RNA. This is probably the reason why earlier studies, using Western blot technique, suggested that dystrophin is not present in glial cells (Hoffman et al., 1988). While PCR analysis of RNA from primary glial cell cultures or a recently isolated cell line, immortalized by SV40 large T antigen revealed the expression of the muscletype promoter only, analysis of the glioma cell line C6 revealed the expression of both isoforms of dystrophin mRNA. Expression of both isoforms was also detected in a number of other neuroblastoma and glioma cell lines (unpublished data). It is possible that the transformation of the cells or their prolonged cultivation in cell culture results in the relaxation of the control of expression of the two promoters. Alternatively, it is possible that the cell lines contain more primitive cells that express both isoforms or give rise to more than one cell type. This question is currently under investigation. The precise role of dystrophin in muscle and brain is unknown. As a result of the use of a different 5’exon in muscle- and brain-type dystrophin mRNAs, the first IO amino acids of the muscle-type dystrophin are replaced by 3 amino acids in the brain-type dystro-
phin (Nude1 et al., 1989). However, it has been reported that in addition to the differences produced by alternative usage of the first exon in brain and muscle, differences in the isoforms may be produced by alternative splicing of exons farther downstream (Feener et al., 1989). Thus, it is possible that in spite of the use of the muscle-type first exon in glial dystrophin mRNA, the glial dystrophin isoform is not identical to the muscle isoform. In view of the obvious big difference in structure and function between glial cells and muscle cells, it will be of great interest to investigate whether the major isoform of dystrophin in glial cells is identical to that in muscle cells. The finding that the DMD gene is expressed in glial cells raises the question whether the misfunction of the gene in these cells is involved in the mental retardation sometimes associated with the muscular dystrophy. Sequence analysis of the muscle-type promoter of the human (Klamut et al., 1990) and rat (Yaffe et al., 1990; and unpublished data) DMD genes reveals several conserved DNA motifs that are present in musclespecific genes. Transfection experiments showed that this region of the DNA can confer developmentally regulated expression of chimeric genes in myogenic cells (Klamut et al., 1990; Yaffe et al., 1990). In view of the finding that the glial dystrophin mRNA contains the muscle-type first exon, it would be of interest to examine the control elements involved in the activation of the gene in the different cell types and in the developmentally regulated expression. While this manuscript was in preparation for publication, Chelly et al. (1990) published data indicating the presence of the muscle-type DMD gene first exon in glial cell mRNA. Experimental
Procedures
RNA Preparation Total RNA was prepared by the LiCI-urea extraction method (Auffray et al., 1980). The integrity and quantity of all RNA samples were verified by electrophoresis on agarose-formaldehyde gels and ethidium bromide staining. RNAase Protection Assay and cRNA Probes The assay was performed essentially as described by Nude1 et al. (1988), except that 15 ug of total RNA was used for each assay and hybridization was done at 4WC. Synthetic oligonucleotides, genomic DNA fragments (of rat and mouse origin), and cDNA fragments (rat origin) were cloned in the polylinker of the vector Gemini 3 (Promega) containing the T7 and SP6 promoters. The following probes were used in this study: -MD-l (Nude1 et al., 1988), which was derived from a rat genomic DNA clone and contains the exon extending from nucleotide 2502 to nucleotide 2588 of the muscle-type dystrophin cDNA. This sequence is present in both the brain- and muscle-type dystrophin mRNAs. -MD-13 (Nude1 et al., 1989), which was derived from a mouse genomic DNA clone and contains the 5’end of the muscle-type dystrophin transcript. -MD-103, which was derived from a brain-type dystrophin cDNA clone and contains a part of the brain-specific first exon, the second exon, and a part of the third exon, in which the sequences of the second and third exons are shared with the muscle-type dystrophin mRNA. -MD-105, which is a shortened version of MD-103 containing 120 nucleotides less at its 5’ end.
Specificity 887
of Expression
of Dystrophin
Promoters
% Brain
?
mRNA Exons:
Figure 9. Identification Brain-Type Dystrophin
v
C
M ,,,,“sc,e
mR,,,A Exons:
.. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .7.’
v
’
2
1
3
v
4 7
5
kiOOnt,
Polymerase Chain Reaction The PCRassaywas performed according to a modification of the procedure described by Saiki et al. (1988). Ten micrograms of total RNA and a primer specific for dystrophin mRNA (Figure 9, primer C) were annealed, and cDNA was generated with reverse transcriptase (Life Sciences). The cDNA was amplified by the PCR, using Taq polymerase (Promega) according to the following program: 1 cycle of 7 min at 95OC (denaturation), 5 min at 56OC (annealing), and 10 min at 72’C (polymerization), followed by 35 cycles of 1,2, and 3 min at the respective temperatures. The last cycle included 10 min at 72OC and 20 min at 56OC. The diagnostic primers for the brainand muscle-type dystrophin mRNAs included a common primer containing sequences of the fourth exon of the gene (this primerwas also used for the synthesis of the cDNA [Figure 9, primer C]) and specific primers containing sequences of either the muscle-type first exon or the brain-type first exon (Figure 9, primers M and B). Samples of the amplified products were size-fractionated on 2% agarose gels and analyzed by the Southern blot technique using a 57 base oligonucleotide end-labeled probe that hybridizes to the common region of the PCR product (Figure 9). Neuronal and Clial Cell Cultures Monolayer cultures of glia and neurons were prepared as detailed by Schwartz and Simantov (1988), except that the whole brain (rather than the striatum) was used to prepare the neuronal cultures. Additionally, 2.5 x 10e6 M cytosine arabinoside was included in the nutritional medium of the neuronal cultures to block the proliferation of, and thus eliminate, the nonneuronal cells. The glial cultures contained type-l and type-2 astrocytes. C6 rat glioma cells were grown in Dulbecco’s minimal eagle’s medium supplemented with 5% horse serum. Confluent cultures were harvested 9 days after plating. Cloning of Skeletal Muscle Myoblasts Primary rat skeletal muscle cell cultures were prepared as previously described (Yaffe, 1973). Forty-eight hours after plating, the cells were suspended by trypsin and subjected to differential plating, which removes most of the nonmyogenic cells (Yaffe, 1968, 1973). The cells in suspension (consisting mainly of myoblasts) were collected and counted. Between 50 and 100 cells per 9 cm petri dish were plated on a feeder layer of X-irradiated (5,000 R) L8 cells (IO5 cells per 100 mm plate) and grown in FE medium, which stimulates cell proliferation without cell fusion (Yaffe, 1973). The lfi cells (Yaffe and Saxel, 1977) used as a feeder layer carry a large deletion in the DMD gene, which includes exon 4 (unpublished data). Therefore, cDNA synthesized on RNA from these cells cannot provide a template for the PCR assay described below. To induce cell fusion, the medium was changed 4-5 days after plating to the fusion-stimulating S-medium (Yaffe, 1973). Fusion started about 18 hr after the change to S-medium. Cultivation and Cloning of 1185 Myogenic The L185 rat myogenic cell line is a recent
Cells subline
of L8, originat-
of Muscleand mRNAs by the PCR
The PCR primers used for the assays are shown. The horizontal bars represent the 5’ regions of the brain- and muscle-type mRNAs. Arrowheads indicate the exon boundaries. Black bar, common sequence; hatched bar, brain-type first exon; dotted bar, muscle-type first exon. C represents the common primer, Band M are the brainand muscle-specific primers, respectively. The open bar represents the oligonucleotide used as a probe for the detection of the PCR products.
325
2
1
v
T
ing from a frozen early passage of L8 cells. Nonconfluent cultures of proliferating mononucleated cells were maintained (as is I& in Waymouth medium supplemented with 15% fetal calf serum and 0.5% chicken embryo extract. To induce cell fusion, cultures approaching confluency were refed with Dulbecco’s modified eagle’s medium supplemented with 2% horse serum and 4 U/100 ml insulin (2HI medium). For isolation of single myogenie clones, L185 cells were plated at a density of 100 cells per plate on an X-irradiated l8 feeder layer, as described above, and grown in Waymouth medium as described. To induce differentiation in the cloned cells, the cultures were refed with 2HI medium 6 days after plating. Fusion started about 2-3 days after the change to 2HI medium. PCR Analysis of Single Clones Plates were appropriately illuminated, and well-isolated single colonies of skeletal muscle myoblasts or L185 cells were marked and carefully inspected on an inverted phase-contrast microscope as described in Results. After aspirating of the medium and washing three times with PBS, selected clones were isolated with glass cylinders (6 mm diameter) fixed to the plate surface with silicon grease. The wells of the cylinder were filled with 20 ~1 of a solution containing 1 mM EDTA and 30 U of RNAsin (Promega). Five microliters of the solution was used in a reverse transcriptase reaction (20 ~1 of 40 mM Tris [pH 8.31, 30 mM KCI, 10 mM M&& 5 mM DTT, 1 mM each of dATP, dCTP, dCTP, and dTTP, 60 ng of specific primer [Figure 9, primer Cl, 15 U of reverse transcriptase [Life Sciences]). Five microliters of this reaction was used in the PCR. Acknowledgments We wish to thank Dr. D. Schubert for the gift of the cell lines of mouse brain tumor origin, Dr. Ora Bernard for providing her protocol for the PCR analysis of single cell RNA, which was adapted with some modifications for the analysis of cloned myogenie cells, Drs. M. Schwartz and 2. Fogel for helpful discussions and antisera, and Dr. M. Raff for the Ran2 antiserum. The valuable technical assistance of Ora Fuchs, Sara Neuman, and Zehava Levi and the secretarial assistance of Naomi Zuri are gratefully acknowledged. This work was supported by research grants from the Muscular Dystrophy Association of the USA, the Association Francaise contre les Myopathies, and the Israel Academy of Sciences. Further support was provided by the Leo and Julia Forchheimer Center for Molecular Genetics, the Kekst Foundation, and the Belle S. and Irving E. Meller Center for the Biology of Aging at the Weizmann Institute of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received
August
17, 1990;
revised
September
17, 1990.
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Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. Schwartz,
J. P., and Siniantov,
R. (1988).
Developmental
Expres-
Yaffe, D. (1968). Retention of differentiation prolonged cultivation of myogenic cells. USA 61, 477-483.
and
in striatal
cul-
Kunkel, L. M. of dystrophin can 372,
potentialities during Proc. Natl. Acad. Sci.
Yaffe, D. (1973). Rat skeletal muscle myoblasts. In Tissue Culture: Methods and Applications, P F. Kruse and M. K. Patterson, eds. (New York: Academic Press, Inc.) pp. 106-114. Yaffe, D., and Saxel, serum requirements 166.
0. (1977). A myogenic cell line with for differentiation. Differentiation
Yaffe, D.,Zuk, D., Einat, f?, Shinar, O., and Nudel, U. (1989). Tissuesion of the Duchenne muscular Must. Dev. 93, pp. 963-975.
altered 7, 159-
D., Levy,Z., Neuman, S., Fuchs, and stage-specificity of expresdystrophy gene. UCLA Symp.
Yaffe, D., Zuk, D., Barnea, E., Bar, S., Makover, A., and Nudel, U. (1990). Promoters and isoforms of transcripts of the DMD gene. In Proc. Uehara Memorial Foundation Symposium on Frontiers in Muscle Research (Tokyo), in press. Zubrycka-Caarn, E. E., Bulman, D. E., Karpati, G., Burghes, A. H. M., Belfall, B., Klamut, H. J., Talbot, J., Hodges, R. S., Ray, P. N., and Worton, R. C. (1988). The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle. Nature 333, 466-469. Note
Added
in Proof
We have recently identified a novel mRNA product of the DMD gene that is only 6.5 kb long. This mRNA differs greatly from the two 14 kb dystrophin mRNAs in its protein coding content and tissue distribution. The abundance of this mRNA in brain is apparently higher than that of the 14 kb DMD mRNA. The cell type distribution of this mRNA is as yet unknown (S. Bar, E. Barnea, D. Yaffe, and U. Nudel, 1990. A novel product of the DMD gene which greatly differs from the known isoforms in its structure and tissue distribution. Biochem. J., in press).
Vol. 5, 881-888,
December,
1990, Copyright
0 1990 by Cell Press
Specificity of Expression of the Muscle and Brain Dystrophin Gene Promoters in Muscle and Brain Cells Efrat Barnes: Dorit Zuk: Rabi Simantov,+ Uri Nudel: and David Yaffe* *Department of Cell Biology +Department of Molecular Genetics and Weizmann Institute of Science Rehovot 76100 Israel
Virology
Summary The gene that is defective in Duchenne and Becker muscular dystrophies is expressed in the muscle and brain. However, the 5’ ends of the 14 kb mRNA in these tissues are derived from two different exons, indicating the involvement of at least two promoters in the regulation of the cell-type and developmental specificities of expression of this gene. In the study presented here, we used the polymerase chain reaction and RNAase protection methods and various cell cultures to investigate the specificities of expression of these promoters. The results indicate a very stringent control of expression of the two promoters. In cloned rat myogenic cells, only the muscle-type dystrophin transcript was detected, and its presence was correlated with the formation of multinucleated fibers. In neuronal cell cultures, the braintype transcript was detected. However, glial cell cultures expressed the muscle transcript only. Some cell lines derived from brain cells expressed both isoforms. Introduction Duchenne muscular dystrophy (DMD) is an X-linked recessive disease that results in a progressive degeneration of the muscle and death in the second or third decade of life. In addition to the severe damage to the muscle, some 30% of the affected patients also suffer various degrees of mental retardation (reviewed in Moser, 1984). The gene that, when defective, is responsible for the disease spans over 2300 kb. Its 14 kb mRNA codes for a 3685 amino acid protein (dystrophin). The predicted amino acid sequence suggests that the protein has a rod shape (Koenig et al., 1988). Electron microscopic and immunofluorescence investigations have indicated that the protein is associated with the cell membrane (Hoffman et al., 1987; Arahata et al., 1988; Sugita et al., 1988; Watkins et al., 1988; Zubrycka-Gaarn et al., 1988.) Early studies suggested that the gene is expressed in muscle only; however, it was later shown that significant amounts of dystrophin mRNA are also found in the brain and to a lesser extent in several other nonmuscle tissues (Nude1 et al., 1988, 1989). It was also demonstrated that the 14 kb isoform of the DMD gene mRNA, which is expressed in the brain, is not identical to the muscle isoform. The Sends of the two mRNAs are derived from different exons, indicating
the involvement of at least two promoters in the control of the tissue and developmental specificities of expression of this gene (Nude1 et al., 1989; Yaffe et al., 1989; Feener et al., 1989). In the current study we have employed the polymerase chain reaction (PCR) and RNAase protection methods and various cell cultures to test more rigorously the cell-type specificities of expression of these promoters. The results indicate a very stringent control of expression of the two promoters. Only the muscle-type DMD transcript was detected in cloned myogenic cells, and it was developmentally regulated. The major DMD isoform detected in neuronal cultures was the brain isoform, whereas primary glial cell cultures expressed the muscle isoform.
Results Expression of the Dystrophin mRNA lsoforms in Muscle and Brain To extend the analysis of the dystrophin gene expression in the brain, we dissected rat brains into various regions. Total RNA was prepared from the cerebellum, medulla, midbrain, thalamus, hypothalamus, hippocampus, striatum, temporal cortex, and occipital cortex. RNA samples were tested with a probe derived from the plasmid pMD-1, using the RNAase protection assay. This probe hybridizes with both muscleand brain-type mRNAs. As demonstrated in Figure 1, all parts of the brain tested contained dystrophin mRNA, with some variation in quantity. When using the RNAase protection assay, probes complementary to the first exon of the muscle-type dystrophin mRNA did not detect dystrophin transcripts in brain RNA (Nude1 et al., 1989). Likewise brain-type dystrophin mRNA was not detected in muscle RNA by probes derived from the first exon of the brain-type DMD gene transcript. However, when we used the extremely sensitive PCR technique and primers diagnostic for the muscleand brain-type DMD first exons (described in Experimental Procedures), we also detected small amounts of the brain isoform in the muscle and of the muscle isoform in brain (e.g., Figures 4 and 5).
Expression of the Dystrophin mRNA lsoforms in Cultured Cells The detection of both dystrophin mRNA isoforms in RNA preparations from muscle and brain tissue could be due to both isoforms being synthesized by the same cells, or a result of expression of the two isoforms in different cell types found in the tissues from which the RNA was prepared. To investigate these possibilities further, we analyzed the expression of the two isoforms in cloned cell populations of muscle and brain origin.
NWMl a82
Cloned
cells: Multi nucleated fibers! Primer:
Sk.muscle I
-
L185 +
II
-
+
I
M 81 M B M 81 M B I
NuC.
Figure Clones
2. PCRAnalysis of Myogenic
of Dystrophin Cells
mRNA
lsoforms
in Isolated
Mononucleated myoblasts from primary skeletal muscle cultures (Sk. muscle) and cells of the myogenic subline Ll85 (L185) were cloned and induced to differentiate as described in Results and Experimental Procedures. Single myogenic clones consisting of only mononucleated cells f-J and clones containing multinucleated fibers (+) were isolated, lysed, and analyzed by the PCR technique using primers diagnostic from the muscle-type first exon (M) or brain-type first exon (B).
:
i (a
Figure 1. Expression the Brain
of the Dystrophin
Gene
in Various
Parts of
The indicated regions were dissected from rat brains, and total RNA was extracted as described in Experimental Procedures. The RNAase protection assay was performed on 20 pg RNA samples. The radiolabeled probe used in this assay was MD-l, which is complementary to nucleotides 2502-2588 of rat dystrophin mRNA (numbering is according to the human dystrophin cDNA; Koenig et al., 1988). The mouse skeletal muscle mRNA (M.Sk. Muscle) protects a shorter stretch of the rat-derived probe, most probably because of some divergence between mouse and rat sequences (Nude1 et al., 1988).
M yogenic Primary pared plating.
Cell Cultures rat
and
skeletal enriched Secondary
muscle cell for myogenic cultures were
cultures were precells by differential plated at cloning
density. Between 4 and 6 days after cloning, the medium in part of the cultures was changed to the fusion-inducing medium (group A); the other cultures were refed with fresh proliferation-supportive medium (group 6). Between 1 and 2 days later, single colonies in group B, containing several dozen typical myogenic cells, were marked and carefully inspected with an inverted phase-contrast microscope to ensure that all ce,lls were mononucleated. Conversely, in group A, colonies containing multinucleated fibers were marked. The colonies were isolated using glass cylinders and lysed for cDNA preparation and PCR analysis as described in Experimental Procedures. The PCR analysis, using primers diagnostic for the first exons of the muscleor brain-type dystrophin mRNAs, did not reveal the DMD gene transcript in extracts from undifferentiated colonies. A clear signal for the muscle-type dystrophin mRNA was obtained in extracts from colonies containing multinucleated fibers; primers for the brain-type dystrophin transcript did not produce a positive signal in this RNA (Figure 2). A similar experiment was done using colonies formed by an established myogenic cell line, L185. As with cloned primary culture cells, only the muscletype dystrophin mRNA was detected, and this finding was correlated with the presence of multinucleated fibers (Figure 2). Moreover, no brain-type dystrophin mRNA was detected by PCR analysis of RNA extracted from differentiated or undifferentiated L185 mass cultures. The muscle-type dystrophin transcript was detected main-
Specificity 883
of Expression
of Dystrophin
Promoters
A Multinucleated
t
fibers: Primer:
+ M
Figure 3. PCR Analysis genie Cell Cultures
B
II B
M
of Dystrophin
RN A: Muscle
l+ln M
B
mRNA
lsoforms
Primer: BM
in Myo-
ly in the differentiated cultures containing multinucleated fibers (Figure 3). The small amount of this mRNA found in the myoblast cultures may be a result of the existence of small numbers of myofibers in mass myoblast cultures.
mRNA:
Figure
4. PCR Analysis
Neur.
Must. Brain
‘MB”MB”M ..
of Dystrophin
-tar
mRNA
lsoform
Glia
‘MBI’M8”M’M
G4 B’
B
Total RNA was extracted from undifferentiated (-) and differentiated (+) mass cultures of L185 cells. (A) Southern blot analysis of the size-fractionated PCR product. The blot was overexposed to allow for the detection of even minor amounts of the brain isoform. (B) Ethidium bromide staining of the size-fractionated PCR product. Lanes labeled M and B represent reactions with primers diagnostic for muscleand brain-type dystrophin mRNAs, respectively.
Primer:
Brain
in Neurons
RNA was extracted from rat skeletal muscle (Must.), rat brain (Brain), and primary neuron cultures (Neur.). Lanes labeled M and B represent reactions with primers diagnostic for muscleand brain-type dystrophin mRNAs, respectively. The blot was overexposed to allow for the detection of even minor amounts of muscle-type dystrophin mRNA in neurons. The upper band in each lane is double-stranded DNA, and the lower band is single-stranded DNA.
Figure Cells
5. PCR Analysis
of Dystrophin
mRNA
lsoforms
in Clial
RNAwas prepared from muscle, brain glial cells, and an immortalized clone isolated from a glial culture (G4). Lanes labeled M and B represent reactions with primers diagnostic for the first exons of muscleand brain-type dystrophin mRNAs, respectively.
Thus, at this level of detection, products of the brain-type promoter are not found in myogenic cultures at all, whereas the products of the muscle-type promoter are detected in colonies containing multinucleated fibers. Neuronal and Clial Cell Cultures Primary rat neuronal and glial cultures were prepared as described in Experimental Procedures. RNA was extracted and analyzed by the PCR using the primers specific for the muscleor brain-type first exons. The brain dystrophin mRNA isoform but not the muscle isoform was reproducibly found in one RNA preparation from neuronal cell cultures (Figure 4). A second RNA preparation from neuronal cultures gave a very weak signal with the primer diagnostic for the muscle-type first exon, in addition to the major signal obtained with the brain-type primers (data not shown). The weak signal obtained with primers diagnostic for the muscle-type dystrophin mRNA in this RNA preparation is probably due to contamination of the primary neuronal cultures with small amount of glial cells (see below). In contrast to the results obtained with the neuronal cell cultures, the muscle-type dystrophin mRNA, but not the brain-type, was reproducibly detected in RNA preparations from glial cell cultures (Figure 5). The presence of the muscle-type dystrophin mRNA isoform in glial cells was also demonstrated by the RNAase protection method (Figure 6). In accordance with the PCR results, the brain-type dystrophin transcript was not detected in the same RNA preparations (data not shown). Comparison of the signal produced by RNAase protection of glial cell RNA and that of muscle RNA (at shorter exposures than that of Figure 6) indicates that the DMD mRNA sequences in glial cells are far less abundant than those in muscle cells.
-nt
- 148
-I03
65 nt
Brain Probe Muscle
Figure6 Brain-Type
RNAase Protection Assay Analysis Dystrophin mRNAs in Clial Cells
of
Muscle-
and
Growth of glial cell cultures, preparation of total RNA, and RNAase protection assay were performed as described in Experimental Procedures. The RNA probewas synthesized on plasmid MD-12 containing sequences from the first and second exons of the muscle-type dystrophin mRNA (nucleotides 221285 according to the numbering of the human dystrophin cDNA by Koenig et al. [1988]). The 65 nucleotide RNA fragment was protected by muscle-type dystrophin mRNA only.
Expression of the Muscle-Type in Clial Cell Lines
Dystrophin
mRNA
To test whether the positive signals in glial cell-cultures obtained with the primer and probe diagnostic for the muscle-type DMD gene first exon were not the result of the presence in the glial cell cultures of some other cell types, we examined the expression of the dystrophin mRNA in cloned glial cell lines. Cg is a glioma cell line established by Benda et al.
Figure 7. RNAase Protection Brain-Type Dystrophin mRNAs
mRNA(l48nt) mRNA(l03nt)
Assay Analysis of Musclein Cloned C6 Cells
and
C6 cells were plated at cloning density. Subclones were isolated and grown to confluency, and total RNA was extracted and analyzed by the RNAase protection assay using probe MD-105 as described in Experimental Procedures. C6-3 and C,-6 are representative of the six subclones assayed. The brain- and muscle-type dystrophin mRNAs protected 148 and 103 nucleotides of the probe, respectively.
(1968). Cells of this line accumulate the glial-specific protein SIOO (Pfeifer et al., 1970) and display an increase in the activity of the glial-specific membrane protein 2’,3’-cyclic nucleotide 3’ phosphohydrolase (Maltese and Volpe, 1979) when reaching high cell density. Preliminary RNAase protection analysis revealed the presence of both dystrophin mRNA isoforms in RNA extracts of these cells (data not shown). As this cell line has been maintained for many passages in cell culture, it is possible that the line consists of a het-
Specificity 885
Figure
of Expression
8. The
Response
of Dystrophin
Promoters
of C4 Ceils
to Dibutyric
CAMP
(A) Cultures of the cloned cell line G4, grown in 33% (B) G4 cells grown CAMP to the nutritional medium. The cells were stained with Ciemsa.
erogeneous cell population and that the two isoforms are produced by different cell types. Alternatively, the results may indicate that the same cell type can produce both isoforms. To examine this question, we plated Cb cells under cloning conditions and six single clones were isolated and analyzed by RNAase protection and the PCR. The results clearly showed that all recloned Cr, cells contained both dystrophin mRNA isoforms, although there was some variation in the relative amounts of the two isoforms in the different clones (Figure 7). This indicates that the activation of both promoters in the same cell lineage is not mutually exclusive. It is of interest to note that comparable levels of the dystrophin mRNA were detected in both confluent and proliferating cultures of C6 cells (data not shown). This indicates that the giant DMD gene can be transcribed in proliferating cell populations. To examine the question of dystrophin mRNA synthesis in glial cells, we attempted to clone cells from primary glial cell cultures. Primary glial cell cultures were infected with the retrovirus RVtsA58 containing the early region of SV40, encoding a temperaturesensitive large T antigen, and a gene conferring resistance to the neomycin derivative C418 Uat and Sharp, 1989). The infected cultures were grown in medium
at 39OC for 6 days,
1 day after
addition
of 1 mM
dibutyric
containing G418, and one of the G418-resistant clones was amplified. When grown at 33%, these cells proliferated as an immortalized cell line, without overt morphological differentiation, and stained positively with the antibody Ran2, typical of progenitors of type-l astrocytes (Miller et al., 1989). Transfer of the cultures to 39OC and subsequent addition of N6,0*‘-dibutyryladenosine 3’,5’-cyclic monophosphate (dibutyric cAMP) resulted in the rapid acquisition of a glia-like morphology (Figure 8). The precise nature of these cells is currently under investigation. In PCR analysis, RNA preparations from either undifferentiated or differentiated cells produced a strong signal with the probes diagnostic for the first exon of the muscle-type dystrophin mRNA. No signal was detected when probes diagnostic for the brain-type dystrophin mRNA were used (Figure 5, G4). The signal observed when RNA from differentiated cultures was used was reproducibly stronger than that seen with RNA from undifferentiated cultures (data not shown). Discussion The investigation presented here the activation of the two DMD gene here brain-type and muscle-type,
demonstrates that promoters, termed can be very strin-
gently controlled. Analysis of cloned myogenic cell populations with the extremely sensitive PCR technique showed that products of the brain-type promoter are undetectable in myogenic cells and that the expression of the muscle-type promoter mRNA product is detectable only in colonies containing multinucleated fibers. Conversely, no muscle-type dystrophin mRNA is detected in an RNA preparation from neuronal cultures depleted of proliferating cells. Glial cells are ontogenetically closely related to neuronal cells and develop from a common precursor cell (e.g., Turner and Cepko, 1987; Holt et al., 1988; Wetts and Fraser, 1988; Frederiksen et al., 1988; Ready et al., 1978). Yet surprisingly, the dystrophin mRNA detectable in primary cultures of these cells differs from the brain-type dystrophin mRNA and contains the muscle-type first exon. The observation that RNA of a cloned population of glial cells immortalized by SV40 large T antigen also reacts only with the muscle-typespecific primers indicates that the presence of the muscle-type diagnostic sequences in mRNA extracted from glial cell cultures is not due to a contaminant of muscle cells, but rather to a genuine product of the glial cell lineage. Thus, the reproducible signal obtained in the PCR of RNA prepared from brain tissue with primers specific for the muscle-type dystrophin mRNA may originate from glial cells. It was previously reported that when a brain cDNA library was screened for DMD cDNA clones, two out of the three isolated cDNA clones had the muscle-type 5’ first exon (Feener et al., 1989). In view of the current findings, it is conceivable that these cDNA sequences were formed on RNA templates originating from glial cells. It should be pointed out that the relative amount of the muscle-type dystrophin mRNA in primary glial cell cultures is much smaller than the relative amount of dystrophin mRNA in total brain RNA. This is probably the reason why earlier studies, using Western blot technique, suggested that dystrophin is not present in glial cells (Hoffman et al., 1988). While PCR analysis of RNA from primary glial cell cultures or a recently isolated cell line, immortalized by SV40 large T antigen revealed the expression of the muscletype promoter only, analysis of the glioma cell line C6 revealed the expression of both isoforms of dystrophin mRNA. Expression of both isoforms was also detected in a number of other neuroblastoma and glioma cell lines (unpublished data). It is possible that the transformation of the cells or their prolonged cultivation in cell culture results in the relaxation of the control of expression of the two promoters. Alternatively, it is possible that the cell lines contain more primitive cells that express both isoforms or give rise to more than one cell type. This question is currently under investigation. The precise role of dystrophin in muscle and brain is unknown. As a result of the use of a different 5’exon in muscle- and brain-type dystrophin mRNAs, the first IO amino acids of the muscle-type dystrophin are replaced by 3 amino acids in the brain-type dystro-
phin (Nude1 et al., 1989). However, it has been reported that in addition to the differences produced by alternative usage of the first exon in brain and muscle, differences in the isoforms may be produced by alternative splicing of exons farther downstream (Feener et al., 1989). Thus, it is possible that in spite of the use of the muscle-type first exon in glial dystrophin mRNA, the glial dystrophin isoform is not identical to the muscle isoform. In view of the obvious big difference in structure and function between glial cells and muscle cells, it will be of great interest to investigate whether the major isoform of dystrophin in glial cells is identical to that in muscle cells. The finding that the DMD gene is expressed in glial cells raises the question whether the misfunction of the gene in these cells is involved in the mental retardation sometimes associated with the muscular dystrophy. Sequence analysis of the muscle-type promoter of the human (Klamut et al., 1990) and rat (Yaffe et al., 1990; and unpublished data) DMD genes reveals several conserved DNA motifs that are present in musclespecific genes. Transfection experiments showed that this region of the DNA can confer developmentally regulated expression of chimeric genes in myogenic cells (Klamut et al., 1990; Yaffe et al., 1990). In view of the finding that the glial dystrophin mRNA contains the muscle-type first exon, it would be of interest to examine the control elements involved in the activation of the gene in the different cell types and in the developmentally regulated expression. While this manuscript was in preparation for publication, Chelly et al. (1990) published data indicating the presence of the muscle-type DMD gene first exon in glial cell mRNA. Experimental
Procedures
RNA Preparation Total RNA was prepared by the LiCI-urea extraction method (Auffray et al., 1980). The integrity and quantity of all RNA samples were verified by electrophoresis on agarose-formaldehyde gels and ethidium bromide staining. RNAase Protection Assay and cRNA Probes The assay was performed essentially as described by Nude1 et al. (1988), except that 15 ug of total RNA was used for each assay and hybridization was done at 4WC. Synthetic oligonucleotides, genomic DNA fragments (of rat and mouse origin), and cDNA fragments (rat origin) were cloned in the polylinker of the vector Gemini 3 (Promega) containing the T7 and SP6 promoters. The following probes were used in this study: -MD-l (Nude1 et al., 1988), which was derived from a rat genomic DNA clone and contains the exon extending from nucleotide 2502 to nucleotide 2588 of the muscle-type dystrophin cDNA. This sequence is present in both the brain- and muscle-type dystrophin mRNAs. -MD-13 (Nude1 et al., 1989), which was derived from a mouse genomic DNA clone and contains the 5’end of the muscle-type dystrophin transcript. -MD-103, which was derived from a brain-type dystrophin cDNA clone and contains a part of the brain-specific first exon, the second exon, and a part of the third exon, in which the sequences of the second and third exons are shared with the muscle-type dystrophin mRNA. -MD-105, which is a shortened version of MD-103 containing 120 nucleotides less at its 5’ end.
Specificity 887
of Expression
of Dystrophin
Promoters
% Brain
?
mRNA Exons:
Figure 9. Identification Brain-Type Dystrophin
v
C
M ,,,,“sc,e
mR,,,A Exons:
.. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .7.’
v
’
2
1
3
v
4 7
5
kiOOnt,
Polymerase Chain Reaction The PCRassaywas performed according to a modification of the procedure described by Saiki et al. (1988). Ten micrograms of total RNA and a primer specific for dystrophin mRNA (Figure 9, primer C) were annealed, and cDNA was generated with reverse transcriptase (Life Sciences). The cDNA was amplified by the PCR, using Taq polymerase (Promega) according to the following program: 1 cycle of 7 min at 95OC (denaturation), 5 min at 56OC (annealing), and 10 min at 72’C (polymerization), followed by 35 cycles of 1,2, and 3 min at the respective temperatures. The last cycle included 10 min at 72OC and 20 min at 56OC. The diagnostic primers for the brainand muscle-type dystrophin mRNAs included a common primer containing sequences of the fourth exon of the gene (this primerwas also used for the synthesis of the cDNA [Figure 9, primer C]) and specific primers containing sequences of either the muscle-type first exon or the brain-type first exon (Figure 9, primers M and B). Samples of the amplified products were size-fractionated on 2% agarose gels and analyzed by the Southern blot technique using a 57 base oligonucleotide end-labeled probe that hybridizes to the common region of the PCR product (Figure 9). Neuronal and Clial Cell Cultures Monolayer cultures of glia and neurons were prepared as detailed by Schwartz and Simantov (1988), except that the whole brain (rather than the striatum) was used to prepare the neuronal cultures. Additionally, 2.5 x 10e6 M cytosine arabinoside was included in the nutritional medium of the neuronal cultures to block the proliferation of, and thus eliminate, the nonneuronal cells. The glial cultures contained type-l and type-2 astrocytes. C6 rat glioma cells were grown in Dulbecco’s minimal eagle’s medium supplemented with 5% horse serum. Confluent cultures were harvested 9 days after plating. Cloning of Skeletal Muscle Myoblasts Primary rat skeletal muscle cell cultures were prepared as previously described (Yaffe, 1973). Forty-eight hours after plating, the cells were suspended by trypsin and subjected to differential plating, which removes most of the nonmyogenic cells (Yaffe, 1968, 1973). The cells in suspension (consisting mainly of myoblasts) were collected and counted. Between 50 and 100 cells per 9 cm petri dish were plated on a feeder layer of X-irradiated (5,000 R) L8 cells (IO5 cells per 100 mm plate) and grown in FE medium, which stimulates cell proliferation without cell fusion (Yaffe, 1973). The lfi cells (Yaffe and Saxel, 1977) used as a feeder layer carry a large deletion in the DMD gene, which includes exon 4 (unpublished data). Therefore, cDNA synthesized on RNA from these cells cannot provide a template for the PCR assay described below. To induce cell fusion, the medium was changed 4-5 days after plating to the fusion-stimulating S-medium (Yaffe, 1973). Fusion started about 18 hr after the change to S-medium. Cultivation and Cloning of 1185 Myogenic The L185 rat myogenic cell line is a recent
Cells subline
of L8, originat-
of Muscleand mRNAs by the PCR
The PCR primers used for the assays are shown. The horizontal bars represent the 5’ regions of the brain- and muscle-type mRNAs. Arrowheads indicate the exon boundaries. Black bar, common sequence; hatched bar, brain-type first exon; dotted bar, muscle-type first exon. C represents the common primer, Band M are the brainand muscle-specific primers, respectively. The open bar represents the oligonucleotide used as a probe for the detection of the PCR products.
325
2
1
v
T
ing from a frozen early passage of L8 cells. Nonconfluent cultures of proliferating mononucleated cells were maintained (as is I& in Waymouth medium supplemented with 15% fetal calf serum and 0.5% chicken embryo extract. To induce cell fusion, cultures approaching confluency were refed with Dulbecco’s modified eagle’s medium supplemented with 2% horse serum and 4 U/100 ml insulin (2HI medium). For isolation of single myogenie clones, L185 cells were plated at a density of 100 cells per plate on an X-irradiated l8 feeder layer, as described above, and grown in Waymouth medium as described. To induce differentiation in the cloned cells, the cultures were refed with 2HI medium 6 days after plating. Fusion started about 2-3 days after the change to 2HI medium. PCR Analysis of Single Clones Plates were appropriately illuminated, and well-isolated single colonies of skeletal muscle myoblasts or L185 cells were marked and carefully inspected on an inverted phase-contrast microscope as described in Results. After aspirating of the medium and washing three times with PBS, selected clones were isolated with glass cylinders (6 mm diameter) fixed to the plate surface with silicon grease. The wells of the cylinder were filled with 20 ~1 of a solution containing 1 mM EDTA and 30 U of RNAsin (Promega). Five microliters of the solution was used in a reverse transcriptase reaction (20 ~1 of 40 mM Tris [pH 8.31, 30 mM KCI, 10 mM M&& 5 mM DTT, 1 mM each of dATP, dCTP, dCTP, and dTTP, 60 ng of specific primer [Figure 9, primer Cl, 15 U of reverse transcriptase [Life Sciences]). Five microliters of this reaction was used in the PCR. Acknowledgments We wish to thank Dr. D. Schubert for the gift of the cell lines of mouse brain tumor origin, Dr. Ora Bernard for providing her protocol for the PCR analysis of single cell RNA, which was adapted with some modifications for the analysis of cloned myogenie cells, Drs. M. Schwartz and 2. Fogel for helpful discussions and antisera, and Dr. M. Raff for the Ran2 antiserum. The valuable technical assistance of Ora Fuchs, Sara Neuman, and Zehava Levi and the secretarial assistance of Naomi Zuri are gratefully acknowledged. This work was supported by research grants from the Muscular Dystrophy Association of the USA, the Association Francaise contre les Myopathies, and the Israel Academy of Sciences. Further support was provided by the Leo and Julia Forchheimer Center for Molecular Genetics, the Kekst Foundation, and the Belle S. and Irving E. Meller Center for the Biology of Aging at the Weizmann Institute of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received
August
17, 1990;
revised
September
17, 1990.
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Added
in Proof
We have recently identified a novel mRNA product of the DMD gene that is only 6.5 kb long. This mRNA differs greatly from the two 14 kb dystrophin mRNAs in its protein coding content and tissue distribution. The abundance of this mRNA in brain is apparently higher than that of the 14 kb DMD mRNA. The cell type distribution of this mRNA is as yet unknown (S. Bar, E. Barnea, D. Yaffe, and U. Nudel, 1990. A novel product of the DMD gene which greatly differs from the known isoforms in its structure and tissue distribution. Biochem. J., in press).
