Download The dystrophin / utrophin homologues in Drosophila and in sea urchin

Gene 263 (2001) 17±29
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The dystrophin / utrophin homologues in Drosophila and in sea urchin q
Sara Neuman a, Alex Kaban a,1, Talila Volk b, David Yaffe a, Uri Nudel a,*
a
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel
b
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
Received 21 August 2000; received in revised form 12 November 2000; accepted 5 December 2000
Received by H. Cedar
Abstract
The gene which is defective in Duchenne muscular dystrophy (DMD) is the largest known gene containing at least 79 introns, some of
which are extremely large. The product of the gene in muscle, dystrophin, is a 427 kDa protein. The same gene encodes at least two additional
non-muscle full length dystrophin isoforms transcribed from different promoters located in the 5 0 -end region of the gene, and four smaller
proteins transcribed from internal promoters located further downstream, and lack important domains of dystrophin. Several other genes,
encoding evolutionarily related proteins, have been identi®ed. To study the evolution of the DMD gene and the signi®cance of its various
products, we have searched for genes encoding dystrophin-like proteins in sea urchin and in Drosophila. We previously reported on the
characterization of a sea urchin gene encoding a protein which is an evolutionary homologue of Dp116, one of the small products of the
mammalian DMD gene, and on the partial sequencing of a large product of the same gene. Here we describe the full-length product which
shows strong structural similarity and sequence identity to human dystrophin and utrophin. We also describe a Drosophila gene closely
related to the human dystrophin gene. Like the human gene, the Drosophila gene encodes at least three isoforms of full length dystrophin-like
proteins (dmDLP1, dmDLP2 and dmDLP3,), regulated by different promoters located at the 5 0 end of the gene, and a smaller product
regulated by an internal promoter (dmDp186). As in mammals, dmDp186 and the dmDLPs share the same C-terminal and cysteine-rich
domains which are very similar to the corresponding domains in human dystrophin and utrophin. In addition, dmDp186 contains four of the
spectrin-like repeats of the dmDLPs and a unique N-terminal region of 512 amino acids encoded by a single exon. The full length products
and the small product have distinct patterns of expression. Thus, the complex structure of the dystrophin gene, encoding several large
dystrophin-like isoforms and smaller truncated products with different patterns of expression, existed before the divergence between the
protostomes and deuterostomes. The conservation of this gene structure in such distantly related organisms, points to important distinct
functions of the multiple products. q 2001 Elsevier Science B.V. All rights reserved.
Keywords: Dystrophin; Utrophin; Gene evolution; Drosophila; Sea urchin
1. Introduction
The gene which is defective in duchenne muscular
dystrophy (DMD) is the largest gene known to date, spanning over 2500 kb of the X chromosome. The product of the
Abbreviations: BAC, bacterial arti®cial chromosome; CNS, central
nervous system; DAPs, dystrophin associated proteins; DMD, Duchenne
muscular dystrophy; dmDLP, drosophila melanogaster dystrophin like
protein; DRP, dystrophin related protein; ORF, open reading frame; PCR,
polymerase chain reaction; RT, reverse transcription; RACE, rapid ampli®cation of cDNA ends; suDLP, sea urchin dystrophin like protein; UTR,
untranslated region
q
Accession numbers of sequences described here are: dmDp186 ±
AF300294; dmDLP2 ± AF297644; the 5 0 end regions of dmDLP1 and
dmDLP3 ± AF300456, AF302236, respectively; suDLP ± AF304204.
* Corresponding author. Tel.: 1972-8-934-2815; fax: 1972-8-934-4125.
E-mail address: [email protected] (U. Nudel).
1
Present address: Kaplan Hospital, Rehovot, Israel.
gene in the muscle, dystrophin, is a 427 kDa rod-shaped
protein consisting of four domains: N-terminal actin binding
domain, 24 triple helix spectrin-like repeats with four hinge
regions, a cysteine-rich domain with two potential calcium
binding motifs, and a unique C-terminal domain (Koenig et
al., 1988). Dystrophin forms a linkage between the cytoskeletal actin and a group of membrane proteins, as well as with
a number of non-membranal proteins (collectively called
dystrophin associated proteins; DAPs) (Yoshida and
Ozawa, 1990; Ervasti and Campbell, 1991). The association
with the DAPs is mediated mainly by the cysteine-rich and
C terminal domains of dystrophin (Suzuki et al., 1994; Jung
et al., 1995). One of the DAPs, a dystroglycan, binds laminin. Thus, in the muscle this complex links the cytoskeleton,
the sarcolemma and the extracellular matrix (Ahn and
Kunkel, 1993; Campbell, 1995; Ozawa et al., 1995).
The DMD gene also codes for two non muscle isoforms
0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0378-111 9(00)00584-9
18
S. Neuman et al. / Gene 263 (2001) 17±29
of dystrophin, each controlled by a different promoter
located in the 5 0 end region of the gene; the brain type
(Nudel et al., 1989; Barnea et al., 1990; Boyce et al.,
1991) and Purkinje cell type dystrophins (Gorecki et al.,
1992). In addition, internal promoters located within introns
further downstream in the huge DMD gene regulate the
expression of smaller products. Dp71, a 70.8 kDa protein,
consists of only the cysteine-rich and C-terminal domains of
dystrophin (Bar et al., 1990; Lederfein et al., 1992). It is the
most abundant non-muscle product of the DMD gene. The
highest levels of Dp71 are found in the brain (Rapaport et
al., 1992; Greenberg et al., 1996). The other known small
products of the DMD gene consist of the cysteine-rich and
C-terminal domains with various extensions into the spectrin like repeats domain (reviewed in Yaffe et al., 1996).
These products are Dp116 (Byers et al., 1993), Dp140
(Lidov et al., 1995) and Dp260 (D'Souza et al., 1995),
which are expressed mainly in Schwann cells, brain, and
retina, respectively, and have molecular weights of 116,
140 and 260 kDa. The functions of the non-muscle dystrophins and of the smaller products of the DMD gene are not
known.
Several genes encoding proteins with various levels of
sequence identity with dystrophin were characterized. Utrophin (DRP1) is encoded by an autosomal gene and consists
of all four domains of dystrophin. At the amino acid level
the two proteins show ,51% identity. The exon/intron
structures of the two genes are also very similar (Love et
al., 1989; Pearce et al., 1993). The utrophin gene also
encodes smaller products transcribed from downstream
promoters (Fabbrizio et al., 1995; Blake et al., 1995; Wilson
et al., 1999). Sequence comparisons suggested that the
dystrophin and utrophin genes were separated by duplication during early evolution of the vertebrates (Wang et al.,
1998; Roberts and Bobrow, 1998).
Bessou et al. (1998) described a gene in C. elegans encoding a dystrophin related protein. Only a single product was
described. Mutations in the gene which is expressed mainly
in muscle resulted in hypersensitivity to acetylcholine and
hyperactivity of the worms.
The other known genes that seem to be evolutionary
related to the DMD gene are much smaller, each encodes
a single protein with low but signi®cant similarity to the
cysteine-rich and C-terminal domains of dystrophin. These
include the torpedo 87 kDa phosphoprotein and its mammalian homologues dystrobrevin (Carr et al., 1989; Blake et al.,
1996; Sadoulet-Puccio et al., 1996) and the Drosophila 75
kDa protein, Dah (Zhang et al., 1996). A small and simple
structured gene encoding a 110 kDa protein (DRP2) which
consist of two spectrin-like repeats, the cysteine-rich and the
C-terminal domains (like in Dp116), has also been characterized in vertebrates (Roberts et al., 1996).
Roberts and Bobrow (1998), using PCR with redundant
primers, cloned fragments from the 3 0 end region of the
mRNAs of dystrophin related proteins from several organisms, including Drosphila, suggesting the widespread exis-
tence of such proteins in invertebrates. In a previous
publication we reported on the identi®cation of a sea urchin
gene encoding a protein which is most likely an evolutionary homologue of Dp116, one of the small products of
human dystrophin gene. The same gene also encoded a
partially sequenced larger product, which we assumed to
be a homologue of the full-length human dystrophin
(Wang et al., 1998). Sea urchins are deuterostomes which
diverged early from the evolutionary branch leading to
primitive chordates and to vertebrates. To get a further
insight into the evolution of the DMD gene and the function
of its products, we attempted to characterize the dystrophin
gene homologue of the protostome Drosophila. The molecular genetic information on Drosophila and the available
genetic methodologies provide an excellent model system
for a structural and functional analysis of the gene and its
products.
In the present communication we report on the characterization of a Drosophila homologue of dystrophin gene and
on the completion of the sequencing of the mRNA encoding
the sea urchin dystrophin-like protein. Both genes encode
large products homologous to the full-length dystrophin and
utrophin, as well as smaller products transcribed from internal promoters. Like the smaller products of dystrophin and
utrophin genes, the small products of the sea urchin and
Drosophila genes lack the N-terminal actin-binding domain
and a substantial part of the spectrin-like repeats. The
complex structure of the gene, encoding both large fulllength dystrophin-like proteins and smaller truncated
products, with different patterns of expression, is very
ancient and existed before the divergence between the
protostomes and deuterostomes.
2. Materials and methods
2.1. Isolation and sequencing of cDNA clones
A Drosophila 8±12 h embryos cDNA library in ggt11
was obtained from K. Zinn (Zinn et al., 1988). The library
was screened using a 32P cDNA probe based on the partial
sequence of Drosophila mRNA encoding a polypeptide
with great similarity to the C-terminal region of dystrophin
(accession no. X99757). Positive phages were puri®ed, their
cDNA inserts were ampli®ed by PCR (expended high ®delity kit, Boeringer) using vector based primers, and cloned
in the plasmid pGEMT (Promega). DNA was sequenced by
the automatic thermocycling DNA sequencing procedure,
using a 377 DNA sequencer or 3700 DNA analyzer
(Perkin±Elmer). Most of the sequences were identical to
the sequences found in the Drosophila data base. In case
of differences, especially in the published sequence of the 3 0
region of the Drosophila gene, corrections were made
according to sequences that were presented in the data
base two or more times, and by sequencing the second
DNA strand. The sea urchin mRNA sequence was deter-
S. Neuman et al. / Gene 263 (2001) 17±29
mined from overlapping cDNA clones isolated from 24±43
h embryos cDNA library (Wang et al., 1998) obtained from
E. Davidson and from cDNA clones obtained by 5 0 RACE.
Both strands of cDNA were sequenced.
2.2. Reverse transcription/Polymerase chain reaction
RNA was prepared from ¯ies, embryos and larvae by the
Lithium chloride/urea extraction methods (Auffray et al.,
1980). For cloning, reverse transcription was performed
with 5 mg of total ¯ies RNA, using speci®c antisense
primers and the enzyme superscript II (Gibco-BRL). The
cDNA was puri®ed using High Pure PCR Product Puri®cation Kit (Boehringer). Nested PCR was performed on 10%
of the cDNA using the expanded high ®delity kit (Boehringer) or AmpliTag Gold Kit (Perkin±Elmer). Cloning into
pGEMT vector and sequencing was done as described
above. In semiquantitative RT/PCR experiments the
cDNA primer was from exon 4. The antisense PCR primer
that consisted of exons 3±4 border sequences, and sense
primers that were speci®c to each dmDLP isoform. The
PCR reaction was stopped after 27 cycles. The products
were size fractionated and transferred to nylon ®lters.
After hybridization to internal 32p-labelled oligonucleotide
probe, the ®lter was autoradiographed. In addition, the
intensity of each band was determined using phosphoimager.
2.3. 5 0 RACE
5 0 RACE was done essentially as described by Frohman
et al., 1988. First strand cDNA was synthesized using an
antisense oligonucleotide from exon 4, and puri®ed as
described above in 2.2. The cDNA was then dG-tailed
using deoxynucleotide terminal transferase (Promega), puri®ed, and subjected to nested PCR using a speci®c antisense
primer from exon 3 or from the isoform speci®c exons, and a
sense primer containing a 3 0 dC tail. The PCR products were
puri®ed and sequenced or cloned into pGEMT vector and
sequenced.
2.4. In situ hybridization
Whole mount in situ hybridization was performed by the
method of Tautz and Pfei¯e (1989) using a Digoxygeninlabeled DNA probe. The DNA fragments were prepared by
PCR. The 2850 bp probe for dmDLP mRNA was from the
region encoding amino acids 1±950. The 1260 bp probe for
dmDP186 mRNA was from the region encoding amino
acids 20±440.
2.5. Protein sequences analysis and comparisons
Comparisons of the deduced amino acid sequences of
various proteins and calculation of percent identity were
done by best ®t, dotplot and prettybox programs of Genetic
Computer Group (GCG10), University Research Park,
Madison, Wisconsin. Comparison of nucleotide sequences
19
and amino acid sequences to Drosophila data base (Berkeley Drosophila Genome Project BDGP) was done using the
Advanced BLAST server of NCBI. Motifs in the proteins
were searched by the Motifs Program, GCG10.
Comparison of the diverged amino acid sequence of
dmDp186 to protein sequences in the non-redundant gene
bank (mngb) was done using Blast Bioaccelerator and
FASTA programs.
2.6. Oligonucleotides
For details about the oligonucleotides used for cDNA
synthesis, PCR reactions, and 5 0 RACE, please contact the
corresponding author.
3. Results
3.1. The Drosophila homologue of the dystrophin gene
Using sequence data on the 3 0 end coding region of a
potential Drosophila homologue of dystrophin (Roberts
and Bobrow, 1998), we isolated from a cDNA library of
8±12 h Drosophila embryos, partially overlapping cDNA
clones of the corresponding mRNA. Among the isolated
cDNA clones, which consisted of the previously described
sequence, two contained additional 5 0 sequences with a
large open reading frame (ORF). The 3 0 part of these two
clones encoded an amino acid sequence with signi®cant
similarity to the corresponding region in human dystrophin.
However, the 5 0 region encoded an amino acids sequence
with no signi®cant similarity to dystrophin. On the basis of
the sequence of the two different cDNA clones, additional
sequences obtained by 5 0 RACE, sequences of a genomic
DNA clone, and RT/PCR experiments, we concluded that
the sequence is of a genuine mRNA encoding a 186 kDa
protein. In accordance with the nomenclature used for the
mammalian dystrophin gene products, we call this protein
dmDp186 (Fig. 1).
The C-terminal two thirds of dmDp186 are homologous
to the corresponding regions of dystrophin and utrophin.
That includes the cysteine-rich and C-terminal domains,
which as in all dystrophin family members are highly
conserved, and a less conserved sequence consisting of
four spectrin-like repeats. The N-terminal third of the
protein (512 amino acids) shows no sequence similarity to
dystrophin or utrophin. The 1536 nucleotides encoding this
unique sequence, together with a 5 0 untranslated region
(UTR) constitute a single large exon (Fig. 2). The amino
acid sequence encoded by this exon shows no signi®cant
similarity to other proteins in the database (except for the
same sequence in the Drosophila database). It contains
several clusters of 4±6 proline residues each, and a stretch
of 15 alanine residues, encoded by a sequence which is not a
perfect trinucleotide repeat, suggesting that this sequence is
conserved on the amino acid level. The oligoproline clusters
may be involved in protein-protein interactions.
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S. Neuman et al. / Gene 263 (2001) 17±29
Fig. 1. Comparison of the amino acid sequences of dmDp186 and human dystrophin. Alignment of the sequences was done by the prettybox program (GCG10).
The known motifs in the cysteine-rich and C-terminal domains of dystrophin are indicated. The arrows indicate the positions of leucine residues in the coiled
region. The amino acid sequence encoded by exon 71 of dystrophin are marked. This exon was not present in the cDNA clones of Dp71, dmDp186 and sea
urchin suDp98 that were sequenced. The open arrow indicates the end of the sequence with no signi®cant similarity to human dystrophin encoded by the unique
®rst exon of Dp186. A substantial part of C-terminal region of the Drosophila sequence was described by Roberts and Bobrow (1998).
S. Neuman et al. / Gene 263 (2001) 17±29
21
3.2. The Drosophila gene also encodes full length
dystrophin-like proteins
3.3. The transcription of the Drosophila mRNA encoding the
dystrophin-like protein is initiated at three different exons
In view of the prevalence in the dystrophin family of
complex genes encoding large and small products (Bar et
al., 1990; Lederfein et al., 1992; Byers et al., 1993; Lidov et
al., 1995; D'Souza et al., 1995; Fabbrizio et al., 1995; Blake
et al., 1995; Wang et al., 1998; Wilson et al., 1999) we
tested whether the gene encoding dmDp186 also encodes
a full-length dystrophin-like protein. To this end, we
screened the Drosophila data base for amino acid sequences
with similarity to the N-terminal actin binding domain of
human dystrophin. We identi®ed such a sequence in a BAC
clone that also contained the sequence encoding dmDp186.
On the basis of this sequence we performed nested RT/PCR
on total ¯y RNA. The antisense primers were taken from the
region with similarity to dystrophin mRNA, and the sense
primer from the BAC sequence encoding the presumptive
actin binding domain. Two bands of 6 and 7 kb of similar
intensities were obtained, cloned and sequenced. The
sequence consisted of a large ORF, encoding a polypeptide
most of which shows signi®cant sequence similarity to the
corresponding region of human dystrophin. The 1 kb difference in size between the two PCR products was due to
alternative splicing of one large exon (exon 9, Fig. 2A).
An additional alternative splicing pattern which was
observed during the process of cloning was of exon 17,
which was absent in some of the sequenced cDNA clones.
This exon is located just downstream from the ®rst unique
exon of dmDp186. Interestingly, in the cDNA clones of
Dp186 that we sequenced, the ®rst exon, which is situated
in intron 16, was spliced to exon 18, thus skipping exon 17
(Fig. 2B).
Using 5 0 RACE on ¯y RNA we extended the cDNA
sequence toward the 5 0 UTR. Two types of clones were
isolated. They were identical at their 3 0 end regions, but
different at their 5 0 end regions. Most of the diverged
sequences in the two types of clones were non-coding.
The two types of clones also contained different short continuation of the ORF, with potential initiator ATG codons.
Analysis of the genomic DNA structure showed that the
unique sequences were separated from the common
sequence by introns, as described in Figs. 2C and 3A.
Based on the Drosophila genomic DNA sequence and
preliminary gene annotation, Adams et al. recently
described the sequence of the 5 0 half of the cDNA that we
cloned (Adams et al., direct submission, accession no.
AE003726). However, due to a 56 bp deletion in their
sequence that changed the ORF, the authors deduced a
single encoded polypeptide of 547 amino acids (Adams et
al. direct submission, accession no. AAF55673). In addition, in the protein sequence reported by Adams et al., the
N-terminal 14 amino acids are different from those in the
two isoforms that we cloned. We found that these fourteen
amino acids are encoded by a single exon, which, as with the
two other isoforms described above, is spliced to exon 3, the
®rst common exon (Figs. 2C and 3A). Sequence analysis of
5 0 RACE products and RT/PCR analysis (not shown) indicated that the unique sequence is not present in the two
isoforms that we cloned. Thus this sequence encodes the
N-terminus of a third full-length gene product (dmDLP3,
Fig. 3A).
As indicated above and shown in Fig. 2, in each isoform a
Fig. 2. (A) The structure of the Drosophila gene encoding dystrophin-like proteins. Bars represent exons, horizontal bold lines ± introns, Bent arrows ±
transcription start sites. Exons numbers are indicated below the scheme. The size of the large introns (not in scale) is indicated above the scheme. ATG ±
translation initiation codon. Fig. 1a±d indicate the ®rst unique exons of dmLDP1, dmLDP2, dmDLP3 and dmDp186, respectively. (B) Alternative splicings in
the region encoding the N-terminus of dmDp186. Legend as in A. Splicing patterns are shown by broken lines. Not in scale. (C) Alternative splicing in the
region encoding the N-terminus of dmDLP1, dmDLP2 and dmDLP3. Legend as in A and B. Not in scale.
22
S. Neuman et al. / Gene 263 (2001) 17±29
Fig. 3. (A) The amino acid sequence of dmDLP2. The amino acid sequence was deduced from the nucleotide sequence of overlapping cDNA clones. The ®rst
seven amino acids sequence is speci®c to dmDLP2. The arrowhead indicates the beginning of the sequence encoded by exon 3, the ®rst common exon to
dmDLP1, dmDLP2, and dmDLP3 (Fig. 2C). Black dots mark the positions of introns 3±34. The deduced speci®c N-terminal sequence of dmDLP1 is M
HDPSDYDSVYSEEDFVDTVRGAATPPLMDY EEFRVKTM. As marked by underlining, this sequence contains three methionine residues. Since the
sequence around the most 5 0 in frame ATG codon does not ®t well with the consensus translation start site sequence, it is possible that translation is initiated
with the second or third methionine. The speci®c N-terminal sequence of dmDLP3 is MAYNLALKPADLAR. (B) Comparison of the actin binding domains of
human dystrophin, the sea urchin dystrophin-like protein (suDLP) and dmDLPs. Alignment was done by the prettybox program (GCG10). The three actin
binding sites (ABS1, ABS2 and ABS3, (Levine et al., 1992; Corrado et al., 1994) are indicated. In the dmDLP sequence, the ®rst seven amino acids are of
dmDLP2.
S. Neuman et al. / Gene 263 (2001) 17±29
23
Table 1
Sequence identities between dystrophin related genes a
Fig. 4. The domain structure of dmDLP and suDLP. The predicted domain
structure of the known products of the genes encoding the Drosophila and
sea urchin dystrophin-like proteins are presented schematically and
compared with that of human dystrophin. Striped bar indicates the amino
acid sequence of dmDp186 encoded by the unique exon 1d (Fig. 2).
unique 5 0 exon is spliced to the common exon 3. RT/PCR
analysis indicates that also the large exon 4 is present in the
three mRNAs (see Fig. 6). As indicated above, RT/PCR
experiments demonstrated the existence of mRNAs extending from exons 3 and 4 (which encode the actin binding
domain) to beyond the divergence point between the large
and small mRNAs (the 6 and 7 kb RT/PCR products
described in 3.2). It is, therefore, most likely that the three
isoforms consist of all the domains of dystrophin (Figs. 3
and 4). They share with the smaller product of the gene,
dmDp186, the last four spectrin-like repeats and the highly
conserved cysteine-rich and C-terminal domains (Fig. 5).
Thus, the Drosophila gene, like the human DMD gene,
has a complex structure. It codes for at least three isoforms
of full-length dystrophin-like proteins (dmDLP1, dmDLP2,
and dmDLP3, sometimes collectively called here dmDLPs)
and at least one smaller product (dmDp186), regulated by an
internal promoter. The gene encoding these proteins is
referred to as the dmDLP gene. The number of gene
products is greater due to alternative splicing possibilities
described above. It is of interest that unlike the situation
with the human dystrophin gene, in which the small
products have at their N-terminus very short unique amino
acid sequences consisting of only a few amino acids, the
unique N-terminus of dmDp186 comprises about one third
of the protein and may have a distinct function (Fig. 4).
Comparison of dmDLPs with human dystrophin shows a
high degree of sequence identity between the cysteine-rich,
the C-terminal and the actin binding domains of the two
proteins (Figs. 1, 3B and 5 and Table 1). Lower, but still
signi®cant, sequence similarity exists between most of the
spectrin-like repeats domain of the two proteins (Fig. 5A).
However, the regions of the proteins between amino acid
1650±2200 show no obvious similarity. While in dystrophin, this region consists of ®ve repeats, in dmDLP this
region contains two or three repeats. Thus, the spectrinlike repeats region in dmDLPs is slightly shorter than that
of dystrophin and is similar to that of utrophin (21 or 22
repeats). In addition, the sequence similarity between the
Domain
Actin
binding
Spectrin-like
Repeats
Cystein-rich and
C-terminal
h. dystrophin suDLP
h. dystrophin dmDLP
DmDLP h.utrophin
h. dystrophin c.ele dys1
h. dyst. Kakapu (dm)
DmDLP kakapu (dm)
DmDLP c. ele. dys1
56.6
48.2
46.2
29.1
50.8
44.5
27.1
31.8
27.8
26.2
24.2
24.0
22.8
24.6
62.1
56.9
58.6
43.3
±
±
42.0
a
Percent identity of the various domains was calculated by the best®t
program, GCG10. h, human; dm, drosophila; c. ele, c. elegans.
various repeats in dmDLPs is lower than between the
repeats in human dystrophin (compare Fig. 5B,C). It should
be pointed out that a large part of the sequence with low
similarity to dystrophin is encoded by the alternatively
spliced exon 9.
3.4. The Drosophila gene encoding the dystrophin-like
protein maps to position 92A in the right arm of
chromosome 3
In situ hybridization to Drosophila polytene chromosomes, using a probe from the 3 0 end region, mapped the
dmDLP gene to the right arm of chromosome 3 at position
91F±92A (not shown). This assignment was con®rmed by
the ®nding that sequences of the gene are present in three
partially sequenced Drosophila BAC clone that were
mapped to position 92A±92B of Drosophilla genome
(BACACRO7118, BACR14H24 and BACR12P10, accession no. ACOO7118, AC008363 and ACOO8192, respectively).
3.5. The Drosophila gene is smaller that the DMD gene
An outstanding feature of the human dystrophin gene is
its enormous size. Most of the gene consists of introns, some
of which are extremely large. The biological function of the
large introns, whose size is conserved in mammals and birds
and partially in the utrophin gene, is not known.
By comparing the Drosophila cDNA sequence to the
genomic DNA sequence in the BAC clones mentioned
above, we identi®ed 34 introns in the coding sequence of
the dmDLP gene (Fig. 2A). In general, the introns are significantly smaller than the introns in the human DMD gene.
The largest four introns range between 11 and 47 kb. One of
the large introns (intron 16) contains the ®rst unique exon
(and most likely, also the promoter) of dmDp186 (Fig. 2B).
The size of the dmDLP gene is approximately 132 kb.
Thus, the Drosophila gene is about 20 times smaller than the
human dystrophin gene. Interestingly, each gene occupies
roughly 0.1% of the respective genome.
The positions of 14 out of the 34 introns in the coding
sequence are conserved between the human DMD gene and
24
S. Neuman et al. / Gene 263 (2001) 17±29
Fig. 5. Dotplot analysis of human dystrophin and dmDLP. The following comparisons are presented: (A) dmDLP/human dystrophin; (B) dmDLP/dmDLP; (C)
human dystrophin/human dystrophin. Dotplot program of GCG, window of 50 amino acids and stringency of 18 points were used.
the Drosophila gene. Interestingly, the introns which in
human DMD gene accommodate the promoters of Dp260,
Dp140 and Dp116 are missing in the Drosophila gene. A
homologue of intron 62, which in the human dystrophin
gene accommodates the promoter and ®rst exon of Dp71,
consists of only 99 bp in dmDLP gene, and therefore it is
unlikely to contain a 5 0 UTR and a promoter of a Dp71-like
protein.
The identi®cation of homologous introns was done on the
basis of amino acid sequences alignment of dmDLP and
dystrophin, and intron positions in relation to the sequences.
Therefore, in the region of the proteins with poor sequence
similarity the alignment and comparison of the positions of
introns may be inaccurate to some extent.
3.6. Does the Drosophila genome contain additional
homologues of the dystrophin gene?
To check whether the Drosophila genome contains additional homologues of the dystrophin gene, we searched in
S. Neuman et al. / Gene 263 (2001) 17±29
Fig. 6. Differential expression of dmDLPs mRNAs during development.
Semiquantitative RT/PCR was performed on total RNA samples (5mg)
from 0±24 h embryos (emb), 1±3 days larvae (lar) and adult ¯ies (¯y) as
described in Section 2. The sense primers were isoform speci®c. To avoid
ampli®cation of genomic DNA, the anti sense primer consisted of
sequences from exons 3±4 border. Twenty seven PCR cycles were
performed using the AmpliTag Gold Kit (Roche). The PCR products
were analyzed by Southern blotting using a common internal oligonucleotide probe. The numbers on the top of the slots 2, 1, 3 indicate the use of
primers speci®c for DLP2, DLP1 and DLP3 mRNAs, respectively.
the Drosophila database for DNA sequences with great
similarity to the region encoding the cysteine-rich and Cterminal domains of human dystrophin. This is the most
conserved region in the dystrophin gene family. The long
genomic DNA sequences found in this search were all of the
BAC clones mapped to the chromosomal location of
dmDLP gene. However, the search also revealed two short
sequences with 100% identity to dmDLP gene in two other
BAC clones. Each sequence consisted of two exons and one
or two introns. No additional related sequences were found
in the same BAC clones. Therefore we assume that these
two sequences are a result of cloning artifacts. Similar
results were obtained when the search was carried out
with the amino acid sequence of the cysteine-rich and Cterminal domains of dmDLP.
Since the Drosophila database consists of the entire
Drosophila genome, it is most likely that dmDLP gene is
the only Drosphila close homologue of dystrophin and utrophin genes.
3.7. Differential expression of the dmDLP product
Preliminary semiquantitative RT/PCR analysis on RNA
from embryos (0±24 h), larvae (1±3 days) and adult ¯ies
showed that each dmDLP had a speci®c temporal pattern of
expression. DmDLP3 mRNA was detected only in ¯ies.
Embryos and ¯ies had 2±4-fold more dmDLP2 mRNA
than dmDLP1 mRNA. Larvae had approximately equal
amounts of dmDLP1 and dmDLP2 mRNAs (Fig. 6).
The tissue distribution of the small and the large transcripts of dmDLP gene was studied by in situ hybridization
of whole mount embryos, using two distinct DNA probes. A
probe for the full-length dmDLPs transcripts stained the
midgut endoderm of stage 12 embryos. In addition, this
probe stained the pericardial cells, which form a support
25
layer for the embryonic heart (dorsal vessel) cardioblasts,
cells at the ectoderm segmental border, and cells along the
midline of the central nervous system (CNS) in stage 16
embryos (Fig. 7A±C). A probe speci®c for the shorter transcript (dmDp186) did not stain stage 12 embryos but
strongly stained neuronal derived tissues, mainly the CNS
and the brain of stage 16 embryos (Fig. 7D±F), and at lower
levels, the sensory organs (not shown). Staining was also
detected in blastoderm embryos, presumably as maternal
contributed mRNA. Thus, the expression pattern of
dmDp186 is distinct from that of the large dmDLP gene
products. A more detailed analysis of the expression of
the various products is in progress.
3.8. Characterization of the sea urchin dystrophin-like
protein
We have previously reported on the cloning of a sea
urchin mRNA encoding a 98 kDa protein (suDp98) with a
high degree of amino acids sequence identity with the Cterminal region of dystrophin and utrophin. On the basis of
additional 5 0 sequencing we suggested that suDp98 is an
evolutionary homologue of human Dp116 and that the sea
urchin gene also encodes a larger protein (Wang et al.,
1998). We have completed the sequencing of the mRNA
encoding a full-length dystrophin-like protein (suDLP,
previously referred to as SuDRP; Wang et al., 1998). The
448 kDa suDLP consists of the four domains of dystrophin:
The actin binding domain (Fig. 3B), 25 spectrin-like
repeats, and the previously reported cysteine-rich and Cterminal domains (Figs. 4 and 8). Interestingly, the N-terminus of the protein contains a sequence of eight amino acids
which is also present in the N-terminus of utrophin. This
sequence is missing from dystrophin (Fig. 9). Sequence
comparisons suggested that the sea urchin gene and the
Drosophila DLP gene are related to an ancestral gene that
gave rise to dystrophin and utrophin genes (Wang et al.,
1998 and Fig. 10). It thus seems that the ®rst exon of the
sea urchin gene is evolutionary related to the ®rst exon of
the utrophin gene. We cannot yet exclude the possibility that
like the dmDLP gene and human and mouse dystrophin
genes, the sea urchin gene contains additional ®rst exons
and promoters which are related to those of the dystrophin
gene.
4. Discussion
4.1. The Drosophila and sea urchin dystrophin-like proteins
The present communication describes a Drosophila gene
which seems to be a genuine homologue of the human
dystrophin and utrophin genes. Three other Drosophila
genes encoding proteins that share sequence similarity
with regions in human dystrophin have been described
earlier. One of them is a small and simple structured gene
encoding a 75 kDa protein (Dah) with low sequence simi-
26
S. Neuman et al. / Gene 263 (2001) 17±29
larity to the C-terminal part of human dystrophin (Zhang et
al., 1996). The other two genes encode large proteins called
Kakapo and MSP300 (Strumpf and Volk, 1998; Volk,
1992). They contain actin-binding domains with signi®cant
sequence similarity to that of human dystrophin, and spectrin-like repeats. However, they do not contain the cysteinerich and C-terminal domains which are highly conserved in
all known dystrophin related proteins. In contrast, the
Drosophila gene described here encodes three isoforms of
full-length dystrophin-like proteins with all the characteristic domains, and at least one smaller product, thus, resembling the dystrophin and utrophin genes in structure.
The overall sequence identity between dmDLP and
dystrophin, dmDLP and utrophin or dmDLP and suDLP is
approximately 33%. As expected from the evolutionary
distances between the organisms, suDLP is closer than
dmDLP to human dystrophin and utrophin (37% sequence
identity). As reported for other dystrophins and related
proteins, the cysteine-rich and the C-terminal domains are
the most conserved part of the proteins (62% identity
between human dystrophin and suDLP, and 57% identity
between human dystrophin and dmDLP). All the motifs in
this region, which in dystrophin are known to bind the
DAPs, are highly conserved in both the Drosophila and
sea urchin proteins (Fig. 1; Wang et al., 1998; Roberts
and Bobrow, 1998). The existence of conserved blocks of
sequences outside the known motifs suggest that this region
of the protein is involved in additional, yet unknown, interactions or other functions. The actin binding domain region
is somewhat less conserved. (57 and 48% identity between
the sea urchin and human proteins, and between the Drosophila and human proteins, respectively). The ®rst and
second actin-binding motifs of dystrophin are highly
conserved in the Drosophila and the sea urchin proteins.
As mentioned above, the sequence of the spectrin-like
repeats domain is less conserved. This region in the
suDLP consists of 25 repeats and in dmDLP of 21 or 22
repeats.
It was recently shown that dystrophin has several positively charged spectrin-like repeats that are probably
involved in association with actin (Amann et al., 1998).
Utrophin does not have such charged repeats (Amann et
Fig. 7. Differential expression of transcripts of dmDLP and dmDP186 in Drosophila embryos. In situ hybridization of wild type embryos with either
digoxigenin-labeled DNA probe for the transcripts of dmDLPs (A±C), or with a probe for the dmDp186 transcript (D±F). In stage 12 embryos (A,D)
dmDLP probe stains the midgut endoderm (A), while no staining with a probe for dmDp186 is detected (D). At the dorsal aspects of stage 16 embryo (B)
dmDLP probe stains the two rows of pericardial cells (arrow) and cells along the ectodermal segmental border. No staining in these tissues is detected when
using a probe for dmDp186 (not shown). At the ventral aspects of stage 16 embryo dmDLP probe stains cells along the midline of the CNS (C). Staining is also
observed in head structures (B,C). dmDp186 is detected at high levels in the CNS and brain of stage 16 embryos as shown in lateral view (E) or in ventral view
(F).
S. Neuman et al. / Gene 263 (2001) 17±29
Fig. 8. Dotplot comparison between suDLP and human dystrophin.
Comparison was done as described in legend to Fig. 4.
al., 1999). dmDLP and suDLP are similar to utrophin with
this regard. Our results also suggest that these proteins are
not muscle speci®c. It has to be clari®ed whether any of the
dmDLP isoforms are expressed in muscle and functions like
muscle dystrophin.
The small product of the Drosophila gene, dmDp186,
contains a unique sequence of 512 amino acid encoded by
a single exon. No similarity between this sequence and other
proteins was found so far. RT/PCR analysis and the in situ
hybridization using probes derived from this sequence
con®rmed that the sequence is expressed in Drosophila
embryos. These experiments also demonstrated that the
full-length products and small product have clearly distinct
patterns of expression during development. Interestingly,
the small product is mainly expressed in the nervous system.
The small products of the human dystrophin gene are also
expressed primarily in various parts of the nervous system.
4.2. Full length dystrophins and the smaller products
Muscle dystrophin plays an essential role in the stabilization of the muscle ®ber during contraction, as a part of a
complex which links the cytoskeletal actin, the sarcolemma
and the extracellular matrix (Suzuki et al., 1994; Jung et al.,
1995; Ahn and Kunkel, 1993; Campbell, 1995; Ozawa et al.,
1995). The function of the smaller products of the gene,
27
Fig. 10. Suggested evolutionary relationship between members of the
dystrophin gene family. The tree was constructed using the neighbor joining method from the DISTANCE program of the GCG package. The
sequences of the C-terminal region of dystrophin and the homologous
regions of the other proteins were used in the comparison. The structures
of genes are presented schematically on the right. Double arrows symbolize
a large complex gene encoding both dystrophin-like proteins and small
products. A single arrow symbolizes a small simple structured gene
(updated and extended from Wang et al., 1998). The question mark is to
indicate that so far there was no report on small products of the gene in C.
elegans.
which lack the actin binding domain and part or most of
the spectrin-like repeats, is yet to be elucidated. Experiments with transgenic mice expressing Dp71 instead of
dystrophin in their muscle, demonstrated that Dp71 could
bind to the DAPs complex and restore normal levels of
DAPs in dystrophin-de®cient mdx mice. However, this did
not alleviate muscle degeneration, indicating that dystrophin and Dp71 differ in their function (Greenberg et al.,
1994; Cox et al., 1994).
The present communication substantiates the view that
the complex structure and pattern of expression of the
DMD gene, i.e. encoding full length dystrophin-like
proteins and smaller proteins lacking the N-terminal regions
of dystrophin, is very ancient (Wang et al., 1998). It existed
in this gene family before the divergence between the protostomes and the deuterosomes which took place approximately 600 million years ago. The conservation and wide
occurrence of the small truncated products encoded by large
complex genes, as well as the existence of relatively small
and simple genes that encode only small products, which are
evolutionary related to the small products of the dystrophin
gene (Fig. 9), strongly suggest the existence of important,
yet unidenti®ed functions, intrinsic to the highly conserved
C-terminal domains of dystrophin and related proteins.
Recent studies suggest that the membranal dystroglycan
complex, which bind to laminin, is involved in cell adhesion
Fig. 9. The N-terminus of suDLP is similar to the corresponding region of utrophin. Alignment of the sequences was done by the prettybox program (GCG10).
Arrowhead indicates the location of the ®rst intron in the dystrophin gene.
28
S. Neuman et al. / Gene 263 (2001) 17±29
and signaling (Cavaldesi et al., 1999; Belkin and Smalheiser, 1996) and in basement membrane assembly (Henry and
Campbell, 1998). It is possible that the non-muscle products
of the DMD gene play a role in the organization and stabilization of the DAPs complex in non-muscle tissues.
4.3. Size and structure conservation among the dystrophinlike proteins
A substantial part of the full-length dystrophin molecule
consists of spectrin-like repeats which form a rod-shaped
domain between the C-terminal domains and the cytoskeletal actin binding domain. There are several examples of
mutations in human which resulted in deletions of parts of
the spectrin like repeats. The phenotype of some of them
was relatively mild (e.g. Love et al., 1991). These observations led to the notion that the size of the rod formed by the
spectrin-like repeats region of the dystrophin is not very
important. However, comparison of the presently available
data on the full length dystrophin and dystrophin-like
proteins shows that while the sequence conservation in the
spectrin-like repeats region is low, the distance between the
actin binding domain and the two C-terminal domains has
been conserved during evolution (Fig. 4C). For example,
while the amino acid sequence similarity in the spectrinlike repeats region between human dystrophin and the
recently reported dystrophin-like protein of c. elegans,
dys1 (Bessou et al., 1998) is barely signi®cant, the size of
the entire molecule is almost identical (only ten amino acid
difference). These data suggest that there has been a strong
selective pressure to conserve the spectrin-like repeat structure and the distance between the actin binding domain and
the C-terminal region. Both are probably essential for the
formation of the proper structure and mechanical properties
of the link between the cytoskeleton and plasma membrane
which is mediated by dystrophin. In contrast, in the smaller
products of the DMD gene family which lack the highly
conserved actin binding domain, the size of the spectrinlike repeats domain is variable, or, as is the case in Dp71,
this domain is missing altogether.
The assignment of mutations in the dmDLP gene will
facilitate the investigation of the function of the various
gene products.
5. Note added in proof
Greener and Roberts (2000) reported recently on the
squencing of a single product of the gene. It is a shorter,
alternatively spliced isoform of dmDLP2 which is described
in the present communication.
Acknowledgements
We wish to thank Dr R.G. Roberts for the unpublished
sequence data (34), Dr Eyal Schejter for drosophila embryos
and ¯ies, Dr Adi Zalzberg for helping with the chromosomal
mapping, Ora Fuchs for technical assistance and Vivienne
Laufer for editorial assistance. This work was supported by
the Israeli Academy of Science and Humanities, the Muscular Dystrophy Association, USA and the AFM, France. U.N.
is the incumbent of the Elias Sourasky Professorial Chair at
the Weizmann Institute of Science.
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