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DMD (dystrophin)

Contents

• Introduction
• Dystrophin
  • the dystrophin gene
  • the dystrophin RNA
  • the dystrophin protein
  • dystrophin expression
• Dystrophin and disease
  • disease-causing mutations
• Table of dystrophin primer sequences
  • DNA-primers
  • RNA-primers
• Dystrophin sequences

Introduction

Dystrophin was discovered by ... .

The dystrophin gene

The dystrophin gene maps to chromosome X (Xp21.2).

Legend:
Exon: numbering of exons and intron/exon boundaries are according to ..., with the first base of the Met-codon counted as position 1 (see ...reference sequence). Exon size: size of exon indicated in basepairs. Intron size: size of intron indicated in kilobasepairs. 5' cDNA position: first base of the exon (according to cDNA sequence ...). Splice after: splicing occurs in between of two coding triplets (0), after the first (1) or the second (2) base of a triplet. Remarks: 5'UTR = 5' untranslated region, 3"UTR = 3' untranslated region.

genomic sequences

• listing of all DMD genomic sequences in GenBank
  • links to the not annotated files
  • order of markers in and around the dystrophin gene
  • introns, CA-repeats, STS, etc
• dystrophin exons: their position in the cDNA, their numbering, sizes and genomic spacing
• cosmids covering the dystrophin gene: details on construction

The dystrophin RNA

GenBank: NM_004006

The DMD gene produces a range of different mRNA-transcripts encoding various dystrophin isoforms, i.e. proteins of varying lengths containing different segments of the basic dystrophin sequence. The isoforms are encoded by a range of different mRNA's which are generated by three processes; (i) the use of different, unique and often tissue-specific promoters, (ii) alternative splicing, and (iii) the use of different polyA-addition signals.

Tennyson et al. (1995) used quantitative RT-PCR to estimate that transcription of the 2.3 Mb DMD-gene requires approximately 16 hours (at an average elongation rate of 2.4 kb/min). Transcripts were spliced at the 5' end before transcription was complete, which provided strong evidence for co-transcriptional splicing. The rate of transcript accumulation was reduced at the 3' end relative to the 5' end, probably due to premature termination of transcription complexes (Tennyson et al. [1995]).

When comparing the chicken and human cDNA sequences Lemaire et al. [1988] observed a striking evolutionary conservation of three segments of the 3' untranslated region (3'UTR, 85% homology over a total of 920 nucleotides). Including the sequences from an amphibian and a cartilaginous fish Greener et al. [2002] refined this observation to two highly conserved regions separated by a non-conserved 700-2,000 nucleotide spacer; 

• 'Lemaire A', 350 nucleotides (11,145-11,503 - cDNA Reference Sequence) with a central stem-loop structure (11,289-11315 )
• 'Lemaire D', 280 nucleotides (13,469-13,749) with a putative 3' vigilin binding site (13,656-13,689, Kanamori et al. [1998]) and a stem-loop structure across the -AATAAA- polyA-addition signal (13,709-13,735)

RNA transcripts

• mRNA transcripts: how they arise and their nomenclature
• dystrophin mRNA's
  Dp427m, Dp427l, Dp427c, Dp427p, Dp260, Dp140, Dp116, Dp71 and Dp40
• Dp427m: cDNA and protein sequence together
• cDNA Reference Sequence  (used for to describe sequence changes)

The dystrophin protein

Dystrophin is a rod-shaped protein, measuring about 150 nm, consisting of 3684 amino acids with a calculated molecular weight of 427 kDa. Dystrophin is predominantly hydrophilic throughout its entire length and 31% of the amino-acids are charged (i.e. Arg, Asp, Glu, His and Lys). A "Chou and Fasman" prediction of secondary structure reveals a very high potential for an alpha-helical formation over the majority of the sequence. Dystrophin can be separated into four domains:

• actin binding domain (amino acids 14-240):
  this region contains a putative actin-binding region and was discovered by its homology with chicken alpha-actinin (Hammonds). alpha-actinin is a normal component of the actin filaments in smooth and skeletal muscle and is probably involved in cross-linking F-actin and thereby connecting the filamentous elements of the cytoskeleton to the cell membrane.
• central rod domain (amino acids 253-3040):
  a large central domain formed by 24 spectrin-like triple-helical elements of about 109 amino acids with homology to alpha-actinin and spectrin. The repeats are interrupted by four proline-rich non-repeat segments, the so called hinge regions. Repeats 15 and 16 are separated by an 18 amino acid stretch which seems a major site for proteolytic cleavage of dystrophin (Koenig). The identity between most repeats ranges from 10-25%.
  One repeat contains three alpha-helices: 1, 2 and 3. Apha-helices 1 and 3 are each formed by 7 helix turns, probably interacting as a coiled-coil through a hydrophobic interface. Alpha-helix 2 has a more complex structure and is formed by segments of four and three helix turns, separated by a Glycine or Proline residue.
  Each repeat is encoded by two exons, always interrupted by an intron exactly between amino acids 47 and 48 in the first part of alpha-helix 2. The other intron is found at different positions in the repeat, usually scattered over helix-3.
• Cysteine-rich domain (amino acids 3080-3360):
  a cysteine-rich segment (i.e. 15 Cysteines in 280 amino acids) showing homology to the C-terminal domain of the slime mold (Dictyostelium discoideum) alpha-actinin. The homologous region, amino acids 3115-3269 with 24% identity over 142 amino acids, includes two potential EF-hand Ca²⁺-binding sites although the dystrophin sequence does not match the consensus sequence for these EF-hands very well.
• Carboxy-terminal domain (amino acids 3361-3685):
  this 325 amino acid C-terminal domain does not show any homology to known proteins, except with the dystrophin related proteins.

Dp71 is phosphorylated in vitro by p34cdc2 protein kinase Milner 93, JBC 21:21901-21905 (consensus site in exon 78 -TPGK-). Exon 1 encodes the 5'-untranslated region and 11 amino acids.

the protein

• the dystrophin glycoprotein complex (DGC): dystrophin associated proteins
• dystrophin isoforms: how they arise and their nomenclature
• dystrophin protein sequences
  Dp427m, Dp427l, Dp427c, Dp427p, Dp260, Dp140, Dp116, Dp71 and Dp40
• dystrophin antibodies and their epitopes
• Dystrophin related sequences
  • dystrophin associated proteins
  • dystrophin related proteins / homologous proteins

Evolution

Function

Dystrophin and disease: DMD, BMD, XLDC

Sequence variants  -  DMD mutation databases

• deletions / duplications  (junctions not sequenced)
  • deletions/duplications from all over the world (per country)
  • deletions/duplications from Leiden (Nederland)
• all sequenced changes (mutation, polymorphisms)

Table of primer sequences

DNA-primers

RNA-primers

Dystrophin sequences

• cDNA-sequence
  • M18533 at GenBank
  • M18533 copy at this site
  • reference sequence, derived from M18533, used for mutation description
  • related cDNA sequences: M20205, X14298
  • other organisms
    • mouse M68859, M18025, X83506 (G-utrophin)
    • rat X69767 (apo3)
    • chicken X13369
    • Torpedo californica M37645
• gene sequence
  • at GenBank
  • copy at this site

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