ANÁLISIS DE LOS PRINCIPALES ESPACIOS PÚBLICOS EN EL CENTRO DE LA CIUDAD.

expression, have so far been identified, including the

muscle and brain type promoters; this is not surprising considering the huge size of the DMD locus. Some of the promoters are at the 5' end of the gene separated by large distances and some are located much further downstream and control the expression of smaller proteins lacking some of the major domains of the muscle and brain type

dystrophins (see table 1.4).

Immunocytochemical and in situ hybridisation studies have identified an isoform of dystrophin which is expressed

specifically in Purkinje neurons, named dystrophin-P [Lidov e t a!., 1990, Gorecki et a!., 1992]. Using PCR the dystrophin-P transcript was characterised and found to have a novel first exon, with exons 2 and 3 identical to the consensus muscle type dystrophin which implies that the transcription of dystrophin-P Is controled by a third promoter [Koenig et al.,

1988, Gorecki et a!., 1992]. Purkinje cells are the major

output neurons of the cerebellar cortex and the fact that there is a specific promoter controlling dystrophin expression in these cells suggests an important role for the protein in brain function [Gorecki et a!., 1992] (see table 1.4).

Using Northern blot analysis and RNase protection assays, a major 6.5Kb mRNA transcript of the DMD locus was identified in liver [Bar et al., 1990]. It was later named apo-dystrophin 1

[Blake et al., 1992] or Dp71 [Lederfein et al., 1993] and the mRNA has been variously described as being 6.5Kb [Bar et al., 1990], 4.5Kb [Hugnot et al., 1992], 4.8kb [Blake et al., 1992] and most recently has been determined as 5Kb [Lederfein et al., 1993]. Apo-dystrophin 1 is expressed most highly in brain and heart and many other non-muscle tissues [Bar et al., 1990, Blake et al., 1992, Hugnot et al., 1992, Cox et al., 1993a, Lederfein et al., 1993, Fabbrizio et al., 1993b] (see table 1.4).

The apo-dystrophin 1 has been described as an 80kDa protein, [Blake et al., 1992], a 77kDa protein [Fabbrizio et al., 1993b], a 75kDa protein [Hugnot et al., 1992] and as a 70.8kDa protein [Rapaport et al., 1992, Leiderfein et al., 1992, 1993], which lacks the actin binding domain and the rod region found in the muscle and brain type DMD but has a contiguous cysteine rich domain, C-terminal domain (although modified by an

alternative splicing event in which muscle type exons 71 and 78 are deleted) and 3' untranslated region [Bar et al., 1990, Barnea et al., 1990, Blake et al., 1992, Hugnot et al., 1992, Lederfein et al., 1992, Rapaport et al., 1992a]. The lack of an actin binding region and rod region implies a very different structure for apo-dystrophin 1 but the conservation of the cysteine rich domain and the C-terminal domain suggest that it is membrane associated and it has been found to be enriched in membranous fractions [Arahata et al., 1988, Rapaport et al., 1992a]. The 5' untranslated region, however, is unique as are the first 7 codons. This sequence is located in the intron between exons 62 and 63 of the consensus muscle DMD

transcript which suggests that this intron may also contain an alternative promoter [Hugnot et al., 1992, Lederfein et al.,

1992, Rapaport et al., 1992a]. Recently the promoter region was characterised. The 5' untranslated region is translated from a single exon and flanked by a GC rich housekeeping

promoter region which has three possible S p l binding sites but lacks TATA and CAAT motifs. Interestingly, the distal

location of this promoter means that most dystrophin

mutations will not affect the apo-dystrophin 1 transcript or protein. This is corroborated by apo-dystrophin 1 expression being detected in the brain of m dx mice, where a point

mutation has precluded the expression of full length

dystrophin [Sicinski et al., 1989, Bar et a!., 1990, Rapaport e t a!., 1992a]. In early rat embryos apo-dystrophin 1 mRNA and protein were assessed as being quantitively the main product of the DMD gene. Using PCR, apo-dystrophin 1 mRNA has been detected in undifferentiated ES (embryonic stem) cells

whereas the 14Kb transcript is undetectable, this result was confirmed for the protein by Western analysis. In fact, in undifferentiated ES cells the level of apo-dystrophin 1 mRNA is greater than the level of dystrophin in muscle although it decreases when cells are induced to differentiate [Rapaport e t

al., 1992b]: this is consistent with the observation that the transcript is detected in foetal gut and muscle and placenta

but not adult skeletal muscle [Bar et al., 1990, Blake et al., 1992]. Western blots on rat bain extracts show that there is only a low level of apo-dystrophin 1 in 18 day embryos and newborns and that this level gradually increases thereafter [Jung et al., 1993]. These data suggest developmental

specificity for the muscle and apo-dystrophin 1 promoters [Rapaport et al., 1992b], see table 1.4. At a subcellular level, apo-dystrophin 1 is found mainly in synaptic plasma

membranes and microsomes. It was also faintly detected in synaptic vesicles and mitochondria, thus suggesting that it has different functions to dystrophin in the central nervous

system [Jung et al., 1993].

The fourth DMD gene product that has been described as possibly transcribed from an alternative promoter has been named Dpi 16 with an mRNA transcript of 5.2Kb in length [Byers at al., 1993]. This seems likely to be the same

transcript as described by Blake et al., who identified a 5.8Kb transcript in Schwannoma cells and glioma cells and named it apo-dystrophin 2 [Blake et al., 1992, Tinsley et al., 1993]. The D pi 16 transcript has a new exon which lies approximately 850bp upstream of the equivalent of muscle DMD exon 56 and possibly a specific promoter upstream of this. There is also evidence for possible 3' splicing of the Dpi 16 mRNA.

Immunoblot assays detected the corresponding 116kDa protein product which encodes the last 946 amino acids of dystrophin

and contains the distal rod region from repeat 22, the cysteine rich region and the C-terminal. The expression of this

transcript and protein product is found exclusively in adult peripheral nerves and Schwann cells and its presence appears to be mutually exclusive of the muscle and brain type

dystrophin and of apo-dystrophin 1 [Blake et al., 1992, Byers a t a!., 1993]. Immunolocalisation showed expression of D pi 16 to be specific to the outside of myelinated peripheral nerve

fibres and the perimeter of the Schwann cell sheath. This subcellular localisation may suggest that the protein has an important role in peripheral nerve conduction but patients with deletions in this region show no obvious peripheral neuropathy [Ahn et a!., 1993 re w ie w , Byers et al., 1993] (see table 1.4).

The most recent isoform described is apo-dystrophin 3 which has a 5' untranslated region and first 7 amino acids identical to that of apo-dystrophin 1 and therefore is almost certainly transcribed from the same promoter [Blake et al.,

1992, Hugnot et al., 1992, Lederfein et al., 1992, Rapaport e t al., 1992a, Tinsley et al., 1993]. The protein encodes the end of repeat 46 of the rod region, the cysteine rich region and the first 48 amino acids of the C-terminus of dystrophin. The 3'

DMA sequence has an in frame stop codon and initial

untranslated region identical to to one of those described by Freener et al., for a human foetal dystrophin truncated at the proximal end of the C-terminal [Freener et al., 1989, Tinsley e t al., 1993]. The transcript is 2.2kb, the protein product

predicted to be 341 amino acids long and 40kOa. Apo-

dystrophin 3 appears to be less abundant in Schwannoma and HeLa cells than apo-dystrophin 1. The mRNA transcript has been detected by PCR in adult and foetal muscle, foetal liver, foetal lung and foetal brain. Mouse and human tissues showed identical distributions. Apo-dystrophin 3 was detected in pluripotent mouse ES cells whereas, in this study, apo-

dystrophin 1 was not. However, although Rapaport et al., did find apo-dystrophin 1 in such cells, this probably due to the

Rapaport et al., primers not differentiating between apo- dystrophin 1 and 3 (Dp70) [Rapaport et al., 1992b, Tinsley e t

al., 1993]. Previously described dystrophin isoforms will have to be reappraised in view of the characterisation of

TABLE 1.4 DYSTROPHIN iSOFORMS WITH ALTERNATIVE PROMOTERS DYSTROPHIN TYPE TISSUE SIZE mRNA (Kb) SIZE PROTEIN (kDa) STRUCTURAL DIFFERENCE to 14Kb muscle transcript Brain type dystrophin cerebral cortex neurons, hippocampus 1 4 427 Novel exon 1

Dystrophin- P Purkinje neurons

( h, m) = 14 (nd) = 400 (nd) Novel exon 1 Apo-dystrophlnl or Dp71 Brain, sp cord, pane, liver, testis, lung, kidney, skin, heart, diaph, spleen, myom. Foetal; brain, heart, liver, stomach, cc; Schwannoma glial, neuronal. Unique 5' UTR, 1st 7 codons+ promoter betw. exon 62 and 63. No actin binding+rod dom. Has cyst rich dom+C- ter (exons 71+78 del.) Apo-dystrophin3 (same promoter as apo- dystrophinl )

Adul t/ foet al;

muscle, liver, lung, brain. cc:schwannoma, HeLa(h,m), ES (foetal). 2.2Kb 40kDa 5' UTR + 1st 7 codons= to apo- dystrophinl. Has pt of rpt 46 rod dom+cyst rich dom+lst 48aa of C-ter Apo-dystrophin2 or Dp116 cczSchwannoma, glioma 5.8 ( 5 . 2 ) 116kDa Starts 850bp upstream of exon 56. Has distal rod dom +cyst rich dom+C-ter.

Table 1.4 h=human, m=mouse, rt=rat, Sk.=skeletal, Sm.=smooth, cc=cultured cells, nd=not determined. cc= cultured cells, ES=embryonic stem cell, sp cord=spinal cord, panc=pancreas, lymphobl=lymphoblastoid, diaph=diaphram, myom=myometrium, skmu=skeletal muscle. aa=amino acids, UTR=untranslated region, btw=between, pt=part, rpt=repeat, cyst=cysteine, C-ter=C-terminal, dom=domain, del=deleted.

apo-dystrophin1 and 3 and apo-dystrophin 2 (Dp116) [Tinsley et al., 1993 and Blake et a!., unpublished]. For example,

immunologically related proteins of 400kDa, 120kDa, IIO kD a and 70-80kDa have been identified at the postsynaptic density of the cerebral cortex, cerebellum and olafactory bulb, these proteins seem likely to be dystrophin isoforms since there is reduction in their expression in the dystrophin deficient m dx mouse [Kim et al., 1992] see table 1.4. The identification of alternative promoters and the multitude of truncated and alternatively spliced dystrophin isoforms has an important impact on diagnosis and prognosis of DMD and BMD patients.

Although the amino-terminal and the rod domain show homology to a-actinin and spectrin [Koenig et al., 1988], the 0- terminal of dystrophin is the only domain that, until recently, was not found to be homologous to any other known proteins (see discussion). The conservation of nucleic acid sequence at the carboxy terminal region amongst the various isoforms discussed above, and amongst dystrophins from different species, infers a functional significance for this region. This function is not tissue specific since the tissue distribution of the various DMD transcripts is very different.