Metodología para aplicación correcta del IPERC

that arise by differential splicing and are transcribed from the brain and muscle type promoters and gives information on the size of the resulting protein product and the position TABLE 1.2 ALTERNATIVE TRANSCRIPTS PART I

Sp. TISSUE SIZE OF PROTEIN PRODUCT (aa) STRUCTURAL DIFFERENCEt exon nos REFS. h m Sk. muscle, brain 3680 71 D [Freener '89, Bies '92a] h Sk. muscle, brain Sm muscle 3670 78 D [Freener '89, Kunkel '91]

ck Sk. muscle 3750 72 and 73 D [Freener '89,

h Sm. muscle 34 1 0 * 70 T [Freener '89,

h Sm. muscle 3 2 80* 68 D [Freener '89,

h brain 3 6 5 0 * 71 D and 78 D [Freener '89,

h brain 3 6 6 5 * 71 and 72 D 78 D [Freener '89, h, m brain, retina, 3 5 9 0 * 71, 72, 73 and 74 D 78 D [Freener '89, Tamura, 93]

Table 1.2 t Comparison to the full length muscle oDNA, exon nos from Roberts et

a/., 1992. ^Possibly transcribed from a smooth muscle specific promoter. * = transcript with brain first exon. Sp.=species, h=human, m=mouse, rt=rat, ck=chicken Sk.=skeletal, Sm.=smooth, cc=cultured cells, D=deietion, Ds=two deletions, position of largest described, T=termination position. Method of detection indicated by: N=Northern blot, S=Southern blot, W=western blot, RNaseP=RNase protection assay, PCR=Polymerase chain reaction.

of internal splice sites relative to the 14kb muscle transcript as described by Freener et a/., [1989].

The polymerase chain reaction technique was used to amplify the DMD cDNA in 1Kb lengths and identied transcripts which showed PCR amplification products different in size to that expected from the consensus skeletal muscle cDNA. It was demonstrated that the 3' end of dystrophin is capable of being alternatively spliced to create isoforms differing at their carboxy terminal domains. Two C-terminal isoforms were detected in skeletal muscle (transcribed from the muscle promoter), three in brain (transcribed from the brain promoter) and two in smooth muscle (possibly transcribed from another promoter) [Freener et a/., 1989]. Existance of one of these isoforms has been corroborated and many more discovered see table 1.2 and 1.3 [Kunkel et a l, 1991, Tamura et a/., 1993].

Further to these studies, multiple novel spliced forms have been identified in mouse brain, skeletal muscle, cardiac

muscle, diaphragm and Purkinje fibres [Bies et a!., 1992a], only one brain specific isoform appearing identical to that

described by Freener et a/.. Interestingly, the greatest number of isoforms have been found in non-skeletal muscle tissue.

Examination of the nucleotide sequence revealed that two of these isoforms were observed in all tissues examined, with several others specific to cardiac muscle and brain. The human cardiac Purkinje fibres express an isoform that is

primarily expressed in the brain (mouse heart tissue, however, does not express a significant amount of this isoform) [Bies e t a!., 1992a]. Another study, examining RNA from mouse tissues using the PCR technique, has shown a DMD transcript to be present in the retina which also occurs in brain and muscle, and has demonstrated two further alternatively spliced species of DMD mRNA present in the brain and retina. Using first exon PCR primers the brain type DMD transcript appears to be more abundant in the retina than muscle type DMD

transcript. The isoforms detected were not tissue specific but an Isoform expressed in the brain and retina, with exons 71, 72, 73 and 74 deleted, is not detectable in the muscle and appears to be the same isoform detected by Freener et al., in

the brain [Freener et al., 1989, Tamura et a!., 1993]. This lends support to the suggestion that the main type of dystrophin in the retina is the brain type DMD [Tamura et a!., 1993].

Further studies using human retina protein extract on Western blots has shown that, using an amino-terminal

antibody, in addition to the 427kDa protein, two smaller major species can be detected of 420kDa and 407kDa. Cross reactive bands of 300kDa, 290kDa, 195kDa were also detected by this antibody and using anti-bodies directed against the rod domain and the C-terminal an additional 260kDa species was detected, but these species were not further characterised [Tamura e t a!., 1993].

Using the PCR technique transcripts of both the brain and the muscle type, were detected in the retina. Transcripts corresponding to the 420kDa and 407kDa dystrophins were identified as lacking exons 71 and exons 71, 72, 73 and 74 respectively, with predicted sizes of 425kDa and 414kDa [Fillers et al., 1993]. It is possible that the isoform lacking exon 71 described by Tamura et al., and by Bies et al., is the same as that identified by Fillers et al., and likewise the isoform with exons 71 to 74 deleted may be the same as one described by Bies, et al., [Bies et al., 1992a, Fillers et al.,

1993, Tamura et al., 1993], see table 1.3. Evidence from these various studies also indicates that most of the isoforms

detected are conserved across species [Bies et al., 1992a, Tamura et al., 1993]. Those isoforms not described by Freener

et al., are detailed in table 1.3.

Developmental and tissue specific alternative splicing patterns appears to be very important in the regulation of expression of DMD mRNA. Alternative splicing at the 3’ end of the gene does not seem to be specific to the c is acting

promoter, for example; the cardiac Purkinje fibre isoform [Bies et al., 1992a] has the muscle first exon (i.e. transcribed from the muscle type promoter) but the same 3' deletion as the brain and retina isoform transcribed from the brain type

promoter [Freener at al., 1989, Tamura et al., 1993] nor is there is there an obvious correlation between promoter use in