Jimenez-Mallebrera, Cecilia; (2001) Complex transcription of the utrophin gene: A study of short utrophin isoforms. Doctoral thesis (Ph.D), UCL (University College London).

Text out.pdf Download (29MB)

Abstract

Utrophin is the autosomal homologue of dystrophin. The aim of this project was to characterise the short utrophin forms at the mRNA and protein levels and to look for other utrophin transcripts. Mice knocked-out for full-length utrophin (UKOex6) were used as an experimental system. These mice lack full- length utrophin but it was expected that they would express all the short forms. In order to examine the distribution of utrophin protein in UKOex6 mice, polyclonal antibodies were raised against different regions of the protein. Western blotting and immunofluorescence studies, using these antibodies, confirmed that full-length utrophin is absent in UKOex6 tissues and identified two novel short forms Up120 and Up109. These studies also showed that while Up140 and Up71 appear not to be translated, the other short forms are translated in a tissue specific fashion. G-utrophin is neuronal specific; Up120 is expressed specifically in kidney glomeruli; Up109 was identified in foetal hands/feet and appears to localise to the epidermis. A cDNA for Up109 was isolated by 5' RACE; this contains 460bp of unique sequence encoding 5'UTR and 17 amino acids. Immunological studies confirmed that the full-length form is the major utrophin in skeletal muscle, and demonstrated that this form is also the most abundant In kidney and testis. In the testis, full-length utrophin localises to intertubular tissue and to Leydig cells. Western blot analysis suggested that a novel isoform(s) is expressed in the testis, however, this could not be confirmed by immuno-histochemistry. Weak signal detected at the neuromuscular junction in UKOex6 adult muscle was interpreted as being due to a short isoform, perhaps Up140. In contrast to the apparently limited distribution of short forms as proteins, mRNA in situ hybridisation experiments to control and UKOex6 embryo sections showed that they are transcribed abundantly throughout development. Short transcripts are first detected by 8.5 dpc in the neural tube and appear to comprise the majority of utrophin expression during development. Sites of high expression of full-length utrophin mRNA during development are tongue, choroid plexus and the outflow of the heart. A comparison of the mRNA and protein distributions suggests that the transcription of short utrophin mRNAs is not matched by their translation (this is particularly true for Up140 and Up71). The significance of this important observation and possible roles for the short transcripts are discussed. The developmental studies also revealed three novel sites of utrophin expression - pyloric sphincter, the urethra and the semicircular canals. Utrophin is the autosomal homologue of dystrophin. Transcription from the utrophin gene involves five promoters that regulate the expression of two full-length and three short transcripts. The skeletal muscle isoform has been well studied because it has a potential role in the treatment of Duchenne Muscular Dystrophy. However, there is little information on the short isoforms, particularly their patterns of expression and function. The aim of this project was to characterise the short utrophin isoforms at the mRNA and protein levels and to look for other utrophin transcripts. Mice knocked-out for full-length utrophin (UKOex6) were used as the experimental model. These mice lack full-length utrophin but it was believed that they would express all the short isoforms. In order to examine the distribution of utrophin protein in UKOex6 mice, polyclonal antibodies were raised against different regions of the protein. Western blotting and immunofluorescence to tissue sections confirmed that full- length utrophin is absent in tissues and showed that while Up140 and Up71 appear not to be translated, some short isoforms are translated in a limited fashion. Two of these isoforms, Up120 and Up109, had not been reported previously. Up120 is a kidney specific isoform and Up109 might be a foetal isoform. Up109 cDNA, which contains novel sequence in intron 55, was isolated by 5' RACE. Immunological analysis of a selection of tissues showed that the full-length form is the major utrophin in skeletal muscle, kidney and testis and revealed that in the latter full-length utrophin localises to the intertubular tissue and to the Leydig cells. Surprisingly, weak utrophin signal could be detected at the neuromuscular junction in UKOex6 adult muscle. In contrast to the restricted expression of short isoforms as proteins, mRNA in situ hybridisation experiments using probes to different regions of utrophin mRNA showed that short utrophin isoforms are transcribed abundantly throughout development. Thus, for these isoforms the abundance of mRNA is not matched by that of the protein. The significance of this observation and possible roles for the short transcripts are discussed. (Abstract shortened by UMI.)

Download activity - last month

Export as XMLCSVJSON

Download activity - last 12 months

Export as CSVXMLJSON

Downloads by country - last 12 months

Export as CSVXMLJSON

Archive Staff Only

View Item