MOLECULAR CLONING OF GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR (GDNF) GENE FROM MOUSE
WANG FU JUN
WANG FU JUN
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Abstract
Recently, exciting and novel observations have implicated a particular factor the glial cell line-derived neurotrophic factor (GDNF), to be a key survival factor for many types of neurons including the doparninergic neurons in the midbrain. These findings have raised hopes that GDNF may serve as a potential therapeutic for Parkinson's disease and other related neurological disorders.
In order to gain a better understanding of the molecular biology of GDNF. the mouse homologues of GDNF have been isolated and the gene partially characterized. Two unique forms of GDNF transcripts ( GDNF-? and GDNF-? from adult mouse brain have been identified and characterized. The largest clone contained an insert size of 3509 bp encoding the GDNF-? form. Another unique clone contained the partial sequence of the GDNF-? form, which is similar to the ? form except for a 78 bp deletion in the open reading frame. The deduced amino acid sequences of the mouse GDNF-? are highly homologous to those of rat and human GDNF-?. Reverse transcription PCR (RT-PCR) from various mouse tissues showed extensive expression of both forms of GDNF.
Genomic clones encoding the 3' exon of GDNF have been isolated and partially characterized. The GDNF-? is encoded by at least two distinct exons. Two consensus polyadenylation signal (AAUAAA) sites and two of the unusual polyadenylation hexamer sites (AAUGAA), are present in the 3'-UTR. The second AAUAAA site in the 3 '-UTR was found to be used as the signal for polyadenylation in normal adult mouse brain. The whole of the 3'-UTR is encoded in a single exon.
The coding The coding region of the mature form of the mouse GDNF-? was expressed in the prokaryotic expression vector pQE-12. The expressed protein was found exclusively in the inclusion body in all the E. coli strains studied.
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Date
1997
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