Abstract
Mutations affecting the peripheral myelin protein-22 (PMP22) gene have been shown to be associated with inherited peripheral neuropathies. To provide the molecular basis for the analysis of such mutations, we have cloned and characterized the human PMP22 gene. It spans approximately 40 kilobases and contains four coding exons. Detailed analysis of its 5'-flanking region suggested the presence of two alternatively transcribed, but untranslated exons. Mapping of separate PMP22 mRNA transcription initiation sites to each of these exons indicates that PMP22 expression is regulated by two alternatively used promoters. In support of this hypothesis, both putative promoter sequences demonstrated the ability to drive expression of reporter genes in transfection experiments. Furthermore, the structures of the 5'-portions of the PMP22 genes appear to be identical in rat and human, supporting the biological significance of the observed arrangement of regulatory regions. The relative expression of the alternative PMP22 transcripts is tissue-specific, and high levels of the exon 1A-containing transcript are tightly coupled to myelin formation. In contrast, exon 1B-containing transcripts are predominant in non-neural tissues and in growth-arrested primary fibroblasts. Interestingly, although a strong upregulation of PMP22 mRNA was observed in cultured Schwann cells in the presence of the adenylate cyclase activator forskolin under various culture conditions, the regulation of the different PMP22 mRNA species did not mimic the regulation that occurs during myelin formation in vivo. The observed regulation of the PMP22 gene by a complex molecular mechanism is consistent with the proposed dual role of PMP22 in neural and non-neural tissue.
Highlights
(PMP22) gene have been shown to be associated with prises about2 4 % of the total proteinfound in isolated myelin inherited peripheral neuropathies
Based on the genomic human PMP22 DNA sequence and Identification of Human PMP22 cDNAs with Different 5’- appropriate cDNA sequences, oligonucleotide primers specific Sequences-We have identified several PMP22 cDNA clones by for either exon 1Aor 1B weresynthesized andused in separate a combination of reverse transcriptasePCR and 5”RACE-PCR primer extension experiments to determine the transcription using RNA derived from human sciatic nerve as a template initiationsites of the two observed mRNA species
Thealignment of theresulting DNA se- experiments, several extended cDNA products were obtained quences with published human PMP22 cDNAs (Patel et al, (Fig. 3,A and C ) .In ordert o eliminate signals duteo premature 1992; Hayasaka et al, 1992; Edomi et al, 1993) revealed the terminations of the reverse transcriptasereactions and potenpresence of three different 5”flanking sequences t h a t diverge tial nonspecific priming of the oligonucleotides used, the results from the common sequence at the same nucleotide position of the primerextension experiments were confirmed by appro
Summary
(PMP22) gene have been shown to be associated with prises about2 4 % of the total proteinfound in isolated myelin inherited peripheral neuropathies. A strong upregulation of PMP22 mRNA was observed in cultured Schwann cellsin the presence of the adenylate cyclase responsible forthe severemyelin deficiencies and theexcessive proliferation of Schwann cells that underlie the profound peripheralneuropathiesinthe neurological mouse mutants, trembler and trembler-J (Suter et al, 1992a, 1992b) Based on these findings and itslocation in a region of conserved synteny, activator forskolin under various culture conditions, PMP22 has been proposed as a candidate gene for the most the regulation of the different PMP22 mRNA species did common inherited peripheral neuropathyin humans, Charcotnot mimic the regulation that occurs during myelin for- Marie-Tooth disease type1A(CMTlA),' and the rarely encounmation in vivo. P.) from the Canadian Network of Centers on Neural Regeneration and Functional ' The abbreviations used are: CMTIA, Charcot-Marie-Tooth disease
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