Tissue-specific expression of connexin32 from alternate promoters
Date of Award
Doctor of Philosophy (Ph.D.)
First Committee Member
Rudolf Werner - Committee Chair
The gene of the rat connexin32, the gap junction protein found in liver, consists of two exons which are separated by a 6.1-kb intron located within the 5$\sp\prime$-untranslated region. The promoter of this gene was studied in tissue-culture cells and transgenic mice using reporter gene constructs. Connexin32 transgenes, containing 2.5-kb of sequence upstream from the promoter, exon I, the entire 6.1-kb intron, and the beginning of the coding sequence linked to the coding sequence of the reporter gene luciferase, were found to be expressed in mouse in the same tissue-specific manner as previously reported for the connexin32 gene. Another reporter gene construct lacking the known promoter, but retaining 1.8-kb from the 3$\sp\prime$-end of the intron, a region containing a potential second promoter, was found to be expressed specifically in the nervous system. This result suggested that a second promoter different from that used in liver drives the expression of the connexin32 gene in nervous tissue. The use of alternate promoters in normal rats was demonstrated by sequence analysis of reverse-transcribed PCR (RT-PCR) products of mRNA obtained from different rat tissues. While an upstream promoter, P1, drives the expression of the gene in liver, the second promoter, P2, drives the synthesis of a second connexin32 mRNA species.In view of the recent finding that mutations in the connexin32 gene have been associated with the X-linked form of the Charcot-Marie-Tooth disease, it was important to determine whether, like in the rat, the human connexin32 gene was transcribed from two alternate promoters. Because the human connexin32 gene had not been isolated, the sequence of the promoter P2 region of the human connexin32 gene was obtained by PCR amplification and sequencing of genomic DNA with primers derived from the known rat sequence. A human brain cDNA library was then screened by PCR using primers that allowed the detection of transcripts originating from either promoter P1 or P2. The analysis showed that the connexin32 mRNA expressed in human brain has a different 5'-end than the mRNA isolated from liver. These results were corroborated by RT-PCR amplification of mRNA isolated from human brain and liver. While the liver connexin32 mRNA is initiated from promoter P1, the brain connexin32 mRNA is initiated from promoter P2, located only 550 bases upstream of the start codon.Seven of the 28 reported families with CMT-X disease, however, exhibit no mutations in the connexin32 coding region. If connexin32 mutations are responsible for the CMT-X phenotype, then one would expect that these families exhibit mutations within the promoter region, and these mutations should be present in the promoter P2. DNA samples from patients with CMT-X disease that do not show mutations in the connexin32 coding sequence were obtained. These patients belonged to five different families. The above hypothesis was tested by PCR amplification of the genomic DNA with primers specific for the promoter P2 region. The analysis showed that one of the patients' DNA did not result in the normal PCR amplification pattern, suggesting that a rearrangement or a deletion in the promoter P2 region in this patient's DNA may have occurred. Sequence analysis of the promoter P2 region in these patients is currently under investigation.
Biology, Molecular; Biology, Neuroscience; Biology, Genetics
Neuhaus, Isaac Moises, "Tissue-specific expression of connexin32 from alternate promoters" (1995). Dissertations from ProQuest. 3342.