Title

Cloning and characterization of a new gap junction gene: Connexin33

Date of Award

1993

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Rudolf K. Werner, Committee Chair

Abstract

Connexins, a family of integral membrane proteins, assemble in multimeric units in apposing cell membranes to form cell-cell channels. In order to study the gene regulation of one of its members, connexin43, the corresponding cDNA was used as a hybridization probe to screen a rat genomic bacteriophage library. Instead of the expected connexin43 gene, the gene of a new gap junction protein, connexin33 was isolated. The coding region of connexin33 is 62% identical to connexin43 at the amino acid level. The gene structure of connexin33 is similar to that of connexin43, connexin32 and connexin26, whose genes have also been cloned. The gene contains two exons, 156 bp and about 0.9 kb, respectively, separated by a 4 kb intron. Exon I constitutes the 5$\sp\prime$ untranslated sequence, while exon II contains the uninterrupted coding sequence. The intron boundaries were mapped by using sequence data, 5$\sp\prime$ RACE cloning (Rapid Amplification of cDNA Ends) and Southern analysis.Northern analysis of various rat tissues showed that connexin33 mRNA is found in low, but clearly detectable levels in testis. It was also found by PCR in heart and uterus. This discrete pattern of expression is typical of the connexins. The connexin33 gene was shown by an S1 nuclease protection assay to possess multiple transcription start sites, also found in other connexin genes.The Xenopus expression system was used to assay for a function of the connexin33 gene. Coding sequences for connexin33 were subcloned into an appropriate vector to synthesize mRNA in vitro. This connexin mRNA was injected into Xenopus laevis oocytes for translation. The junctional conductance across paired oocytes is proportional to the number of open cell-cell channels. Data from this assay indicate that connexin33 does not form channels with itself, nor with several other connexins that have been tested. However, it possesses the unique capability to specifically inhibit the formation or open channels by connexin37 and connexin43 in the oocyte, while it has no effect on connexin32 channel formation. The possible physicological significance of this property of connexin33 is discussed.

Keywords

Biology, Molecular; Chemistry, Biochemistry

Link to Full Text

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