Two gap junction genes, connexin 31.1 and 30.3, are closely linked on mouse chromosome 4 and preferentially expressed in skin.

H Hennemann,E Dahl,J.B White,H.J Schwarz,P.A Lalley,S Chang,B.J Nicholson,K Willecke

doi:10.1016/s0021-9258(18)41916-3

Abstract

Two new gap junction genes isolated from the mouse genome code for connexin homologues of 271 and 266 amino acids, designated here Cx31.1 and Cx30.3, respectively. The two open reading frames, oriented in the same direction, are only 3.4 kb apart on mouse chromosome 4. Within the connexin family, these two proteins are most closely related to one another (70% amino acid sequence identity) and to Cx31 (65 and 68% identity, respectively). Comparison of the Cx31.1 mouse gene with a Cx31.1 cDNA showed a similar genomic organization to that found with other members of the connexin gene family, i.e. the coding and 3'-untranslated regions are contained within a single exon, which is preceded by an intron, less than 25 bases upstream of the ATG start codon. Northern blot hybridization revealed highly tissue-specific coexpression of the 1.6-kb Cx31.1 mRNA and two Cx30.3 transcripts of 1.9- and 3.2-kb size, predominantly in skin and two related mouse keratinocyte cell lines. Minor levels of Cx31.1 mRNA were detected in testis. Microinjection of Cx30.3, but not Cx31.1 cRNA, into Xenopus oocyte pairs induced formation of functional gap junction channels with unique voltage-gated parameters compared to other connexins expressed similarly.

Highlights

Two new gap junctiognenes isolated from the mouse function is thoughtto be involved in tissue homeostasis, genome code for connexin homologues o2f71 and 266 coordinated physiological response, metabolic cooperation, amino acids, designated hereCx31.1 and Cx30.3, re- growth control, and regulation of development as well as spectively
Tween connexins expressed in apposed cells (Swenson et d . , 1989;Barrio et al, 1991; Hennemann et al, 1992~)A. lthough connexin genes are expressed in a cell type-specific manner, several cell types tested contain at least two different con
Nucleotide sequence and predicted amino acid sesize of the Cx31.1 transcript in F9 cells is approximately 1.6 kb ascalculated from Northern blot analyses, Using an RNase protection experiment it was demonstrated that the5' 1360bp of this clone could not be protectedby F9 RNA and were not part of the genuine F9 Cx31.1 cDNA

Summary

MATERIALS AND METHODS

Isolation of Mouse Genomic and cDNA Clones-Recombinant genomic phage clones were isolated from a Charon 4A library, described previously by Hynes et al (1981), constructed from partially EcoRI digested liver DNA of GR mice A total of4.6 X lo phages was plated on agar plates, lifted onto nylon membranes (Hybond-N, Amersham, United Kingdom) as described by the manufacturer, and hybridized to Cx26 rat liver cDNA (Cx26-1, Zhang and Nicholson, 1989). One phage clone, which did not hybridize to Cx26 rat liver cDNA under high stringency conditions, was used for further examination. Southern and NorthernBlot Analysis-Genomic mouse liver DNA was prepared by a standard procedure (Sambrook et al, 1989) and digested with different restriction endonucleases in 10-pg aliquots. High stringency hybridizations (55% formamide, 42 "C, 5 x SSC), filter washing, and autoradiography were performed as described (Willecke et al, 1991b). SSC) either to the Cx31.1 probe (1300-bp BsmI fragment, extending downstream of the BsmI site at position 445 (see Fig. 2)) or to the

RESULTS

CXII I

Mouse chromosome

DISCUSSION