Previous studies have shown that chemical regulation of connexin43 (Cx43) depends on the presence of the carboxyl terminal (CT) domain. A particle-receptor (or "ball-and-chain") model has been proposed to explain the mechanism of gating. We tested whether the CT region behaved as a functional domain for other members of the connexin family. The pH sensitivity of wild-type and Ct-truncated connexins was quantified by use of electrophysiological and optical techniques and the Xenopus oocyte system. The CT domain of Cx45 had no role in pH regulation, although a partial role was shown for Cx37 and Cx50. A prominent effect was observed for Cx40 and Cx43. In addition, we found that the CT domain of Cx40 that was expressed as a separate fragment rescued the pH sensitivity of the truncated Cx40 (Cx40tr), which was in agreement with a particle-receptor model. Because Cx40 and Cx43 often colocalize and possibly heteromerize, we tested the pH sensitivity of Cx40tr when coexpressed with the CT domain of Cx43 (hetero-domain interactions). We found that the CT domain of Cx43 enhanced the pH sensitivity of Cx40tr; similarly, the CT domain of Cx40 restored the pH sensitivity of the truncated Cx43. In addition, the CT domain of Cx43 granted insulin sensitivity to the otherwise insulin-insensitive Cx26 or Cx32 channels. These data show that the particle-receptor model is preserved in Cx40 and the regulatory domain of one connexin can specifically interact with a channel formed by another connexin. Hetero-domain interactions could be critical for the regulation of heteromeric channels.

BackgroundMobile group II introns insert site-specifically into DNA target sites by a mechanism termed retrohoming in which the excised intron RNA reverse splices into a DNA strand and is reverse transcribed by the intron-encoded protein. Retrohoming is mediated by a ribonucleoprotein particle that contains the intron-encoded protein and excised intron RNA, with target specificity determined largely by base pairing of the intron RNA to the DNA target sequence. This feature enabled the development of mobile group II introns into bacterial gene targeting vectors (“targetrons”) with programmable target specificity. Thus far, however, efficient group II intron-based gene targeting reactions have not been demonstrated in eukaryotes.Methodology/Principal FindingsBy using a plasmid-based Xenopus laevis oocyte microinjection assay, we show that group II intron RNPs can integrate efficiently into target DNAs in a eukaryotic nucleus, but the reaction is limited by low Mg2+ concentrations. By supplying additional Mg2+, site-specific integration occurs in up to 38% of plasmid target sites. The integration products isolated from X. laevis nuclei are sensitive to restriction enzymes specific for double-stranded DNA, indicating second-strand synthesis via host enzymes. We also show that group II intron RNPs containing either lariat or linear intron RNA can introduce a double-strand break into a plasmid target site, thereby stimulating homologous recombination with a co-transformed DNA fragment at frequencies up to 4.8% of target sites. Chromatinization of the target DNA inhibits both types of targeting reactions, presumably by impeding RNP access. However, by using similar RNP microinjection methods, we show efficient Mg2+-dependent group II intron integration into plasmid target sites in zebrafish (Danio rerio) embryos and into plasmid and chromosomal target sites in Drosophila melanogster embryos, indicating that DNA replication can mitigate effects of chromatinization.Conclusions/SignificanceOur results provide an experimental foundation for the development of group II intron-based gene targeting methods for higher organisms.

Mobile group II introns retrohome by an RNP-based mechanism in which the excised intron lariat RNA fully reverse splices into a DNA site via 2 sequential transesterification reactions and is reverse transcribed by the associated intron-encoded protein. However, linear group II intron RNAs, which can arise by either hydrolytic splicing or debranching of lariat RNA, cannot carry out both reverse-splicing steps and were thus expected to be immobile. Here, we used facile microinjection assays in 2 eukaryotic systems, Xenopus laevis oocyte nuclei and Drosophila melanogaster embryos, to show that group II intron RNPs containing linear intron RNA can retrohome by carrying out the first step of reverse splicing into a DNA site, thereby ligating the 3 end of the intron RNA to the 5 end of the downstream exon DNA. The attached linear intron RNA is then reverse transcribed, yielding an intron cDNA whose free end is linked to the upstream exon DNA. Some of these retrohoming events result in the precise insertion of full-length intron. Most, however, yield aberrant 5 junctions with 5 exon resections, 5 intron truncations, and/or extra nucleotide residues, hallmarks of nonhomologous end-joining. Our findings reveal a mobility mechanism for linear group II intron RNAs, show how group II introns can co-opt different DNA repair pathways for retrohoming, and suggest that linear group II intron RNAs might be used for site-specific DNA integration in gene targeting.DNA repair ͉ gene targeting ͉ retrotransposition ͉ reverse transcriptase ͉ ribozyme M obile group II introns found in bacterial and organellar genomes are retroelements that consist of an autocatalytic intron RNA and an intron-encoded protein (IEP) with reverse transcriptase (RT) activity (1, 2). The intron RNA and IEP function together in a ribonucleoprotein particle (RNP) to promote the integration of the intron into specific DNA sites by a mechanism in which the intron RNA reverse splices directly into a DNA strand and is then reverse transcribed by the IEP (1). Group II introns use this mechanism both to retrohome to the ligated-exon junction (homing site) in intronless alleles at high frequency and to retrotranspose to ectopic sites that resemble the normal homing site at low frequency. These processes enabled the dispersal of mobile group II introns to diverse bacteria and may have been used for the invasion and proliferation of group II introns in the nuclear genomes of early eukaryotes, where they evolved into spliceosomal introns (1,3,4).Like spliceosomal introns, most group II introns splice via 2 sequential transesterification reactions that result in the formation of an intron lariat RNA (2). For mobile group II introns, the splicing reactions are assisted by the IEP, which binds specifically to the intron RNA to stabilize the catalytically active RNA structure (5). The IEP then remains bound to the excised intron lariat RNA in an RNP that promotes intron mobility (6). RNPs initiate intron mobility by recognizing DNA target sites using both the IEP and base p...

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