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- Materials and methods
The cloning and sequencing of two bovine connexin32 cDNAs are reported. Comparative analysis with known corresponding mammalian cDNA and protein sequences, besides confirming a high degree of similarity among these proteins, allowed us to identify some specific features of the bovine connexin32 gene. The latter include: the presence of a novel exon in the 5′ UTR which is alternatively spliced, giving rise to a new mRNA species; the presence of two potential hairpin loops in the 5′ and 3′ UTR; and the presence of an additional amino acid, glycine235, in the C-terminal domain of the 284 residue protein. Among the common features, the presence of polypyrimidine clusters within the 3′ UTR, containing a consensus sequence for a cis-acting element, is noteworthy. Expression of connexin32 mRNAs was analysed in 16 bovine tissues. Transcript analysis suggests the presence, in cattle, of an alternative downstream promoter.
Gap junctions are specialized clusters of transmembrane channels that mediate intercellular communications, allowing small molecules and ions to move between neighbouring cells without exposure to the extracellular space [1–3]. Gap junctions appear to be dynamic structures, exhibiting short half-lives (5–6 h). They are functionally controlled by channel opening and closing and therefore play an important role in the regulation of intercellular communications [1,3,4].
The major structural components of gap junctions are integral membrane proteins, connexins, which belong to a multigene family [1,3,5,6]. In rodents 13 different members of this family, as well as their complex and often overlapping expression patterns , have so far been described. Genes belonging to this family share a similar structure consisting of two exons separated by a large intron (5–11 kb in size) interrupting the 5′ UTR. The entire coding region and the 3′ UTR are, therefore, part of the same exon (exon 2). This feature allowed connexin genes in different species to be mapped using rather short cDNA probes [8–11].
The structure of the connexin32 gene appears to be more complex, because, in addition to the upstream promoter P1, an alternative promoter P2, close to the 3′ end of the large intron, is present [12–14]. Connexin32 messengers are therefore formed by exon 2 linked either to exon 1, transcribed from the upstream promoter P1, or to exon 1b transcribed from the downstream promoter P2. The latter transcript is at least partially specific for the nervous system both in rodents and in humans [12–14]. This is noteworthy since mutations in the human connexin32 gene have been associated with an X-linked form of Charcot-Marie-Tooth disease (CMTX 1) which shows nervous-tissue-specific symptoms without affecting other connexin32-expressing organs .
The cloning and sequencing of bovine connexin32 cDNA reported here, led to the isolation of a new mRNA species derived from the transcript controlled by the upstream promoter P1 through alternative splicing. We demonstrated that the 5′ UTR of this new mRNA comprises a new exon, located within the large intron of the gene. Expression-pattern analysis of connexin32 mRNAs in 16 bovine tissues, besides allowing the detection of a transcript originating from the promoter P2, confirmed the existence of two alternatively spliced transcripts under the control of promoter P1.
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- Materials and methods
The bovine connexin32 cDNA sequences reported here were obtained from overlapping DNA fragments, isolated by different approaches from RNA and from genomic DNA.
The 284 amino-acid-encoded connexin32 protein is almost identical to other known mammalian connexin32s, showing 97–98% identity [18,21]. The only noteworthy difference is the presence of an additional glycine at position 235 lying in a glycine-rich context (four glycines in an exapeptide) within the C-terminal cytoplasmic domain . A number of potential phosphorylation sites, conserved among mammals, were identified within this terminal portion of the protein (Fig. 1). Saez et al.  showed that in mouse and rat liver, two of these sites (Ser 229 and Ser 233) can be phosphorylated by protein kinase C and the latter (Ser 233) by protein kinase A as well.
The structure of the bovine gene for connexin32 appears to be more complex than in other mammals, since an additional exon (1a) was discovered. The bovine connexin32 gene consists of four exons (1, 1a, 1b and 2) and four introns (1, 1a1, 1a2 and 1b). Exons 1, 1a and 1b contain exclusively 5′ UTR sequences. The data reported here are consistent with the presence, also in the bovine gene, of two alternative promoters: P1, upstream of exon 1 and P2, upstream of exon 1b.
We isolated from bovine liver mRNAs two cDNAs derived, through alternative splicing, from promoter P1. Transcript A includes exon 1 and exon 2, while transcript B includes exon 1, the new exon 1a and exon 2. These two cDNAs show a high level of overall similarity (between 80% and 90%) with other mammalian connexin32 cDNAs [18,21]. The new exon in transcript B, which is discussed below, does not display any sequence similarity with the known connexin32 5′ UTRs [12–14]. It will be interesting to verify whether this exon is also present in other members of this gene family, whose intronic sequences are only partially known. Transcript C, originating from promoter P2, exhibited a high degree of identity (96.6%) with the corresponding sequences of the other mammalian species [12–14].
The 5′ UTRs of these transcripts do not show any similarity with a further connexin32 transcript (Dahl. E., accession number X84214), suggested to originate from a third promoter, whose presence in mouse has been reported, with no experimental data, by Söhl et al. .
A feature specific for bovine connexin32 cDNAs, is the presence of two potential secondary structures within the 5′ and 3′ UTRs. The calculated free energy value (−82.75 kJ) for the hairpin loop at the very beginning of the 5′ UTR of transcripts A and B suggests that this structure might be stable enough to restrict translation . The second hairpin loop is located within the 3′ UTR (positions 1076–1107). Its calculated free energy value (– 127.1 kJ) could explain the cessation of sequencing reactions from the 3′ end of the 1035 bp fragment (see Results) exactly at nucleotide 1107. It has been reported that stem-loop elements located within 3′ UTR could be involved in the regulation of mRNA half-life, usually through binding of specific proteins .
A feature conserved among mammalian connexin32 cDNAs, including bovine, is the presence of an upstream open reading frame (uORF) beginning at position – 10 (i.e. in exon 2), out-of-frame with respect to the connexin32 start codon, extending 134 nucleotides into the coding region and encoding very similar peptides of unknown function constituted by 48 amino acids (43 residues in mouse). These peptides do not show sequence similarity with known proteins. This uORF lies in a context rather unfavourable for translation [27,28]. However, its constant presence in connexin32 cDNAs suggests a possible role, either disturbing translation start at the correct AUG or mediating regulatory effects through the encoded peptide .
Transcript B showed a unique feature, i.e. the presence of two additional in-frame overlapping uORFs starting at nucleotides – 63 and – 45, respectively, and comprising 18 and 12 codons. Their starting codons are located in functionally different contexts, only the first being favourable for translation [27,28]. They share the same stop codon (TGA) located six nucleotides upstream of the ATG start codon of connexin32 and partially overlapping the start codon of the common uORF, this stop signal is therefore present within the common exon 2. A functional role of short uORFs terminating before the main ATG codon has been demonstrated for some genes such as murine complement factor B and CCAAT/enhancer-binding protein, where the uORFs act in cis[30,31], and β2 adrenergic receptor mRNA, where the encoded peptide itself is able to inhibit protein expression .
Pyrimidine-rich sequences and polypyrimidine-binding proteins are important functional elements in RNA metabolism . A putative cis-element, present within the first (positions 873–932) of the three pyrimidine clusters of bovine connexin32 mRNA, fits the consensus (C/U)CCANXCCC(U/A)YXUC(C/U)CC perfectly and is found in some stable eukaryotic mRNAs . This sequence has been reported to bind cytosolic proteins able to form an RNA–protein complex (α-complex) involved in mRNA stabilization . An almost identical sequence at corresponding positions is also present in the other mammalian connexin32 mRNAs, except for human where the consensus is only partially recognizable.
Tissue distribution of the connexin32 mRNA is in substantial agreement with the expression patterns detected in other mammals [35–38], including its prevalence in liver and kidney. RT–PCR analyses with primers specific for the different connexin32 mRNA species were carried out in order to assess the tissue distribution of transcripts A, B and C.
Transcripts A and B share the same qualitative expression pattern, are absent from nervous tissue and are detectable in liver, kidney, intestine, uterus, abomasum, testis, ovary and pancreas. As PCR efficiency was shown to be identical for these two transcripts, their co-expression was further investigated by densitometric measurements of the corresponding amplification bands. Although transcript B exhibits low expression levels, our results indicate tissue differences in its relative abundance. The meaning of these differences is unknown, however, they could be part of a subtle transcriptional and/or translational control in physiological and/or pathological conditions.
Transcript C, which is under the control of promoter P2, is present in nervous tissue (brain and spinal cord) thus confirming its expression specificity, already determined in humans and rodents [12–14].
The generation of transcripts differing only in their 5′ regions has been described for many unrelated genes and mostly interpreted as being an evolutionary gain to refine transcriptional and translational control. A relationship between these alternatively spliced transcripts and a particular function has been suggested for a minority of them. Examples are mouse heat shock protein 47 (HSP 47) gene  and human lactoferrin gene . In the first case, one of the alternative transcripts acquires an increased translatability at high temperatures, while in the second case it might play an important role in the regulation of cell growth.
When alternative transcripts arise from distinct promoters, they usually show tissue-specific or cell-specific expression, as in the case of mouse leukaemia inhibitory factor receptor (LIF-R)  and mammalian connexin32 transcript controlled by promoter P2 (refs 12–14 and present paper). Indeed it is more difficult to understand the significance of 5′ UTR alternative transcripts originating from the same promoter, since they often do not display clear-cut tissue specificity. Among a number of published examples showing co-expression of these transcripts are: muscle-specific enolase , human vigilin , human CC chemokine receptor 5 (CCR5)  and bovine prion protein . The most shared hypothesis suggests that their functional role might be related to physiological or metabolic changes. Because no quantitation of the relative abundance between the transcripts has been performed in the above cases, the existence of some differences cannot be excluded, as reported here for connexin32. Subtle transcriptional and post-transcriptional regulations have already been argued for connexin32 and other connexin mRNAs in both normal and regenerating rat liver after partial hepatectomy [46, 47], but the mechanisms by which regulation is achieved have not been elucidated. The new connexin32 transcript reported here may represent a further step in identifying the components of such complex mechanisms. Additional information could be acquired through a better knowledge of the large intron 1 interrupting the 5′ UTR and harbouring both promoter P2 and exons 1a and 1b. Structural and functional characterization of this intron is in progress in our laboratory.