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Expression of connexin genes in the human retina.

Söhl G, Joussen A, Kociok N, Willecke K - BMC Ophthalmol (2010)

Bottom Line: Here we report that Northern blot hybridization signals of the orthologuous hCx36 and hCx45 were found in human retinal RNA.Cx59 mRNA, however, with high sequence identity to zebrafish Cx55.5 was only weakly detectable by RT-PCR in cDNA of human retina.Appropriate antibodies against Cx59 and Cx62 protein will clarify expression of these proteins in future studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Genetik der Universität Bonn, Römerstr, Bonn, Germany. sekretariat@martinus-gymnasium.de

ABSTRACT

Background: Gap junction channels allow direct metabolically and electrical coupling between adjacent cells in various mammalian tissues. Each channel is composed of 12 protein subunits, termed connexins (Cx). In the mouse retina, Cx43 could be localized mostly between astroglial cells whereas expression of Cx36, Cx45 and Cx57 genes has been detected in different neuronal subtypes. In the human retina, however, the expression pattern of connexin genes is largely unknown.

Methods: Northern blot hybridizations, RT-PCR as well as immunofluorescence analyses helped to explore at least partially the expression pattern of the following human connexin genes GJD2 (hCx36), GJC1 (hCx45), GJA9 (hCx59) and GJA10 (hCx62) in the human retina.

Results: Here we report that Northern blot hybridization signals of the orthologuous hCx36 and hCx45 were found in human retinal RNA. Immunofluorescence signals for both connexins could be located in both inner and outer plexiform layer (IPL, OPL). Expression of a third connexin gene denoted as GJA10 (Cx62) was also detected after Northern blot hybridization in the human retina. Interestingly, its gene structure is similar to that of Gja10 (mCx57) being expressed in mouse horizontal cells. RT-PCR analysis suggested that an additional exon of about 25 kb further downstream, coding for 12 amino acid residues, is spliced to the nearly complete reading frame on exon2 of GJA10 (Cx62). Cx59 mRNA, however, with high sequence identity to zebrafish Cx55.5 was only weakly detectable by RT-PCR in cDNA of human retina.

Conclusion: In contrast to the neuron-expressed connexin genes Gjd2 coding for mCx36, Gjc1 coding for mCx45 and Gja10 coding for mCx57 in the mouse, a subset of 4 connexin genes, including the unique GJA9 (Cx59) and GJA10 (Cx62), could be detected at least as transcript isoforms in the human retina. First immunofluorescence analyses revealed a staining pattern of hCx36 and hCx45 expression both in the IPL and OPL, partially reminiscent to that in the mouse, although additional post-mortem material is needed to further explore their sublamina-specific distribution. Appropriate antibodies against Cx59 and Cx62 protein will clarify expression of these proteins in future studies.

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Phylogenetic tree deduced from a multiple protein sequence alignment revealed the phylogenetical relationship of mouse Cx57, porcine Cx60 and human Cx62. Instead, human Cx59 (GJA9) seems much more related to both zebrafish connexins Cx55.5 and Cx52.6. Hypothetical shuffling of genomic fragments could occur between mCx57, hCx59 and hCx62. Human-mouse alignments with BLASTZ indicate that human Cx62 (GJA10) (Chr. 6) might be the orthologuous connexin gene to mouse Cx57 (Gja10) (Chr. 4). N; N-terminus, C*; C-terminus * indicates that this stop codon is not used. SD; splice donor site.
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Figure 1: Phylogenetic tree deduced from a multiple protein sequence alignment revealed the phylogenetical relationship of mouse Cx57, porcine Cx60 and human Cx62. Instead, human Cx59 (GJA9) seems much more related to both zebrafish connexins Cx55.5 and Cx52.6. Hypothetical shuffling of genomic fragments could occur between mCx57, hCx59 and hCx62. Human-mouse alignments with BLASTZ indicate that human Cx62 (GJA10) (Chr. 6) might be the orthologuous connexin gene to mouse Cx57 (Gja10) (Chr. 4). N; N-terminus, C*; C-terminus * indicates that this stop codon is not used. SD; splice donor site.

Mentions: Deduced from comparisons of connexin gene pairs, human Cx62 (GJA10) is most likely the orthologuous connexin gene to mouse Cx57 (Gja10) (82% nucleotide identity; 78% amino acid identity; see [20]. A direct comparison between mouse Cx57 (Gja10) and human Cx59 (GJA9) instead yielded much lower sequence identities, suggesting that the Cx59 (GJA9) gene has no mouse ortholog. Results from a phylogenetic alignment, however, indicated that hCx59 (GJA9) might belong to a group of connexin open reading frames (ORFs) of relatively high molecular weight having its closest sequence relationship to zebrafish Cx55.5, with which it was tightly clustered. Both connexin genes are further aligned to a subgroup containing mCx57 (Gja10), porcine Cx60 [55] and hCx62 (GJA10) (dendrogram in Figure 1; accession numbers in Methods).


Expression of connexin genes in the human retina.

Söhl G, Joussen A, Kociok N, Willecke K - BMC Ophthalmol (2010)

Phylogenetic tree deduced from a multiple protein sequence alignment revealed the phylogenetical relationship of mouse Cx57, porcine Cx60 and human Cx62. Instead, human Cx59 (GJA9) seems much more related to both zebrafish connexins Cx55.5 and Cx52.6. Hypothetical shuffling of genomic fragments could occur between mCx57, hCx59 and hCx62. Human-mouse alignments with BLASTZ indicate that human Cx62 (GJA10) (Chr. 6) might be the orthologuous connexin gene to mouse Cx57 (Gja10) (Chr. 4). N; N-terminus, C*; C-terminus * indicates that this stop codon is not used. SD; splice donor site.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2984586&req=5

Figure 1: Phylogenetic tree deduced from a multiple protein sequence alignment revealed the phylogenetical relationship of mouse Cx57, porcine Cx60 and human Cx62. Instead, human Cx59 (GJA9) seems much more related to both zebrafish connexins Cx55.5 and Cx52.6. Hypothetical shuffling of genomic fragments could occur between mCx57, hCx59 and hCx62. Human-mouse alignments with BLASTZ indicate that human Cx62 (GJA10) (Chr. 6) might be the orthologuous connexin gene to mouse Cx57 (Gja10) (Chr. 4). N; N-terminus, C*; C-terminus * indicates that this stop codon is not used. SD; splice donor site.
Mentions: Deduced from comparisons of connexin gene pairs, human Cx62 (GJA10) is most likely the orthologuous connexin gene to mouse Cx57 (Gja10) (82% nucleotide identity; 78% amino acid identity; see [20]. A direct comparison between mouse Cx57 (Gja10) and human Cx59 (GJA9) instead yielded much lower sequence identities, suggesting that the Cx59 (GJA9) gene has no mouse ortholog. Results from a phylogenetic alignment, however, indicated that hCx59 (GJA9) might belong to a group of connexin open reading frames (ORFs) of relatively high molecular weight having its closest sequence relationship to zebrafish Cx55.5, with which it was tightly clustered. Both connexin genes are further aligned to a subgroup containing mCx57 (Gja10), porcine Cx60 [55] and hCx62 (GJA10) (dendrogram in Figure 1; accession numbers in Methods).

Bottom Line: Here we report that Northern blot hybridization signals of the orthologuous hCx36 and hCx45 were found in human retinal RNA.Cx59 mRNA, however, with high sequence identity to zebrafish Cx55.5 was only weakly detectable by RT-PCR in cDNA of human retina.Appropriate antibodies against Cx59 and Cx62 protein will clarify expression of these proteins in future studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Genetik der Universität Bonn, Römerstr, Bonn, Germany. sekretariat@martinus-gymnasium.de

ABSTRACT

Background: Gap junction channels allow direct metabolically and electrical coupling between adjacent cells in various mammalian tissues. Each channel is composed of 12 protein subunits, termed connexins (Cx). In the mouse retina, Cx43 could be localized mostly between astroglial cells whereas expression of Cx36, Cx45 and Cx57 genes has been detected in different neuronal subtypes. In the human retina, however, the expression pattern of connexin genes is largely unknown.

Methods: Northern blot hybridizations, RT-PCR as well as immunofluorescence analyses helped to explore at least partially the expression pattern of the following human connexin genes GJD2 (hCx36), GJC1 (hCx45), GJA9 (hCx59) and GJA10 (hCx62) in the human retina.

Results: Here we report that Northern blot hybridization signals of the orthologuous hCx36 and hCx45 were found in human retinal RNA. Immunofluorescence signals for both connexins could be located in both inner and outer plexiform layer (IPL, OPL). Expression of a third connexin gene denoted as GJA10 (Cx62) was also detected after Northern blot hybridization in the human retina. Interestingly, its gene structure is similar to that of Gja10 (mCx57) being expressed in mouse horizontal cells. RT-PCR analysis suggested that an additional exon of about 25 kb further downstream, coding for 12 amino acid residues, is spliced to the nearly complete reading frame on exon2 of GJA10 (Cx62). Cx59 mRNA, however, with high sequence identity to zebrafish Cx55.5 was only weakly detectable by RT-PCR in cDNA of human retina.

Conclusion: In contrast to the neuron-expressed connexin genes Gjd2 coding for mCx36, Gjc1 coding for mCx45 and Gja10 coding for mCx57 in the mouse, a subset of 4 connexin genes, including the unique GJA9 (Cx59) and GJA10 (Cx62), could be detected at least as transcript isoforms in the human retina. First immunofluorescence analyses revealed a staining pattern of hCx36 and hCx45 expression both in the IPL and OPL, partially reminiscent to that in the mouse, although additional post-mortem material is needed to further explore their sublamina-specific distribution. Appropriate antibodies against Cx59 and Cx62 protein will clarify expression of these proteins in future studies.

Show MeSH