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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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Hypothetical and real transcript isoforms of hCx62. Schematic drawing of the hCx62 connexin gene (GJA10). The gray hatched boxes represent the coding region of hCx62 which is linked by the roof-like black line. The first exon contains 1440 nucleotides coding for 480 amino acid residues and the second exon comprises at least the 36 nucleotides coding for the C-terminal amino acid residues. Both exons are flanked by an intron of about 25 kb. The dashed boxes and lines only represent hypothetical splice isoforms which could not be proven by RT-PCR analyses. The arrows indicate the primers used for RT-PCR analyses. USP; upstream-primer, DSP; downstream-primer. The sequences of the splice donor sites are listed separately.
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Figure 2: Hypothetical and real transcript isoforms of hCx62. Schematic drawing of the hCx62 connexin gene (GJA10). The gray hatched boxes represent the coding region of hCx62 which is linked by the roof-like black line. The first exon contains 1440 nucleotides coding for 480 amino acid residues and the second exon comprises at least the 36 nucleotides coding for the C-terminal amino acid residues. Both exons are flanked by an intron of about 25 kb. The dashed boxes and lines only represent hypothetical splice isoforms which could not be proven by RT-PCR analyses. The arrows indicate the primers used for RT-PCR analyses. USP; upstream-primer, DSP; downstream-primer. The sequences of the splice donor sites are listed separately.

Mentions: In a previous study, the C-terminal end of mCx57 (12aa) was demonstrated to be encoded on a putative third exon approx. 9 kb further downstream of the major coding region (480 amino acid residues) that is functionally spliced after transcription [30]. RT-PCR primer combinations between upstream primer (USP) 1 to 4 and downstream primer (DSP) 1 to 2 confirmed the uninterrupted transcription of the major part of the mCx57 coding region on exon2 (data not shown). However, after application of downstream primer DSP3, -4, -5 and -6, the corresponding RT-PCR amplicons failed to emerge in each case (data not shown), thus indicating that neither the C-terminus (25 amino acid residues) is encoded on exon2 (as predicted by [41]), nor that the hypothetical transcript isoforms, deduced from data base predictions, are transcribed in figure 2. Only downstream primer DSP7 yielded an amplicon that was cloned and sequenced [30]. The position of primer DSP7 further downstream of a fourth predicted small reading frame and splice acceptor site concomitantly excluded their usage but instead confirmed the transcription of a small reading frame (36 nucleotides; 12 amino acid residues) and its upstream located splice acceptor site (figure 2).


Expression of connexin genes in the human retina.

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

Hypothetical and real transcript isoforms of hCx62. Schematic drawing of the hCx62 connexin gene (GJA10). The gray hatched boxes represent the coding region of hCx62 which is linked by the roof-like black line. The first exon contains 1440 nucleotides coding for 480 amino acid residues and the second exon comprises at least the 36 nucleotides coding for the C-terminal amino acid residues. Both exons are flanked by an intron of about 25 kb. The dashed boxes and lines only represent hypothetical splice isoforms which could not be proven by RT-PCR analyses. The arrows indicate the primers used for RT-PCR analyses. USP; upstream-primer, DSP; downstream-primer. The sequences of the splice donor sites are listed separately.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2984586&req=5

Figure 2: Hypothetical and real transcript isoforms of hCx62. Schematic drawing of the hCx62 connexin gene (GJA10). The gray hatched boxes represent the coding region of hCx62 which is linked by the roof-like black line. The first exon contains 1440 nucleotides coding for 480 amino acid residues and the second exon comprises at least the 36 nucleotides coding for the C-terminal amino acid residues. Both exons are flanked by an intron of about 25 kb. The dashed boxes and lines only represent hypothetical splice isoforms which could not be proven by RT-PCR analyses. The arrows indicate the primers used for RT-PCR analyses. USP; upstream-primer, DSP; downstream-primer. The sequences of the splice donor sites are listed separately.
Mentions: In a previous study, the C-terminal end of mCx57 (12aa) was demonstrated to be encoded on a putative third exon approx. 9 kb further downstream of the major coding region (480 amino acid residues) that is functionally spliced after transcription [30]. RT-PCR primer combinations between upstream primer (USP) 1 to 4 and downstream primer (DSP) 1 to 2 confirmed the uninterrupted transcription of the major part of the mCx57 coding region on exon2 (data not shown). However, after application of downstream primer DSP3, -4, -5 and -6, the corresponding RT-PCR amplicons failed to emerge in each case (data not shown), thus indicating that neither the C-terminus (25 amino acid residues) is encoded on exon2 (as predicted by [41]), nor that the hypothetical transcript isoforms, deduced from data base predictions, are transcribed in figure 2. Only downstream primer DSP7 yielded an amplicon that was cloned and sequenced [30]. The position of primer DSP7 further downstream of a fourth predicted small reading frame and splice acceptor site concomitantly excluded their usage but instead confirmed the transcription of a small reading frame (36 nucleotides; 12 amino acid residues) and its upstream located splice acceptor site (figure 2).

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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