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Expressed sequence tag analysis of guinea pig (Cavia porcellus) eye tissues for NEIBank.

Simpanya MF, Wistow G, Gao J, David LL, Giblin FJ, Mitton KP - Mol. Vis. (2008)

Bottom Line: Approximately 60% of the total gene clusters were novel cDNA sequences and had significant homologies to other mammalian sequences in GenBank.For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin.In the 'rest-of-eye' library, the most abundant transcripts included decorin and keratin 12, representative of the cornea.

View Article: PubMed Central - PubMed

Affiliation: Eye Research Institute, Oakland University, Rochester, MI 48309, USA.

ABSTRACT

Purpose: To characterize gene expression patterns in guinea pig ocular tissues and identify orthologs of human genes from NEIBank expressed sequence tags.

Methods: RNA was extracted from dissected eye tissues of 2.5-month-old guinea pigs to make three unamplified and unnormalized cDNA libraries in the pCMVSport-6 vector for the lens, retina, and eye minus lens and retina. Over 4,000 clones were sequenced from each library and were analyzed using GRIST for clustering and gene identification. Lens crystallin EST data were validated using two-dimensional electrophoresis (2-DE), matrix assisted laser desorption (MALDI), and electrospray ionization mass spectrometry (ESIMS).

Results: Combined data from the three libraries generated a total of 6,694 distinctive gene clusters, with each library having between 1,000 and 3,000 clusters. Approximately 60% of the total gene clusters were novel cDNA sequences and had significant homologies to other mammalian sequences in GenBank. Complete cDNA sequences were obtained for many guinea pig lens proteins, including alphaA/alphaAinsert-, gammaN-, and gammaS-crystallins, lengsin and GRIFIN. The ratio of alphaA- to alphaB-crystallin on 2-DE gels was 8: 1 in the lens nucleus and 6.5: 1 in the cortex. Analysis of ESTs, genome sequence, and proteins (by MALDI), did not reveal any evidence for the presence of gammaD-, gammaE-, and gammaF-crystallin in the guinea pig. Predicted masses of many guinea pig lens crystallins were confirmed by ESIMS analysis. For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin. The guinea-pig ortholog of NRL, a key rod photoreceptor-specific transcription factor, was also represented in EST data. In the 'rest-of-eye' library, the most abundant transcripts included decorin and keratin 12, representative of the cornea.

Conclusions: Genomic analysis of guinea pig eye tissues provides sequence-verified clones for future studies. Guinea pig orthologs of many human eye specific genes were identified. Guinea pig gene structures were similar to their human and rodent gene counterparts. Surprisingly, no orthologs of gammaD-, gammaE-, and gammaF-crystallin were found in EST, proteomic, or the current guinea pig genome data.

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Alternate splicing in the guinea pig S-antigen gene. A: Predicted guinea pig S-antigen gene structure from guinea pig retina S-antigen ESTs. B: ESTs aligned to guinea pig genome (scaffold_13) and guinea pig mRNA from GenBank for S-antigen (Sag, Arrestin) (EF457996) generated from NEIBank ESTs. Sag shows that some exons are absent, for example: EST (i) and (iii) are missing exons 6, 7, 8, 9, and 10, EST (ii) and (iv) may terminate early at exons 8 and 5. Note: arrows show introns in the direction of sequence reads.
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f6: Alternate splicing in the guinea pig S-antigen gene. A: Predicted guinea pig S-antigen gene structure from guinea pig retina S-antigen ESTs. B: ESTs aligned to guinea pig genome (scaffold_13) and guinea pig mRNA from GenBank for S-antigen (Sag, Arrestin) (EF457996) generated from NEIBank ESTs. Sag shows that some exons are absent, for example: EST (i) and (iii) are missing exons 6, 7, 8, 9, and 10, EST (ii) and (iv) may terminate early at exons 8 and 5. Note: arrows show introns in the direction of sequence reads.

Mentions: The percentage of gene clusters of the guinea pig retina cDNA library having significant homology to mammalian (non-guinea pig) GenBank sequences was 56%. In comparison, human and mouse retina cDNA libraries had 80% [40] and 85% [54] significant GenBank homology, respectively. While the majority of ESTs correspond to canonical gene transcripts, some genes show evidence of relatively frequent alternative (or aberrant) splicing. As an example from retina, S-antigen (Arrestin) spliced ESTs and guinea pig mRNAs are shown aligned to the guinea pig genome scaffold_13 (Figure 6). A surprising number of alternatively spliced transcripts are evident, in particular different patterns of exclusion of exons 6–10. For example, EST (i) and (iii) are missing exons 6, 7, 8, 9, and 10 while EST (ii) and (iv) may terminate early at exons 8 and 5, respectively. Whether this has functional significance remains to be seen, but since several of the variants interrupt the open reading frame and have stop codons ahead of the last exon, they would be subject to nonsense-mediated decay and would not produce proteins. This might have a regulatory role for levels of S-antigen or may simply reflect inefficient splicing of an abundant mRNA. Such exon skipping is also apparent in human S-antigen ESTs, but at a lower frequency.


Expressed sequence tag analysis of guinea pig (Cavia porcellus) eye tissues for NEIBank.

Simpanya MF, Wistow G, Gao J, David LL, Giblin FJ, Mitton KP - Mol. Vis. (2008)

Alternate splicing in the guinea pig S-antigen gene. A: Predicted guinea pig S-antigen gene structure from guinea pig retina S-antigen ESTs. B: ESTs aligned to guinea pig genome (scaffold_13) and guinea pig mRNA from GenBank for S-antigen (Sag, Arrestin) (EF457996) generated from NEIBank ESTs. Sag shows that some exons are absent, for example: EST (i) and (iii) are missing exons 6, 7, 8, 9, and 10, EST (ii) and (iv) may terminate early at exons 8 and 5. Note: arrows show introns in the direction of sequence reads.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2605723&req=5

f6: Alternate splicing in the guinea pig S-antigen gene. A: Predicted guinea pig S-antigen gene structure from guinea pig retina S-antigen ESTs. B: ESTs aligned to guinea pig genome (scaffold_13) and guinea pig mRNA from GenBank for S-antigen (Sag, Arrestin) (EF457996) generated from NEIBank ESTs. Sag shows that some exons are absent, for example: EST (i) and (iii) are missing exons 6, 7, 8, 9, and 10, EST (ii) and (iv) may terminate early at exons 8 and 5. Note: arrows show introns in the direction of sequence reads.
Mentions: The percentage of gene clusters of the guinea pig retina cDNA library having significant homology to mammalian (non-guinea pig) GenBank sequences was 56%. In comparison, human and mouse retina cDNA libraries had 80% [40] and 85% [54] significant GenBank homology, respectively. While the majority of ESTs correspond to canonical gene transcripts, some genes show evidence of relatively frequent alternative (or aberrant) splicing. As an example from retina, S-antigen (Arrestin) spliced ESTs and guinea pig mRNAs are shown aligned to the guinea pig genome scaffold_13 (Figure 6). A surprising number of alternatively spliced transcripts are evident, in particular different patterns of exclusion of exons 6–10. For example, EST (i) and (iii) are missing exons 6, 7, 8, 9, and 10 while EST (ii) and (iv) may terminate early at exons 8 and 5, respectively. Whether this has functional significance remains to be seen, but since several of the variants interrupt the open reading frame and have stop codons ahead of the last exon, they would be subject to nonsense-mediated decay and would not produce proteins. This might have a regulatory role for levels of S-antigen or may simply reflect inefficient splicing of an abundant mRNA. Such exon skipping is also apparent in human S-antigen ESTs, but at a lower frequency.

Bottom Line: Approximately 60% of the total gene clusters were novel cDNA sequences and had significant homologies to other mammalian sequences in GenBank.For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin.In the 'rest-of-eye' library, the most abundant transcripts included decorin and keratin 12, representative of the cornea.

View Article: PubMed Central - PubMed

Affiliation: Eye Research Institute, Oakland University, Rochester, MI 48309, USA.

ABSTRACT

Purpose: To characterize gene expression patterns in guinea pig ocular tissues and identify orthologs of human genes from NEIBank expressed sequence tags.

Methods: RNA was extracted from dissected eye tissues of 2.5-month-old guinea pigs to make three unamplified and unnormalized cDNA libraries in the pCMVSport-6 vector for the lens, retina, and eye minus lens and retina. Over 4,000 clones were sequenced from each library and were analyzed using GRIST for clustering and gene identification. Lens crystallin EST data were validated using two-dimensional electrophoresis (2-DE), matrix assisted laser desorption (MALDI), and electrospray ionization mass spectrometry (ESIMS).

Results: Combined data from the three libraries generated a total of 6,694 distinctive gene clusters, with each library having between 1,000 and 3,000 clusters. Approximately 60% of the total gene clusters were novel cDNA sequences and had significant homologies to other mammalian sequences in GenBank. Complete cDNA sequences were obtained for many guinea pig lens proteins, including alphaA/alphaAinsert-, gammaN-, and gammaS-crystallins, lengsin and GRIFIN. The ratio of alphaA- to alphaB-crystallin on 2-DE gels was 8: 1 in the lens nucleus and 6.5: 1 in the cortex. Analysis of ESTs, genome sequence, and proteins (by MALDI), did not reveal any evidence for the presence of gammaD-, gammaE-, and gammaF-crystallin in the guinea pig. Predicted masses of many guinea pig lens crystallins were confirmed by ESIMS analysis. For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin. The guinea-pig ortholog of NRL, a key rod photoreceptor-specific transcription factor, was also represented in EST data. In the 'rest-of-eye' library, the most abundant transcripts included decorin and keratin 12, representative of the cornea.

Conclusions: Genomic analysis of guinea pig eye tissues provides sequence-verified clones for future studies. Guinea pig orthologs of many human eye specific genes were identified. Guinea pig gene structures were similar to their human and rodent gene counterparts. Surprisingly, no orthologs of gammaD-, gammaE-, and gammaF-crystallin were found in EST, proteomic, or the current guinea pig genome data.

Show MeSH
Related in: MedlinePlus