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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 of the guinea pig Decorin gene. A: Predicted guinea pig Decorin (Dcn) gene structure from guinea pig “rest of the eye” Decorin ESTs. B: ESTs aligned to guinea pig genome (scaffold_9) and guinea pig mRNA from GenBank for Dcn generated from NEIBank ESTs. Dcn gene shows a minor alternative first exon (alternative transcription start site; a black asterisk shows two cDNAs which contain an alternative exon with a potential protein coding open reading frame) that is also seen in other species [45]. Note: arrows show introns in the direction of sequence reads.
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f7: Alternate splicing of the guinea pig Decorin gene. A: Predicted guinea pig Decorin (Dcn) gene structure from guinea pig “rest of the eye” Decorin ESTs. B: ESTs aligned to guinea pig genome (scaffold_9) and guinea pig mRNA from GenBank for Dcn generated from NEIBank ESTs. Dcn gene shows a minor alternative first exon (alternative transcription start site; a black asterisk shows two cDNAs which contain an alternative exon with a potential protein coding open reading frame) that is also seen in other species [45]. Note: arrows show introns in the direction of sequence reads.

Mentions: Eye tissue for the guinea pig eye minus lens and retina library (nba) consisted of the cornea, iris, ciliary body, trabecular meshwork, choroid, sclera, and RPE (Figure 1B-D). This library contained some retinal content, including clones for Rhodopsin (8 clones in this library versus 64 in the retina library), and S-antigen (3 clones versus 24 in retina). This results from the difficulty in avoiding some neural retina contamination in the dissection. Most of the abundantly expressed genes identified were logical markers for cornea, RPE/choroid, and sclera. Several genes for extracellular matrix and glycoproteins, which are abundant in cornea, sclera, and choroid, were also observed (Table 5). Examples include decorin, annexin A1 (Lipocortin-like protein 39 kDa), Collagen alpha-2 type I, Sparc and Keratin 12. For Decorin (Dcn), EST evidence indicated an alternative 5′-exon and as a possible alternative transcription start site (Figure 7B, shown with a black asterisk) which is also apparent in some human mRNAs [73,74]. Decorin ESTs and mRNA were aligned with scaffold_9 of the guinea pig genome (Figure 7). No guinea pig reference mRNAs were available during production of this alignment.


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 of the guinea pig Decorin gene. A: Predicted guinea pig Decorin (Dcn) gene structure from guinea pig “rest of the eye” Decorin ESTs. B: ESTs aligned to guinea pig genome (scaffold_9) and guinea pig mRNA from GenBank for Dcn generated from NEIBank ESTs. Dcn gene shows a minor alternative first exon (alternative transcription start site; a black asterisk shows two cDNAs which contain an alternative exon with a potential protein coding open reading frame) that is also seen in other species [45]. 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

f7: Alternate splicing of the guinea pig Decorin gene. A: Predicted guinea pig Decorin (Dcn) gene structure from guinea pig “rest of the eye” Decorin ESTs. B: ESTs aligned to guinea pig genome (scaffold_9) and guinea pig mRNA from GenBank for Dcn generated from NEIBank ESTs. Dcn gene shows a minor alternative first exon (alternative transcription start site; a black asterisk shows two cDNAs which contain an alternative exon with a potential protein coding open reading frame) that is also seen in other species [45]. Note: arrows show introns in the direction of sequence reads.
Mentions: Eye tissue for the guinea pig eye minus lens and retina library (nba) consisted of the cornea, iris, ciliary body, trabecular meshwork, choroid, sclera, and RPE (Figure 1B-D). This library contained some retinal content, including clones for Rhodopsin (8 clones in this library versus 64 in the retina library), and S-antigen (3 clones versus 24 in retina). This results from the difficulty in avoiding some neural retina contamination in the dissection. Most of the abundantly expressed genes identified were logical markers for cornea, RPE/choroid, and sclera. Several genes for extracellular matrix and glycoproteins, which are abundant in cornea, sclera, and choroid, were also observed (Table 5). Examples include decorin, annexin A1 (Lipocortin-like protein 39 kDa), Collagen alpha-2 type I, Sparc and Keratin 12. For Decorin (Dcn), EST evidence indicated an alternative 5′-exon and as a possible alternative transcription start site (Figure 7B, shown with a black asterisk) which is also apparent in some human mRNAs [73,74]. Decorin ESTs and mRNA were aligned with scaffold_9 of the guinea pig genome (Figure 7). No guinea pig reference mRNAs were available during production of this alignment.

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