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

Mukoma F. Simpanya, Graeme Wistow, James Gao, Larry David, Frank J. Giblin, Kenneth P. Mitton

Research output: Contribution to journalArticle

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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 αA/αAinsert-, γN-, and γS-crystallins, lengsin and GRIFIN. The ratio of αA- to αB-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 γD-, γE-, and γF-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 γD-, γE-, and γF- crystallin were found in EST, proteomic, or the current guinea pig genome data.

Original languageEnglish (US)
Pages (from-to)2413-2427
Number of pages15
JournalMolecular Vision
Volume14
StatePublished - Dec 19 2008

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Expressed Sequence Tags
Sequence Analysis
Crystallins
Guinea Pigs
Lenses
Libraries
Genes
Retina
Electrospray Ionization Mass Spectrometry
Multigene Family
Keratin-12
Complementary DNA
Clone Cells
Genome
Transducin
Light Signal Transduction
Decorin
Arrestin
Retinal Rod Photoreceptor Cells
Crystalline Lens

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Simpanya, M. F., Wistow, G., Gao, J., David, L., Giblin, F. J., & Mitton, K. P. (2008). Expressed sequence tag analysis of guinea pig (Cavia porcellus) eye tissues for NEIBank. Molecular Vision, 14, 2413-2427.

Expressed sequence tag analysis of guinea pig (Cavia porcellus) eye tissues for NEIBank. / Simpanya, Mukoma F.; Wistow, Graeme; Gao, James; David, Larry; Giblin, Frank J.; Mitton, Kenneth P.

In: Molecular Vision, Vol. 14, 19.12.2008, p. 2413-2427.

Research output: Contribution to journalArticle

Simpanya, MF, Wistow, G, Gao, J, David, L, Giblin, FJ & Mitton, KP 2008, 'Expressed sequence tag analysis of guinea pig (Cavia porcellus) eye tissues for NEIBank', Molecular Vision, vol. 14, pp. 2413-2427.
Simpanya MF, Wistow G, Gao J, David L, Giblin FJ, Mitton KP. Expressed sequence tag analysis of guinea pig (Cavia porcellus) eye tissues for NEIBank. Molecular Vision. 2008 Dec 19;14:2413-2427.
Simpanya, Mukoma F. ; Wistow, Graeme ; Gao, James ; David, Larry ; Giblin, Frank J. ; Mitton, Kenneth P. / Expressed sequence tag analysis of guinea pig (Cavia porcellus) eye tissues for NEIBank. In: Molecular Vision. 2008 ; Vol. 14. pp. 2413-2427.
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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 αA/αAinsert-, γN-, and γS-crystallins, lengsin and GRIFIN. The ratio of αA- to αB-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 γD-, γE-, and γF-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 γD-, γE-, and γF- crystallin were found in EST, proteomic, or the current guinea pig genome data.",
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AU - Simpanya, Mukoma F.

AU - Wistow, Graeme

AU - Gao, James

AU - David, Larry

AU - Giblin, Frank J.

AU - Mitton, Kenneth P.

PY - 2008/12/19

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N2 - 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 αA/αAinsert-, γN-, and γS-crystallins, lengsin and GRIFIN. The ratio of αA- to αB-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 γD-, γE-, and γF-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 γD-, γE-, and γF- crystallin were found in EST, proteomic, or the current guinea pig genome data.

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