Section on Molecular Structure and Functional Genomics
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A major emphasis of this group is to move beyond single gene, single technology studies in an attempt to integrate genomics, structure and function and evolutionary perspectives to obtain a wider systems view of the molecular biology of the eye. We have made extensive use of genomics and bioinformatics (largely through NEIBank) to gain new insights into the molecular composition of the eye and to identify sets of specific changes in sequence and expression that distinguish the human eye from those of other mammals, opening the possibility of improving animal models of human eye disease. We are now applying the tools of functional genomics to these discoveries, using techniques including microarrays, yeast 2-hybrid analysis and knock-out/knock-in mouse models to delineate the roles of key proteins. We also pursue direct biophysical studies of recombinant proteins of interest through collaborations with X-ray, NMR and Electron microscopy structural biology labs.
Current focus is on novel eye-specific genes with potential involvement in homeostasis and aging in the human eye and on mapping functional interactions related to important eye diseases, particularly age-related macular degeneration.NEIBank and Functional Genomics
NEIBank is project for producing, organizing and disseminating genomics resources and expression and sequence data for eye research. The web site is at http://neibank.nei.nih.gov. Collaborators from many institutions have contributed freshly dissected tissues for cDNA libraries of many eye tissues from many species. These are used for high throughput sequencing. Analyzed data are displayed in the web site, submitted to GenBank and described in peer-reviewed publications. Clones are made available through OpenBiosystems. Eye-related data from other sources are also gathered and displayed through the web site. Researchers throughout the world visit the web site and cite NEIBank in publications.
Key features of NEIBank include:
- Sequence and expression data for human eye tissues and for a wide range of important animal models including mouse and zebrafish.
- A cDNA library comparison tool that compares two libraries (within a species or between species for which homologous gene data available) side by side with frequency of expression of each gene. This can help identify major differences or similarities among tissues.
- SAGE gene expression data for human retina and RPE through EyeSAGE, a collaboration with researchers at Duke University.
- Human eye disease database that includes all genes that have known associations with human eye-related disease with links to OMIM, Entrez, SNP and other databases.
- A database of mapped regions of the human genome containing as yet unidentified disease genes.
- EyeBrowse, an eye-centric whole genome browser that integrates all the data on cDNA and expression data as well as disease genes and candidate disease gene regions available for human, mouse, rat, chicken, zebrafish and several other species. It gives a rapid overview of expression patterns for particular genes; shows available clones; identifies variant transcripts; allows display of expression patterns for candidate genes in defined gene linkage loci. http://eyebrowse.cit.nih.gov/
NEIBank also includes yeast 2-hybrid libraries for adult mouse lens and retina and for adult human retina and RPE/choroid. These are being used to search for protein interactions involved in age related macular degeneration (AMD) as well as in normal lens and retina function and have been shared with several other groups.
NEIBank clones and sequence data have been used to construct cDNA microarrays for eye expressed genes from human and mouse and an oligo-based array for rabbit (at the University of Florida). A project to collect data for a similar microarray for studies of cone-cell function in the ground squirrel is underway.
The data from NEIBank are a rich source for gene discovery and for insights into species and tissue comparisons. Recent examples of this include the following:
- Discovery of many novel genes, including retbindin (an abundant, novel component of human retina), lengsin (a marker for terminal differentiation in lens cells) and PDGFD (a growth factor related to the PDGF and VEGF families).
- Identification of tissue and disease specific markers in human keratoconus cornea.
- Analysis of human pterygium showing characteristics of both conjunctiva and corneal epithelium, with makers for the former predominating. Potential targets for therapeutic intervention have been identified.
Crystallins and the Lens
Crystallins are the major structural proteins of the eye lens. They are involved in cataract, which is still a major cause of vision loss world wide, and are fascinating models for processes of molecular evolution and protein interaction. Recent observations have also implicated roles for ß - and γ -crystallins in retina and retinal pigment epithelium. For example they are (unexpectedly) detectable in Drusen, deposits associated with age-related macular degeneration.
We have long standing interests in the genomics, expression, structure and function and evolution of crystallins, including collaborative projects for x-ray and NMR structure analyses. Recent work has focused on the analysis of crystallin mutations in cataract and the effects of these mutations as well as differences amongst wild type proteins on stability and solubility. Crystallin mutants provide insights into the general problems of ‘protein-unfolding’ or’ amyloid’ diseases (such as Alzheimer’s disease). A knock out mouse model is providing new insights into the functional role of the highly conserved γ S-crystallin in the eye.
We have also used genomics, structural methods, yeast 2-hybrif and KO mice to discover and define the role of lengsin in the maturation of the lens. Lengsin has a fascinating evolutionary history and is a candidate for involvement in age-related cortical cataract.
Wistow, G. (1995) Molecular Biology and Evolution of Crystallins: Gene Recruitment and Multifunctional Proteins in the Eye Lens. Molecular Biology Intelligence Unit, R.G. Landes Co., Austin TX and Springer, New York. [see: http://neibank.nei.nih.gov/Publications.shtml.]
Wistow, G. (2006). The NEIBank Project for Ocular Genomics. Data-mining gene expression in human and rodent eye tissues. Prog. Ret. Eye Res. 25(1):43-77.
Wyatt, K., White, H.E., Wang, L., Bateman, O., Slingsby, C., Orlova, E. and Wistow, G. (2006). Lengsin is a survivor of an ancient family of class I glutamine synthetases re-engineered by evolution for a role in the vertebrate lens. Structure. 14:1823-34.
Nag, N., Peterson, K. Wyatt, K., Hess, S., Ray, S., Favor, J., Bogani, D., Lyon, M. and Wistow, G. (2007). The murine No3 cataract is the result of endogenous retroviral insertion in Cryge. Genomics 89:512-20.
Popp, M.P., Liu, L., Timmers, A., Esson, W.E. Shiroma, L., Meyers, C., Berceli, S., Tao, M., Wistow, G., Schultz, G.S. and Sherwood, B. (2007). Development of a Microarray Chip for Rabbit Ocular Research. Mol. Vis. 13:164-173.
Purkiss, A.G., Bateman, O.A., Wyatt, K., Wilmarth, P.A., David, L.L., Wistow, G.J. and Slingsby, C. (2007) Biophysical Properties of γC-Crystallin in Human and Mouse Eye Lens: The Role of Molecular Dipoles. J.Mol. Biol. 372:205-22.
Wyatt, K., Gao, C., Tsai, J.Y., Fariss, R., Ray, S. and Wistow, G. (2008). A Role for Lengsin, a Recruited Enzyme, in Terminal Differentiation in the Vertebrate Lens. J. Biol. Chem. 283:6607-15.
Aravind, P., Wistow, G., Sharma, Y. and Sankaranarayanan, R. (2008). Exploring the limits of sequence and structure in a variant ßγ-crystallin domain of AIM1. J. Mol. Biol 381:509-18.
Wistow, G., Peterson, K., Gao, J., Buchoff, P., Bowes Rickman, C., Ebright, J.N., Hauser, M. and Hoover, D. (2008). NEIBank: Genomics and Bioinformatics Resources for Vision Research. Mol. Vis.14:1327-37.
Simpanya, M.F., Wistow, G., Gao, J., David, L.L, Giblin, F.J, Mitton, K.P. (2008). Expressed sequence tag analysis of guinea pig (Cavia porcellus) eye tissues for NEIBank. Mol. Vis. 14:2413-2427.
Bernstein, S.L., Guo, Y., Peterson, K. and Wistow, G. (2009). Expressed sequence tag analysis of adult human Optic Nerve for NEIBank: Identification of cell type and tissue markers. BMC Neurosci (in press).
|Graeme J. Wistow Ph.D.
|Katherine Peterson Ph.D.||Staff Fellow||Petersonk@nei.nih.gov||301-402-3452|
|Jianguo Fan Ph.D.||Staff Fellowfirstname.lastname@example.org||301-402-4812|
|Sanghamitra Mishra Ph.D.||Visiting Fellowemail@example.com||301-402-5351|
|Yingwei Chen Ph.D.||Visiting Fellowfirstname.lastname@example.org||301-402-8649|
|Hoay-Shuen Len B.Sc.||IRTA email@example.com||301-402-5682|