Transparency and optical function of the human lens depend to a great extent on the biochemical properties of the lens-specific crystallins. Normally synthesized in a highly regulated fashion during lens cell differentiation, the crystallins create the refractive gradient necessary for optical function. Currently, the molecular mechanisms regulating expression of the crystallin genes are largely unknown. However, some congenital cataracts may result from an alteration in levels of gene expression and biosynthesis of the crystallin proteins. Using regulatory sequences of bovine gamma-crystallin genes, the primary objective of this study is to identify and characterize nuclear regulatory proteins that modulate the transcription of gamma-crystallin genes. Comparisons of the nucleotide sequences for the promoter regions of bovine gamma-C and gamma-D crystallin genes to analogous sequences reported for other gamma-crystallin genes show significant homology with transcriptional regulatory sequences identified in mouse and human genes. These bovine regulatory sequences will be used to identify and characterize nuclear protein factors that bind to them in mobility-shift assays or Southwestern blots. Nuclear extracts from both lens and non- lens tissues will be examined to identify factors that may be involved in tissue-specific regulation of gamma-crystallin genes. Specific regions of interaction between nuclear proteins and substrate DNA sequences will be identified by DNase footprinting and methylation protection assays. Nucleotides critical to factor binding will be identified by both deletion analysis and site-directed mutagenesis. The effect of mutations within the controlling regions will also be determined in transient expression assays. Expression libraries will then be screened to identify cDNA clones for transcriptional regulatory proteins. Transcriptional regulatory proteins identified by the above methods will be further characterized and partially purified using affinity chromatography. Development of a cell-free, in vitro assay system that may exhibit tissue- specific transcription of crystallin genes will be explored.
| Norledge, B V; Hay, R E; Bateman, O A et al. (1997) Towards a molecular understanding of phase separation in the lens: a comparison of the X-ray structures of two high Tc gamma-crystallins, gammaE and gammaF, with two low Tc gamma-crystallins, gammaB and gammaD. Exp Eye Res 65:609-30 |
| Hay, R E; Andley, U P; Petrash, J M (1994) Expression of recombinant bovine gamma B-, gamma C- and gamma D-crystallins and correlation with native proteins. Exp Eye Res 58:573-84 |
