Sox9 is a transcription factor required for cartilage formation and testis determination in mammals. We have cloned from zebrafish two sox9 genes, termed sox9a and sox9b. Gene phylogenies showed that both genes are orthologous to tetrapod SOX9 genes. Genetic mapping showed that these two loci reside on chromosome segments that were apparently duplicated in a large-scale genomic duplication event in ray fin fish phylogeny. Both Sox9a and Sox9b proteins bind to the HMG consensus DNA sequences in vitro. We tested different domains for transactivation potential and identified a potential activation domain located in the middle of both Sox9a and Sox9b. During embryogenesis, sox9a and sox9b expression patterns are distinct but overlap in some regions of the brain, head skeleton, and fins. Expression of sox9a/b correlates well with that of col2a1 in chondrogenic elements. In the adults, sox9a is expressed in many tissues including brain, muscle, fin, and testis, whereas sox9b expression is restricted to previtellogenic oocytes of the ovary. This expression pattern predicts that sox9a and sox9b may have unique functions in some specific tissues during development. The role of gene duplication for the evolution of developmental gene function is discussed.
Opacities in the crystalline lens of eye appear with high frequency in the general population. Dominantly inherited cataracts with differing clinical features were found in two families carrying different point mutations in the gene encoding lens water channel protein AQP0 (major intrinsic protein, MIP). Families with E134G have a uni-lamellar cataract which is stable after birth, whereas families with T138R have multi-focal opacities which increase throughout life. To establish pathophysiological relevance of cataract formation, the Xenopus laevis oocyte expression system was employed to evaluate functional defects in the mutant proteins, E134G and T138R. Both substitutions cause loss of membrane water channel activity due to impaired trafficking of the mutant proteins to the oocyte plasma membrane. Although missense mutations in AQP1 and AQP2 proteins are known to result in recessive traits in vivo and in vitro, when E134G or T138R are co-expressed with wild-type AQP0 protein, the mutant proteins exhibit dominant negative behaviour. To our knowledge, these studies represent the first in vitro demonstration of functionally defective AQP0 protein from humans with congenital cataracts. Moreover, these observations predict that less severe defects in the AQP0 protein may contribute to lens opacity in patients with common, less fulminant forms of cataracts.
In Opj, an inherited cataract in mice, opacity is associated with a mutation in Crygs, the gene for ␥S-crystallin, the first mutation to be associated with this gene. A single base change causes replacement of Phe-9, a key hydrophobic residue in the core of the N-terminal domain, by serine. Despite this highly non-conservative change, mutant protein folds normally at low temperature. However, it exhibits a marked, concentration-dependent decrease in solubility, associated with loss of secondary structure, at close to physiological temperatures. This is reminiscent of processes thought to occur in human senile cataracts in which normal proteins become altered and aggregate. The Opj cataract is progressive and more severe in Opj/Opj than in Opj/؉. Lens histology shows that whereas fiber cell morphology in Opj/؉ mice is essentially normal, in Opj/Opj, cortical fiber cell morphology and the loss of maturing fiber cell nuclei are both severely disrupted from early stages. This may indicate a loss of function of ␥S-crystallin which would be consistent with ideas that members of the ␥-crystallin superfamily may have roles associated with maintenance of cytoarchitecture.
Age-related macular degeneration (AMD) is a major cause of vision loss. It is associated with development of characteristic plaque-like deposits (soft drusen) in Bruch’s membrane basal to the retinal pigment epithelium (RPE). A sequence variant (Y402H) in short consensus repeat domain 7 (SCR7) of complement factor H (CFH) is associated with risk for “dry” AMD. We asked whether the eye-targeting of this disease might be related to specific interactions of CFH SCR7 with proteins expressed in the aging human RPE/choroid that could contribute to protein deposition in drusen. Yeast 2-hybrid (Y2H) screens of a retinal pigment epithelium/choroid library derived from aged donors using CFH SCR7 baits detected an interaction with EFEMP1/Fibulin 3 (Fib3), which is the locus for an inherited macular degeneration and also accumulates basal to macular RPE in AMD. The CFH/Fib3 interaction was validated by co-immunoprecipitation of native proteins. Quantitative Y2H and ELISA assays with different recombinant protein constructs both demonstrated higher affinity for Fib3 for the disease-related CFH 402H variant. Immuno-labeling revealed colocalization of CFH and Fib3 in globular deposits within cholesterol-rich domains in soft drusen in two AMD donors homozygous for CFH 402H (H/H). This pattern of labeling was quite distinct from those seen in examples of eyes with Y/Y and H/Y genotypes. The CFH 402H/Fib3 interaction could contribute to the development of pathological aggregates in soft drusen in some patients and as such might provide a target for therapeutic intervention in some forms of AMD.
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