Regulation of TIGR/MYOC gene expression in human trabecular meshwork cells.

Glucocorticoid (GC) treatment of human trabecular meshwork (HTM) cells produces delayed, progressive cellular and extracellular protein/glycoprotein inductions with characteristics matching those for intraocular pressure elevation with corticosteroid eyedrops. The cloning of the Trabecular Meshwork Inducible Glucocorticoid Response (TIGR) gene from this system has suggested possible environmental and genetic influences in relation to glaucoma mechanisms. As reported here, the major GC-induced increase of TIGR expression in HTM cells is reduced approximately 4-fold by basic fibroblast growth factor (bFGF, 100-1000 pM), with a somewhat smaller inhibition noted with the thyroid hormone triiodothyronine (T3, 100 nM). Such endogenous 'protective' factors could help balance stimulatory effects on TIGR gene expression from 'stress' and/or mechanical perturbations in the trabecular meshwork. TIGR coding region mutations affecting the gene's olfactomedin (OLF) homology domain may also perturb biosynthetic pathways and cellular homeostatic functions. Our recent studies have shown the OLF domain corresponds to a major translocational 'pause', an area where critical processes for normal TIGR biogenesis are expected to take place. Observations that Glu323Lys (and other mutations early in the OLF domain) altered the pattern of paused protein intermediates provide possible clues to previously unexplained pathogenetic mechanisms. HTM cell transfection studies using TIGR-green fluorescent protein (GFP) fusions showed increased and altered distribution of the expressed protein with constructs missing the OLF domain, an effect also found with the Pro370 Leu mutation for early-onset glaucoma. The data suggest an activation of stress/apoptotic pathways in HTM cells as a potential mechanism for environmental/genetic interactions in glaucoma pathogenesis.

A trabecular meshwork glucocorticoid response (TIGR) gene mutation affects translocational processing.

PURPOSE: To examine possible effects of the E323K mutation in the trabecular meshwork glucocorticoid response (TIGR) gene (also known as myocilin [MYOC]), using assays of translocational processing through the endoplasmic reticulum (ER). The E323K mutation was of particular interest, since the mutation shows a strong association with early onset open-angle glaucoma, but has a minimal predicted effect on protein structure. METHODS: Normal and mutant TIGR cDNA constructs were used to generate protein products in the presence of endoplasmic reticulum (ER) membranes, using an assay previously developed to detect alterations in the ER translocation function. "Paused" regions for potential protein modifications were defined by proteinase K (PK) sensitivity in the presence of ER membranes, with the ability to restart translocation when treated with EDTA. The effects of the E323K mutation were evaluated, as well as mutations located on either side of E323K (G246R, G364V, P370L) as the other mutations had substantial predicted structural changes in addition to clear disease associations. RESULTS: The native TIGR molecule was observed to have a paused region that corresponds to the region of highest olfactomedin (OLF) homology. The E323K mutation, located near the beginning of this region, dramatically altered the normal pattern of nascent proteins observed in the translocational pausing assay. A prominent band appeared with the E323K mutation, which could represent a new product or a marked enhancement of a faint band normally seen, approximately 3 kDa higher than the major paused band. The other TIGR mutants examined did not show this effect. CONCLUSIONS: The major translocational pause that starts near the beginning of the region of high OLF homology may help to explain the high frequency of glaucoma-associated mutations in this area. The observed effect of the E323K mutation on the products of translocational processing suggests a delay in the normal pausing process of TIGR biogenesis. This delay points to a potentially distinct pathogenic mechanism for E323K as compared with the other TIGR mutations so far evaluated.

Identification of novel Drosophila meiotic genes recovered in a P-element screen.

The segregation of homologous chromosomes from one another is the essence of meiosis. In many organisms, accurate segregation is ensured by the formation of chiasmata resulting from crossing over. Drosophila melanogaster females use this type of recombination-based system, but they also have mechanisms for segregating achiasmate chromosomes with high fidelity. We describe a P-element mutagenesis and screen in a sensitized genetic background to detect mutations that impair meiotic chromosome pairing, recombination, or segregation. Our screen identified two new recombination-deficient mutations: mei-P22, which fully eliminates meiotic recombination, and mei-P26, which decreases meiotic exchange by 70% in a polar fashion. We also recovered an unusual allele of the ncd gene, whose wild-type product is required for proper structure and function of the meiotic spindle. However, the screen yielded primarily mutants specifically defective in the segregation of achiasmate chromosomes. Although most of these are alleles of previously undescribed genes, five were in the known genes alphaTubulin67C, CycE, push, and Trl. The five mutations in known genes produce novel phenotypes for those genes.