The Optx2 homeobox gene is expressed in early precursors of the eye and activates retina-specific genes

Vertebrate eye development begins at the gastrula stage, when a region known as the eye field acquires the capacity to generate retina and lens. Optx2, a homeobox gene of the sine oculis-Six family, is selectively expressed in this early eye field and later in the lens placode and optic vesicle. The distal and ventral portion of the optic vesicle are fated to become the retina and optic nerve, whereas the dorsal portion eventually loses its neural characteristics and activates the synthesis of melanin, forming the retinal pigment epithelium. Optx2 expression is turned off in the future pigment epithelium but remains expressed in the proliferating neuroblasts and differentiating cells of the neural retina. When an Optx2-expressing plasmid is transfected into embryonic or mature chicken pigment epithelial cells, these cells adopt a neuronal morphology and express markers characteristic of developing neural retina and photoreceptors. One explanation of these results is that Optx2 functions as a determinant of retinal precursors and that it has induced the transdifferentiation of pigment epithelium into retinal neurons and photoreceptors. We also have isolated optix, a Drosophila gene that is the closest insect homologue of Optx2 and Six3. Optix is expressed during early development of the fly head and eye primordia.

Organization and expression of canine olfactory receptor genes.

Four members of the canine olfactory receptor gene family were characterized. The predicted proteins shared 40-64% identity with previously identified olfactory receptors. The four subfamilies identified in Southern hybridization experiments had as few as 2 and as many as 20 members. All four genes were expressed exclusively in olfactory epithelium. Expression of multiple members of the larger subfamilies was detected, suggesting that most if not all of the cross-hybridizing bands in genomic Southern blots represented actively transcribed olfactory receptor genes. Analysis of large DNA fragments using Southern blots of pulsed-field gels indicated that subfamily members were clustered together, and that two of the subfamilies were closely linked in the dog genome. Analysis of the four olfactory receptor gene subfamilies in 26 breeds of dog provided evidence that the number of genes per subfamily was stable in spite of differential selection on the basis of olfactory acuity in scent hounds, sight hounds, and toy breeds.

Engineering actin-resistant human DNase I for treatment of cystic fibrosis.

Human deoxyribonuclease I (DNase I), an enzyme recently approved for treatment of cystic fibrosis (CF), has been engineered to create two classes of mutants: actin-resistant variants, which still catalyze DNA hydrolysis but are no longer inhibited by globular actin (G-actin) and active site variants, which no longer catalyze DNA hydrolysis but still bind G-actin. Actin-resistant variants with the least affinity for actin, as measured by an actin binding ELISA and actin inhibition of [33P] DNA hydrolysis, resulted from the introduction of charged, aliphatic, or aromatic residues at Ala-114 or charged residues on the central hydrophobic actin binding interface at Tyr-65 or Val-67. In CF sputum, the actin-resistant variants D53R, Y65A, Y65R, or V67K were 10-to 50-fold more potent than wild type in reducing viscoelasticity as determined in sputum compaction assays. The reduced viscoelasticity correlated with reduced DNA length as measured by pulsed-field gel electrophoresis. In contrast, the active site variants H252A or H134A had no effect on altering either viscoelasticity or DNA length in CF sputum. The data from both the active site and actin-resistant variants demonstrate that the reduction of viscoelasticity by DNase I results from DNA hydrolysis and not from depolymerization of filamentous actin (F-actin). The increased potency of the actin-resistant variants indicates that G-actin is a significant inhibitor of DNase I in CF sputum. These results further suggest that actin-resistant DNase I variants may have improved efficacy in CF patients.