Cell Recognition And Synapse Formation

Heart, Lung, And Blood Institute

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Abstract

1. Genes that Affect the Assembly of the Nervous System in Drosophila Embryos. Screening Gene Function by RNA Interference. Methods were devised for rapidly synthesizing double-stranded RNA preparations, injection of embryos with dsRNA, incubation, fixation, and immunostaining many sets of embryos. Thus far, 5800 dsRNA preparations were synthesized, and about 1000 dsRNA preparations were injected into embryos and screened. Seven genes were found that yield mutant phenotypes that affect the structure of the nervous system. 2. vnd/NK-2 Homeobox Gene. A. Neuroblast pattern formation. vnd/NK-2 protein was detected in eleven neuroblasts per hemisegment of the ventral nerve cord of Drosophila embryos as follows: 1-1, 1-2, 2-1, 3-1, MP-2, 4-1, 5-1, 5-2, 6-1, 7-1, and 7-2. Fragments of DNA from the 5'-flanking region of the vnd/NK-2 were inserted upstream of an enhancerless Beta-Galactosidase gene in P-elements and the constructs were used to generate transgenic fly lines. Antibodies directed against Vnd/NK-2 and Beta-Galactosidase proteins then were used in double-label experiments to correlate the expression of Vnd/NK-2 and Beta-Galactosidase proteins in identified neuroblasts. Three regions of DNA were found to be involved in activation of the vnd/NK-2 gene. DNA region A, which corresponds to the -4.0 to -2.8 kb fragment of DNA from the 5'-flanking region of the vnd/NK-2 gene, contains strong enhancers for vnd/NK-2 gene expression in ten neuroblasts. DNA region B (-5.3 to -4.0 kb) contains moderately strong enhancers for vnd/NK-2 gene expression in neuroblasts 1-2 and 3-1, and weaker expression in 2-1 and 4-1, and hypothesized DNA region C, whose location was not identified, contains enhancers that activate vnd/NK-2 gene expression only in neuroblast 7-2. These results show that at least three regions of DNA regulate the expression of the vnd/NK-2 gene, that the vnd/NK-2 gene can be activated in different ways in different neuroblasts, and that the pattern of vnd/NK-2 gene expression in neuroblasts of the ventral nerve cord is the sum of partial patterns. B. Backbone Dynamics for the Wild Type and a Double H52R/T56W Mutant of the vnd/NK-2 Homeodomain. In collaboration with James Ferretti and his colleagues the protein backbone dynamics of the wild type and H52R/T56W double mutant vnd/NK-2 homeodomain in both the free and DNA-bound states were determined by NMR spectroscopy. An important effect of the conservative H52R mutation in position 52 of the homeodomain was shown to be stabilization of the tertiary structure of the homeodomain by the formation of a salt bridge with E17. The T56W replacement was shown to be critical to the elongation and stabilization of a Helix III of the homeodomain.