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. RNA interference (RNAi) was used to screen approximately 5,400 Drosophila double-stranded RNAs, corresponding to approximately 38 percent of Drosophila genes, for genes that affect the structure of the embryonic nervous system. Loss of function mutant phenotypes for 52 genes were found that markedly affect the structure of the developing nervous system. Genes that are required for the development of the nervous system were found that encode transcription factors, protein kinases, signaling proteins, motor proteins, and proteins with unknown functions. 2. vnd/NK-2 Homeobox Gene. vnd/NK-2 homeodomain affinity column chromatography was used to purify Drosophila DNA fragments bound by a protein fragment that contains the vnd/NK-2 homeodomain with some flanking amino acid residues. The purified genomic DNA fragments were cloned. Seventy-seven DNA clones were sequenced, which revealed 42 vnd/NK-2 homeodomain binding loci. Most loci were within upstream or intron regions, especially first introns. Nineteen of the cloned DNA fragments correspond to part of the first intron of the Beadex gene, which contains 5 vnd/NK-2 homeodomain core consensus binding sites, 5'-AAGTG. Four of the vnd/NK-2 homeodomain binding sites in the first intron of the Beadex gene were analyzed by mobility-shift assay, DNase I footprinting, methylation interference, and ethylation interference. Three high affinity and one weaker affinity vnd/NK-2 homeodomain binding sites were found. Similar studies revealed two high affinity and five weaker affinity vnd/NK-2 binding sites in the DNA fragment between -385 and -675 bp from the transcription start site of the vnd/NK-2 gene. HSQC proton-deuterium exchange spectra were obtained for the wild-type and a Y54M mutant vnd/NK-2 homeodomain bound to a sixteen base-pair fragment of DNA that contains the consensus core vnd/NK-2 binding site, CAAGTG. The helix III average amide proton lifetimes of the wild-type vnd/NK-2 homeodomain protein was nine days; whereas, the value for the Y54M mutant homeodomain was 3.7 hours. 3. Longitudinals Lacking (lola) Gene Null mutations in lola result in Drosophila embryos that lack longitudinal nerve fibers in the ventral nerve cord that carry information from the body to or from the brain. Hypomorphic mutations result in embryos with mistakes in axon pathfinding of longitudinal neurons. lola encodes C2H2 zinc finger protein transcription factors. Many species of lola mRNA are formed by alternative splicing. Seventeen isoforms of lola protein encoded by different species of mRNA have an invariant, constant N-terminal amino acid sequence, but each isoform differs in the amino acid sequence of the zinc fingers, which are in the C-terminal portion of the proteins. Since lola proteins are thought to function as transcription factors, each of the 17 isoforms of lola protein would be expected to bind to different nucleotide sequences in DNA. Double-stranded RNAs encoding the variable C-terminal regions of the 17 isoforms of lola mRNA were synthesized and each RNA preparation was injected into early Drosophila embryos to decrease the level of the corresponding species of lola mRNA by RNA interference. Embryos were incubated to allow development to proceed. Then RNA from each set of injected embryos was isolated, labeled, and hybridized to Affymetrix Drosophila nucleic acid microarray chips. The results showed that inactivation of different isoforms of lola mRNA by RNA interference resulted in activation or repression of different sets of genes. 4. Genes Expressed by Undifferentiated versus Differentiated Drosophila Neural Cells A method was found to shift cells from a Drosophila neural cell line from a relatively undifferentiated state to a differentiated state. RNA was prepared from populations of undifferentiated and differentiated neural cells. Nucleic acid microarray anaylsis resulted in the identification of approximately 250 genes whose rates of expression either increased or decreased due to shifting neural cells from an undifferentiated to a differentiated state.