The dam DNA methyltransferase gene from Escherichia coli was introduced into Drosophila in order to probe chromatin structure in vivo. Expression of the gene caused no visible defects or developmental delay even at high levels of active methylase. About half of each target site was found to be methylated in vivo, apparently reflecting a general property of chromatin packaged in nucleosomes. Although site-specific differences were detected, most euchromatic and heterochromatic sites showed comparable degrees of methylation, at least at high methylase levels. Methylase accessibility of a lacZ reporter gene subject to position-effect variegation throughout development was only slightly reduced, consistent with studies of chromatin accessibility in vitro. Silencing of lacZ during development differed from silencing of an adjacent white eye pigment reporter gene in the adult, as though chromatin structure can undergo dynamic alterations during development.
The subject of this study is the organization of essential genes in the 2 map-unit unc-22 IV region of the Caenorhabditis elegans genome. With the goal of achieving mutational saturation of essential genes in this region, 6491 chromosomes mutagenized with ethyl methanesulfonate (EMS) were screened for the presence of lethal mutations in the unc-22 region. The genetic analysis of 21 lethal mutations in the unc-22 region resulted in the identification of 6 new essential genes, making a total of 36 characterized to date. A minimum of 49 essential genes are estimated to lie in this region. A set of seven formaldehyde-induced deficiencies of unc-22 and surrounding loci were isolated to facilitate the positioning of essential genes on the genetic and physical maps. In order to study essential genes at the molecular level, our approach was to rescue lethal mutations by the injection of genomic DNA in the form of cosmid clones into the germ-line of balanced heterozygotes carrying a lethal mutation. The cosmid clones containing let-56 and let-653 were identified by this method.
PRAT (phosphoribosylamidotransferase; E.C. 2.4.2.14) catalyzes the first reaction in de novo purine nucleotide biosynthesis. In Drosophila melanogaster, the Prat and Prat2 genes are both highly conserved with PRAT sequences from prokaryotes and eukaryotes. However, Prat2 organization and expression during development is different from Prat. We used RNA interference (RNAi) to knock down expression of both Prat and Prat2 to investigate their functions. Using the GAL4-UAS system, Prat RNAi driven by Act5c-GAL4 or tubP-GAL4 causes variable pupal lethality (48-100%) and 50% female sterility, depending on the transgenic strains and drivers used. This observation agrees with the phenotype previously observed for Prat EMSinduced mutations. Prat2 RNAi driven by Act5C-GAL4 or tubP-GAL4 also results in variable pupal lethality (61-93%) with the different transgenic strains, showing that Prat2 is essential for fly development. However, Prat2 RNAi-induced arrested pupae have a head eversion defect reminiscent of the ''cryptocephal'' phenotype, whereas Prat RNAi-induced arrested pupae die later as pharate adults. We conclude that Prat2 is required during the prepupal stage while Prat is more important for the pupal stage. In addition, Prat and Prat2 double RNAi results in more severe pupal lethal phenotypes, suggesting that Prat and Prat2 have partially additive functions during Drosophila metamorphosis.
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