Genetically altering the disease vector status of insects using recombinant DNA technologies is being considered as an alternative to eradication efforts. Manipulating the endogenous immune response of mosquitoes such as the temporal and special expression of antimicrobial peptides like cecropin may result in a refractory phenotype. Using transgenic technology a unique pattern of expression of cecropin A (cecA) in Anopheles gambiae was created such that cecA was expressed beginning 24 h after a blood meal in the posterior midgut. Two independent lines of transgenic An. gambiae were created using a piggyBac gene vector containing the An. gambiae cecA cDNA under the regulatory control of the Aedes aegypti carboxypeptidase promoter. Infection with Plasmodium berghei resulted in a 60% reduction in the number of oocysts in transgenic mosquitoes compared with nontransgenic mosquitoes. Manipulating the innate immune system of mosquitoes can negatively affect their capacity to serve as hosts for the development of disease-causing microbes.
DNA methylation at CpG residues is closely associated with a number of biological processes during vertebrate development. Unlike the vertebrates, however, several invertebrate species, including the Drosophila, do not have apparent DNA methylation in their genomes. Nor have there been reports on a DNA (5-cytosine) methyltransferase (CpG MTase) found in these invertebrates. We now present evidence for two CpG MTase-like proteins expressed in Drosophila cells. One of these, DmMTR1, is a protein containing peptide epitopes immunologically related to the conserved motifs I and IV in the catalytic domain of the mammalian dnmt1. DmMTR1 has an apparent molecular mass of 220 kDa and, similar to mammalian dnmt1, it also interacts in vivo with the proliferating cell nuclear antigen. During interphase of the syncytial Drosophila embryos, the DmMTR1 molecules are located outside the nuclei, as is dnmt1 in the mouse blastocyst. However, DmMTR1 appears to be rapidly transported into, and then out of the nuclei again, as the embryos undergo mitotic waves. Immunofluorescent data indicate that DmMTR1 molecules ''paint'' the whole set of condensed Drosophila chromosomes throughout the mitotic phase, suggesting they may play an essential function in the cell-cycle regulated condensation of the Drosophila chromosomes. Through search in the genomic database, we also have identified a Drosophila polypeptide, DmMT2, that exhibits high sequence homology to the mammalian dnmt2 and the yeast CpG MTase homolog pmt1. The expression of DmMT2 appears to be developmentally regulated. We discuss the evolutionary and functional implications of the discovery of these two Drosophila proteins related to mammalian CpG MTases.epitope detection ͉ early embryo ͉ cell cycle ͉ chromatin structure ͉ database
A multiple protein-DNA complex formed at a human ␣-globin locus-specific regulatory element, HS-40, confers appropriate developmental expression pattern on human embryonic -globin promoter activity in humans and transgenic mice. We show here that introduction of a 1-bp mutation in an NF-E2͞AP1 sequence motif converts HS-40 into an erythroid-specific locus-control region. Cis-linkage with this locus-control region, in contrast to the wild-type HS-40, allows erythroid lineage-specific derepression of the silenced human -globin promoter in fetal and adult transgenic mice. Furthermore, -globin promoter activities in adult mice increase in proportion to the number of integrated DNA fragments even at 19 copies͞genome. The mutant HS-40 in conjunction with human -globin promoter thus can be used to direct position-independent and copy number-dependent expression of transgenes in adult erythroid cells. The data also supports a model in which competitive DNA binding of different members of the NF-E2͞AP1 transcription factor family modulates the developmental stage specificity of an erythroid enhancer. Feasibility to reswitch on embryonic͞fetal globin genes through the manipulation of nuclear factor binding at a single regulatory DNA motif is discussed.
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