We used a recently developed method to produce mutant alleles of five endogenous Drosophila genes, including the homolog of the p53 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-SceI endonuclease generates extrachromosomal linear DNA molecules in vivo. These molecules undergo homologous recombination with the corresponding chromosomal locus to generate targeted alterations of the host genome. The results address several questions about the general utility of this technique. We show that genes not near telomeres can be efficiently targeted; that no knowledge of the mutant phenotype is needed for targeting; and that insertional mutations and allelic substitutions can be easily produced. We recently described a method for targeted modification of the Drosophila genome through homologous recombination (HR). The ability to engineer specific changes into the genome is a highly useful adjunct to genetic investigation in any organism, but especially in a species with a completely determined genome sequence such as Drosophila melanogaster (Adams et al. 2000). This procedure had, until recently, been lacking in Drosophila. In our previous reports, we targeted two genes, rescuing a mutant allele of the first and generating a mutant allele of the second (Rong and Golic 2000, 2001). At this time there is a clear need for demonstrations of the generality of this technique. That is, can a variety of genes in different locations be modified by HR? There is also a need for the development of techniques that can produce mutant alleles of target genes. In this work, we address both issues by applying new methods for targeted mutagenesis of five autosomal genes.A variety of schemes has been produced for targeted gene modification in organisms such as yeast and mice (Rothstein 1991;Muller 1999). However, these methods rely critically on the ability to culture single cells and carry out selections for rare events. Because the targeting technique we use occurs in whole animals, we devised variant approaches for introducing mutations into chromosomal genes. The methods are mechanistically similar to those developed for yeast and mouse, but procedurally quite different, as they do not rely on chemical selections. Instead, at each step, arbitrary genetic markers with simple visible phenotypes are used for genetic screening. In our previous experiments, the frequency of targeted gene modification through HR varied from ∼ 1 in 500 gametes to ∼ 1 in 30,000 gametes. These frequencies are easily within reach of the power provided by genetic screening.To perform gene targeting in flies we use transgenic expression of FLP site-specific recombinase and I-SceI endonuclease to generate a targeting donor molecule in vivo. This donor molecule is derived from a third transgenic element: a P element carrying DNA homologous to the target locus. Within the P element, FLP Recombinase Target sites (FRTs) flank a segment of DNA from the target locus, and an I-SceI recognition site is placed within the target-homolog...
mutant mice show compulsive behavior similar to trichotillomania, a human obsessive-compulsive-spectrum disorder. The only lineage-labeled cells in the brains of mice are microglia, suggesting that defective microglia caused the disorder. What is the source of the microglia? It has been posited that all microglia progenitors arise at embryonic day (E) 7.5 during yolk sac hematopoiesis, and colonize the brain at E9.5. In contrast, we show the presence of two microglia subpopulations: canonical, non- microglia and microglia. Unlike non- microglia, microglia progenitors appear to be generated during the second wave of yolk sac hematopoiesis, then detected in the aorto-gonad-mesonephros (AGM) and fetal liver, where they are greatly expanded, prior to infiltrating the E12.5 brain. Further, we demonstrate that hematopoietic progenitor cells taken from fetal liver are competent to give rise to microglia Although the two microglial subpopulations are very similar molecularly, and in their response to brain injury and participation in synaptic pruning, they show distinct brain distributions which might contribute to pathological specificity. Non- microglia significantly outnumber microglia, but they cannot compensate for the loss of function in microglia, suggesting further crucial differences between the two subpopulations.
Telomere loss was produced during development of Drosophila melanogaster by breakage of an induced dicentric chromosome. The most prominent outcome of this event is cell death through Chk2 and Chk1 controlled p53-dependent apoptotic pathways. A third p53-independent apoptotic pathway is additionally utilized when telomere loss is accompanied by the generation of significant aneuploidy. In spite of these three lines of defense against the proliferation of cells with damaged genomes a small fraction of cells that have lost a telomere escape apoptosis and divide repeatedly. Evasion of apoptosis is accompanied by the accumulation of karyotypic abnormalites that often typify cancer cells, including end-to-end chromosome fusions, anaphase bridges, aneuploidy, and polyploidy. There was clear evidence of bridge-breakage-fusion cycles, and surprisingly, chromosome segments without centromeres could persist and accumulate to highcopy number. Cells manifesting these signs of genomic instability were much more frequent when the apoptotic mechanisms were crippled. We conclude that loss of a single telomere is sufficient to generate at least two phenotypes of early cancer cells: genomic instability that involves multiple chromosomes and aneuploidy. This aneuploidy may facilitate the continued escape of such cells from the normal checkpoint mechanisms.
The mechanisms that cells use to monitor telomere integrity, and the array of responses that may be induced, are not fully defined. To date there have been no studies in animals describing the ability of cells to survive and contribute to adult organs following telomere loss. We developed assays to monitor the ability of somatic cells to proliferate and differentiate after telomere loss. Here we show that p53 and Chk2 limit the growth and differentiation of cells that lose a telomere. Furthermore, our results show that two copies of the genes encoding p53 and Chk2 are required for the cell to mount a rapid wildtype response to a missing telomere. Finally, our results show that, while Chk2 functions by activating the p53-dependent apoptotic cascade, Chk2 also functions independently of p53 to limit survival. In spite of these mechanisms to eliminate cells that have lost a telomere, we find that such cells can make a substantial contribution to differentiated adult tissues.
Previously, we observed that heterochromatic 4 and Y chromosomes that had experienced breakage in the male germline were frequently transmitted to progeny. Their behavior suggested that they carried functional telomeres. Here we show that efficient healing by de novo telomere addition is not unique to heterochromatic breaks.
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