DNA Replication Control Is Linked to Genomic Positioning of Control Regions in Escherichia coli

Chromosome replication in Escherichia coli is in part controlled by three non-coding genomic sequences, DARS1, DARS2, and datA that modulate the activity of the initiator protein DnaA. The relative distance from oriC to the non-coding regions are conserved among E. coli species, despite large variations in genome size. Here we use a combination of i) site directed translocation of each region to new positions on the bacterial chromosome and ii) random transposon mediated translocation followed by culture evolution, to show genetic evidence for the importance of position. Here we provide evidence that the genomic locations of these regulatory sequences are important for cell cycle control and bacterial fitness. In addition, our work shows that the functionally redundant DARS1 and DARS2 regions play different roles in replication control. DARS1 is mainly involved in maintaining the origin concentration, whether DARS2 is also involved in maintaining single cell synchrony.     

A NuRD Complex from Xenopus laevis Eggs Is Essential for DNA Replication during Early Embryogenesis

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Topoisomerase II Is Required for the Proper Separation of Heterochromatic Regions during Drosophila melanogaster Female Meiosis

Heterochromatic homology ensures the segregation of achiasmate chromosomes during meiosis I in Drosophila melanogaster females, perhaps as a consequence of the heterochromatic threads that connect achiasmate homologs during prometaphase I. Here, we ask how these threads, and other possible heterochromatic entanglements, are resolved prior to anaphase I. We show that the knockdown of Topoisomerase II (Top2) by RNAi in the later stages of meiosis results in a specific defect in the separation of heterochromatic regions after spindle assembly. In Top2 RNAi-expressing oocytes, heterochromatic regions of both achiasmate and chiasmate chromosomes often failed to separate during prometaphase I and metaphase I. Heterochromatic regions were stretched into long, abnormal projections with centromeres localizing near the tips of the projections in some oocytes. Despite these anomalies, we observed bipolar spindles in most Top2 RNAi-expressing oocytes, although the obligately achiasmate 4^th chromosomes exhibited a near complete failure to move toward the spindle poles during prometaphase I. Both achiasmate and chiasmate chromosomes displayed defects in biorientation. Given that euchromatic regions separate much earlier in prophase, no defects were expected or observed in the ability of euchromatic regions to separate during late prophase upon knockdown of Top2 at mid-prophase. Finally, embryos from Top2 RNAi-expressing females frequently failed to initiate mitotic divisions. These data suggest both that Topoisomerase II is involved in the resolution of heterochromatic DNA entanglements during meiosis I and that these entanglements must be resolved in order to complete meiosis.     

Global Assessment of Genomic Regions Required for Growth in Mycobacterium tuberculosis

Identifying genomic elements required for viability is central to our understanding of the basic physiology of bacterial pathogens. Recently, the combination of high-density mutagenesis and deep sequencing has allowed for the identification of required and conditionally required genes in many bacteria. Genes, however, make up only a part of the complex genomes of important bacterial pathogens. Here, we use an unbiased analysis to comprehensively identify genomic regions, including genes, domains, and intergenic elements, required for the optimal growth of Mycobacterium tuberculosis, a major global health pathogen. We found that several proteins jointly contain both domains required for optimal growth and domains that are dispensable. In addition, many non-coding regions, including regulatory elements and non-coding RNAs, are critical for mycobacterial growth. Our analysis shows that the genetic requirements for growth are more complex than can be appreciated using gene-centric analysis.     

Metabolome Analysis of Drosophila melanogaster during Embryogenesis

The Drosophila melanogaster embryo has been widely utilized as a model for genetics and developmental biology due to its small size, short generation time, and large brood size. Information on embryonic metabolism during developmental progression is important for further understanding the mechanisms of Drosophila embryogenesis. Therefore, the aim of this study is to assess the changes in embryos’ metabolome that occur at different stages of the Drosophila embryonic development. Time course samples of Drosophila embryos were subjected to GC/MS-based metabolome analysis for profiling of low molecular weight hydrophilic metabolites, including sugars, amino acids, and organic acids. The results showed that the metabolic profiles of Drosophila embryo varied during the course of development and there was a strong correlation between the metabolome and different embryonic stages. Using the metabolome information, we were able to establish a prediction model for developmental stages of embryos starting from their high-resolution quantitative metabolite composition. Among the important metabolites revealed from our model, we suggest that different amino acids appear to play distinct roles in different developmental stages and an appropriate balance in trehalose-glucose ratio is crucial to supply the carbohydrate source for the development of Drosophila embryo.     

Singlet Oxygen-Mediated Oxidation during UVA Radiation Alters the Dynamic of Genomic DNA Replication

UVA radiation (320–400 nm) is a major environmental agent that can exert its deleterious action on living organisms through absorption of the UVA photons by endogenous or exogenous photosensitizers. This leads to the production of reactive oxygen species (ROS), such as singlet oxygen (¹O₂) and hydrogen peroxide (H₂O₂), which in turn can modify reversibly or irreversibly biomolecules, such as lipids, proteins and nucleic acids. We have previously reported that UVA-induced ROS strongly inhibit DNA replication in a dose-dependent manner, but independently of the cell cycle checkpoints activation. Here, we report that the production of ¹O₂ by UVA radiation leads to a transient inhibition of replication fork velocity, a transient decrease in the dNTP pool, a quickly reversible GSH-dependent oxidation of the RRM1 subunit of ribonucleotide reductase and sustained inhibition of origin firing. The time of recovery post irradiation for each of these events can last from few minutes (reduction of oxidized RRM1) to several hours (replication fork velocity and origin firing). The quenching of ¹O₂ by sodium azide prevents the delay of DNA replication, the decrease in the dNTP pool and the oxidation of RRM1, while inhibition of Chk1 does not prevent the inhibition of origin firing. Although the molecular mechanism remains elusive, our data demonstrate that the dynamic of replication is altered by UVA photosensitization of vitamins via the production of singlet oxygen.     

Arrest of Micronuclear DNA Replication during Genomic Exclusion in Tetrahymena Produces Haploid Strains

Diploid cells of Tetrahymena thermophila were crossed to strain A*V, whose micronucleus is defective, to induce the unilateral transfer of gametic nuclei from the diploid cells to the A*V cells (round I of genomic exclusion). These haploid nuclei presumably undergo one endomitotic cycle and then become diploid with a G1 (2C) DNA content. However, further DNA replication from 2C to 4C was transiently arrested until the pairs separated. When endomitosis was blocked by treatment with cycloheximide during 6-8 hours of conjugation, the exconjugants of round I of genomic exclusion remained haploid. Competence for diploidization is apparently limited to some period of time after nuclear transfer. Blocking of diploidization during round I of genomic exclusion can be used as an efficient way to induce haploid strains in Tetrahymena.     

DNA Replication is Required for Tissue-Specific Enzyme Development in Ascidian Embryos

Tyrosinase which is a tissue-specific enzyme in the pigment cells of the brain of the ascidian embryo, is thought to be synthesized with activation of appropriate genes, and the enzyme synthesis begins at the early tailbud stage. If embryos at early cleavage stages up to the 64-cell stage are continuously treated with aphidicolin (a specific inhibitor of DNA synthesis), cleavage of the embryos is arrested and they do not differentiate the enzyme. However, the early gastrulae and embryos at later stages that have been permanently arrested with aphidicolin do produce the enzyme. Alkaline phosphatase, a tissue-specific enzyme of the endodermal cells, has been shown to be synthesized by a preformed maternal mRNA and is first detected histochemically at the late gastrula stage. If embryos at early cleavage stages up to the 16-cell stage are prevented from undergoing further divisions with aphidicolin, the arrested embryos do not form the enzyme. However, embryos at the 32-cell and later stages that have been permanently arrested with aphidicolin are able to differentiate the enzyme activity. These results suggest that several DNA replications are required for the histospecific enzyme development in ascidian embryos.     

Replication Stress Shapes a Protective Chromatin Environment across Fragile Genomic Regions

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Differential Replication of DNA Sequences in Drosophila Chromosomes

During the DNA replications involved in polyploidization orpolytenization in Drosophila cells, not all DNA sequences arereplicated to the same extent. Cytological studies have demonstratedthat certain chromosome regions, such as the a-heterochromatin,are in some cells under-replicated and in other cells not replicatedat all. Similarly, such DNA fractions as the highly repeatedsatellite DNAs are also under- or non-replicated in polyploidand polytene cells. The genes for rRNA in polytene cells replicateone to three rounds less than the euchromatic DNA and are capableof differential synthesis to compensate for deficiencies. Thedifferential replication of DNA sequences indicates that thereis regulation of DNA replication at a level intermediate betweenthe replicon and the entire genome. Chromosomes of terminallydifferentiated polyploid or polytene cells have several domainsof DNA sequences, which in replication are controlled as units.This paper, which reviews the literature on differential replicationof DNA in Drosophila, discusses possible controls over thisprocess.     

Functional Genomic Identification of Genes Required for Male Gonadal Differentiation in Caenorhabditis elegans

The Caenorhabditis elegans somatic gonad develops from a four-cell primordium into a mature organ that differs dramatically between the sexes in overall morphology (two arms in hermaphrodites and one in males) and in the cell types comprising it. Gonadal development in C. elegans is well studied, but regulation of sexual differentiation, especially later in gonadal development, remains poorly elucidated. To identify genes involved in this process, we performed a genome-wide RNAi screen using sex-specifically expressed gonadal GFP reporters. This screen identified several phenotypic classes, including ∼70 genes whose depletion feminized male gonadal cells. Among the genes required for male cell fate specification are Wnt/β-catenin pathway members, cell cycle regulators, and genes required for mitotic spindle function and cytokinesis. We find that a Wnt/β-catenin pathway independent of extracellular Wnt ligand is essential for asymmetric cell divisions and male differentiation during gonadal development in larvae. We also find that the cell cycle regulators cdk-1 and cyb-3 and the spindle/cytokinesis regulator zen-4 are required for Wnt/β-catenin pathway activity in the developing gonad. After sex is determined in the gonadal primordium the global sex determination pathway is dispensable for gonadal sexual fate, suggesting that male cell fates are promoted and maintained independently of the global pathway during this period.THE Caenorhabditis elegans gonad derives from a simple primordium of four cells that coalesces during embryogenesis and contains two somatic gonad precursors (SGPs), Z1 and Z4, flanking two germline precursors, Z2 and Z3 (Kimble and Hirsh 1979). The SGPs undergo very different developmental programs in each sex, involving sexually dimorphic cell lineages and migrations and sex-specific cellular differentiation. The result is a two-armed bilaterally symmetrical gonad in the adult hermaphrodite or a single-armed asymmetric gonad in the adult male. The high degree of sexual dimorphism of the mature organ and variety of cellular events that occur sex specifically during its development make the C. elegans gonad an outstanding model for sex-specific organogenesis.Development of the somatic gonad occurs in two phases. The early phase defines the gonadal axes and establishes the precursors of the major gonadal cell types. This takes place during the first larval stage (L1), beginning shortly after hatching with the first division of the SGPs. In both sexes SGP division is asymmetric in terms of both the sizes and the fates of the daughter cells, and establishes the proximal/distal axis of the gonad (Hirsh et al. 1976; Kimble and Hirsh 1979). The global sex determination pathway establishes the future sex of the gonad around the time of hatching (Klass et al. 1976; Nelson et al. 1978), and sexual dimorphism is already apparent when the SGPs divide: the size asymmetry of the SGP daughters is much more pronounced in males than hermaphrodites. In both sexes the asymmetry of the first SGP division requires a Wnt/β-catenin pathway. Mutations compromising this pathway cause a “symmetrical sisters” phenotype in which both daughters adopt the same fate (Miskowski et al. 2001; Siegfried and Kimble 2002; Phillips and Kimble 2009). Sex specificity is imposed on the SGPs by the global sex determining gene tra-1 (Hodgkin 1987) and the gonad-specific sex determining gene fkh-6 (Chang et al. 2004). These genes play opposing roles in SGP sex determination, with tra-1 feminizing and fkh-6 masculinizing the somatic gonad, and they also act redundantly to promote mitotic proliferation of the SGP lineage (Chang et al. 2004). SGP sex determination is linked to cell cycle progression by cyclin D, which is required to overcome repression of fkh-6 expression in the SGPs by E2F (Tilmann and Kimble 2005).The later phase of gonadal development involves the elongation of the gonad, together with cellular proliferation and differentiation, and lasts from L2 to adulthood. During L2 the somatic cells enlarge and leader cells (distal tip cells in the hermaphrodite, linker cell in the male) begin long-range migrations that extend the gonad. During L3, somatic gonad cell division resumes in both sexes, leading to the formation of differentiated somatic cell types by the end of L3 or beginning of L4. Gonadal morphogenesis is completed and gametogenesis begins during L4 (Kimble and Hirsh 1979).Although SGP division and much of hermaphrodite gonadal development have been well studied (Hubbard and Greenstein 2000), sexual cell fate specification in the somatic gonad is more poorly understood, particularly after the L1 stage. Despite the importance of fkh-6 in promoting male differentiation, it is expressed in males only during early L1 and null mutants have incomplete gonadal sex reversal. We have therefore performed a genome-wide RNAi screen to identify additional genes required after hatching for gonadal development in each sex. Among the advantages of this approach is the ability to identify gonadal regulators that also are essential for embryonic development. To our knowledge this is the first functional genomic study of gonadal sex differentiation.The screen identified many genes whose depletion disrupts gonadogenesis in each sex and nearly 70 genes whose depletion causes gonadal feminization in males. Prominent among this latter class were components of a Wnt/β-catenin pathway, cell cycle regulators, and genes involved in mitotic spindle function and cytokinesis. We find that Wnt/β-catenin activity continues in both sexes after SGP division and is required for male cell fate commitment in the gonad. We also find that the cyclin-dependent kinase cdk-1 and its cognate cyclin cyb-3 as well as the mitotic spindle regulator zen-4 are required for gonadal Wnt/β-catenin pathway activity, providing a potential new link between the cell cycle, asymmetric division, and sexual differentiation. The feminization caused by depletion of Wnt/β-catenin pathway components or cdk-1 is independent of the global sex determination pathway, suggesting that sexual fates in the male gonad remain plastic after the primary sex determination decision.     

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare ''professional'' phagocytes - macrophages and dendritic cells to ''non-professional'' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: ''professional'' macrophages and ''semi-professional'' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.     

A Screen for Genetic Loci Required for Body-Wall Muscle Development during Embryogenesis in Caenorhabditis Elegans

We have used available chromosomal deficiencies to screen for genetic loci whose zygotic expression is required for formation of body-wall muscle cells during embryogenesis in Caenorhabditis elegans. To test for muscle cell differentiation we have assayed for both contractile function and the expression of muscle-specific structural proteins. Monoclonal antibodies directed against two myosin heavy chain isoforms, the products of the unc-54 and myo-3 genes, were used to detect body-wall muscle differentiation. We have screened 77 deficiencies, covering approximately 72% of the genome. Deficiency homozygotes in most cases stain with antibodies to the body-wall muscle myosins and in many cases muscle contractile function is observed. We have identified two regions showing distinct defects in myosin heavy chain gene expression. Embryos homozygous for deficiencies removing the left tip of chromosome V fail to accumulate the myo-3 and unc-54 products, but express antigens characteristic of hypodermal, pharyngeal and neural development. Embryos lacking a large region on chromosome III accumulate the unc-54 product but not the myo-3 product. We conclude that there exist only a small number of loci whose zygotic expression is uniquely required for adoption of a muscle cell fate.     

Cellular Proteins Required for Adeno-Associated Virus DNA Replication in the Absence of Adenovirus Coinfection

We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system. The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded proteins were absolutely essential for AAV DNA replication and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DNA replication. Using specific chemical inhibitors and monoclonal antibodies, as well as the fractionation of uninfected HeLa extracts, we identified several of the cellular enzymes involved in AAV DNA replication. They were the single-stranded DNA binding protein, replication protein A (RFA), the 3′ primer binding complex, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA). Consistent with the current model for AAV DNA replication, which requires only leading-strand DNA synthesis, we found no requirement for DNA polymerase α-primase. AAV DNA replication could be reconstituted with purified Rep78, RPA, RFC, and PCNA and a phosphocellulose chromatography fraction (IIA) that contained DNA polymerase activity. As both RFC and PCNA are known to be accessory proteins for polymerase δ and , we attempted to reconstitute AAV DNA replication by substituting either purified polymerase δ or polymerase for fraction IIA. These attempts were unsuccessful and suggested that some novel cellular protein or modification was required for AAV DNA replication that had not been previously identified. Finally, we also further characterized the in vitro DNA replication assay and demonstrated by two-dimensional (2D) gel electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accumulation of single-stranded DNA in vivo that was not seen in vitro. The 2D data also suggested that although both Rep78 and Rep68 can generate dimeric intermediates in vitro, Rep68 is more efficient in processing dimers to monomer duplex DNA. Regardless of the Rep that was used in vitro, we found evidence of an interaction between the elongation complex and the terminal repeats. Nicking at the terminal repeats of a replicating molecule appeared to be inhibited until after elongation was complete.