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1.
JIMD Rep ; 65(4): 280-294, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38974607

ABSTRACT

Classic galactosemia (CG) arises from loss-of-function mutations in the Galt gene, which codes for the enzyme galactose-1-phosphate uridylyltransferase (GALT), a central component in galactose metabolism. The neonatal fatality associated with CG can be prevented by galactose dietary restriction, but for decades it has been known that limiting galactose intake is not a cure and patients often have lasting complications. Even on a low-galactose diet, GALT's substrate galactose-1-phosphate (Gal1P) is elevated and one hypothesis is that elevated Gal1P is a driver of pathology. Here we show that Gal1P levels were elevated above wildtype (WT) in Galt mutant mice, while mice doubly mutant for Galt and the gene encoding galactokinase 1 (Galk1) had normal Gal1P levels. This indicates that GALK1 is necessary for the elevated Gal1P in CG. Another hypothesis to explain the pathology is that an inability to metabolize galactose leads to diminished or disrupted galactosylation of proteins or lipids. Our studies reveal that levels of a subset of cerebrosides-galactosylceramide 24:1, sulfatide 24:1, and glucosylceramide 24:1-were modestly decreased compared to WT. In contrast, gangliosides were unaltered. The observed reduction in these 24:1 cerebrosides may be relevant to the clinical pathology of CG, since the cerebroside galactosylceramide is an important structural component of myelin, the 24:1 species is the most abundant in myelin, and irregularities in white matter, of which myelin is a constituent, have been observed in patients with CG. Therefore, impaired cerebroside production may be a contributing factor to the brain damage that is a common clinical feature of the human disease.

2.
Neuron ; 110(19): 3186-3205.e7, 2022 10 05.
Article in English | MEDLINE | ID: mdl-35961319

ABSTRACT

Structural plasticity in the brain often necessitates dramatic remodeling of neuronal processes, with attendant reorganization of the cytoskeleton and membranes. Although cytoskeletal restructuring has been studied extensively, how lipids might orchestrate structural plasticity remains unclear. We show that specific glial cells in Drosophila produce glucocerebrosidase (GBA) to locally catabolize sphingolipids. Sphingolipid accumulation drives lysosomal dysfunction, causing gba1b mutants to harbor protein aggregates that cycle across circadian time and are regulated by neural activity, the circadian clock, and sleep. Although the vast majority of membrane lipids are stable across the day, a specific subset that is highly enriched in sphingolipids cycles daily in a gba1b-dependent fashion. Remarkably, both sphingolipid biosynthesis and degradation are required for the diurnal remodeling of circadian clock neurites, which grow and shrink across the day. Thus, dynamic sphingolipid regulation by glia enables diurnal circuit remodeling and proper circadian behavior.


Subject(s)
Circadian Clocks , Drosophila Proteins , Animals , Circadian Clocks/physiology , Circadian Rhythm/physiology , Drosophila/metabolism , Drosophila Proteins/metabolism , Glucosylceramidase , Membrane Lipids , Neuroglia/metabolism , Protein Aggregates , Sphingolipids/metabolism
3.
Sci Signal ; 11(547)2018 09 11.
Article in English | MEDLINE | ID: mdl-30206138

ABSTRACT

A major limitation of targeted cancer therapy is the rapid emergence of drug resistance, which often arises through mutations at or downstream of the drug target or through intrinsic resistance of subpopulations of tumor cells. Medulloblastoma (MB), the most common pediatric brain tumor, is no exception, and MBs that are driven by sonic hedgehog (SHH) signaling are particularly aggressive and drug-resistant. To find new drug targets and therapeutics for MB that may be less susceptible to common resistance mechanisms, we used a developmental phosphoproteomics approach in murine granule neuron precursors (GNPs), the developmental cell of origin of MB. The protein kinase CK2 emerged as a driver of hundreds of phosphorylation events during the proliferative, MB-like stage of GNP growth, including the phosphorylation of three of the eight proteins commonly amplified in MB. CK2 was critical to the stabilization and activity of the transcription factor GLI2, a late downstream effector in SHH signaling. CK2 inhibitors decreased the viability of primary SHH-type MB patient cells in culture and blocked the growth of murine MB tumors that were resistant to currently available Hh inhibitors, thereby extending the survival of tumor-bearing mice. Because of structural interactions, one CK2 inhibitor (CX-4945) inhibited both wild-type and mutant CK2, indicating that this drug may avoid at least one common mode of acquired resistance. These findings suggest that CK2 inhibitors may be effective for treating patients with MB and show how phosphoproteomics may be used to gain insight into developmental biology and pathology.


Subject(s)
Casein Kinase II/metabolism , Cerebellar Neoplasms/metabolism , Hedgehog Proteins/metabolism , Medulloblastoma/metabolism , Phosphoproteins/metabolism , Proteomics/methods , Signal Transduction , Anilides/pharmacology , Animals , Casein Kinase II/antagonists & inhibitors , Casein Kinase II/genetics , Cell Line, Tumor , Cerebellar Neoplasms/drug therapy , Cerebellar Neoplasms/genetics , Gene Expression Regulation, Neoplastic/drug effects , Hedgehog Proteins/antagonists & inhibitors , Hedgehog Proteins/genetics , Humans , Kaplan-Meier Estimate , Medulloblastoma/drug therapy , Medulloblastoma/genetics , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, Nude , Mice, SCID , NIH 3T3 Cells , Naphthyridines/pharmacology , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Phenazines , Phosphoproteins/genetics , Pyridines/pharmacology , Xenograft Model Antitumor Assays
4.
G3 (Bethesda) ; 8(7): 2193-2204, 2018 07 02.
Article in English | MEDLINE | ID: mdl-29735526

ABSTRACT

N-glycanase 1 (NGLY1) Deficiency is a rare monogenic multi-system disorder first described in 2014. NGLY1 is evolutionarily conserved in model organisms. Here we conducted a natural history study and chemical-modifier screen on the Drosophila melanogaster NGLY1 homolog, Pngl We generated a new fly model of NGLY1 Deficiency, engineered with a nonsense mutation in Pngl at codon 420 that results in a truncation of the C-terminal carbohydrate-binding PAW domain. Homozygous mutant animals exhibit global development delay, pupal lethality and small body size as adults. We developed a 96-well-plate, image-based, quantitative assay of Drosophila larval size for use in a screen of the 2,650-member Microsource Spectrum compound library of FDA approved drugs, bioactive tool compounds, and natural products. We found that the cholesterol-derived ecdysteroid molting hormone 20-hydroxyecdysone (20E) partially rescued the global developmental delay in mutant homozygotes. Targeted expression of a human NGLY1 transgene to tissues involved in ecdysteroidogenesis, e.g., prothoracic gland, also partially rescues global developmental delay in mutant homozygotes. Finally, the proteasome inhibitor bortezomib is a potent enhancer of global developmental delay in our fly model, evidence of a defective proteasome "bounce-back" response that is also observed in nematode and cellular models of NGLY1 Deficiency. Together, these results demonstrate the therapeutic relevance of a new fly model of NGLY1 Deficiency for drug discovery and gene modifier screens.


Subject(s)
Congenital Disorders of Glycosylation/genetics , Drosophila/genetics , Genetic Association Studies , Neurodevelopmental Disorders/genetics , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/deficiency , Alleles , Animals , Animals, Genetically Modified , Congenital Disorders of Glycosylation/metabolism , Disease Models, Animal , Drosophila/metabolism , Female , Genes, Lethal , Genotype , Humans , Larva , Mutation , Neurodevelopmental Disorders/metabolism , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/genetics , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism , Phenotype , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology
5.
Cell ; 163(4): 934-46, 2015 Nov 05.
Article in English | MEDLINE | ID: mdl-26544940

ABSTRACT

Chemical cross-linking and DNA sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed with regions of little or no interaction, in interphase nuclei. We find that TADs and the regions between them correspond with the bands and interbands of polytene chromosomes of Drosophila. We further establish the conservation of TADs between polytene and diploid cells of Drosophila. From direct measurements on light micrographs of polytene chromosomes, we then deduce the states of chromatin folding in the diploid cell nucleus. Two states of folding, fully extended fibers containing regulatory regions and promoters, and fibers condensed up to 10-fold containing coding regions of active genes, constitute the euchromatin of the nuclear interior. Chromatin fibers condensed up to 30-fold, containing coding regions of inactive genes, represent the heterochromatin of the nuclear periphery. A convergence of molecular analysis with direct observation thus reveals the architecture of interphase chromosomes.


Subject(s)
Drosophila melanogaster/genetics , Polytene Chromosomes/chemistry , Animals , Cell Nucleus/chemistry , Cell Nucleus/genetics , Chromosomal Puffs , Diploidy , Drosophila melanogaster/chemistry , Drosophila melanogaster/cytology , Drosophila melanogaster/growth & development , Genetic Techniques , Larva/chemistry
6.
Elife ; 42015 Sep 15.
Article in English | MEDLINE | ID: mdl-26371509

ABSTRACT

Alterations in Hedgehog (Hh) signaling lead to birth defects and cancers including medulloblastoma, the most common pediatric brain tumor. Although inhibitors targeting the membrane protein Smoothened suppress Hh signaling, acquired drug resistance and tumor relapse call for additional therapeutic targets. Here we show that phosphodiesterase 4D (PDE4D) acts downstream of Neuropilins to control Hh transduction and medulloblastoma growth. PDE4D interacts directly with Neuropilins, positive regulators of Hh pathway. The Neuropilin ligand Semaphorin3 enhances this interaction, promoting PDE4D translocation to the plasma membrane and cAMP degradation. The consequent inhibition of protein kinase A (PKA) enhances Hh transduction. In the developing cerebellum, genetic removal of Neuropilins reduces Hh signaling activity and suppresses proliferation of granule neuron precursors. In mouse medulloblastoma allografts, PDE4D inhibitors suppress Hh transduction and inhibit tumor growth. Our findings reveal a new regulatory mechanism of Hh transduction, and highlight PDE4D as a promising target to treat Hh-related tumors.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Hedgehogs/metabolism , Medulloblastoma/pathology , Neuropilin-1/metabolism , Neuropilin-2/metabolism , Signal Transduction , Animals , Cell Line , Cell Proliferation , Humans , Mice , Mice, Knockout
7.
Methods ; 68(1): 199-206, 2014 Jun 15.
Article in English | MEDLINE | ID: mdl-24556557

ABSTRACT

Hedgehog (Hh) signal transduction is necessary for the development of most mammalian tissues and can go awry and cause birth defects or cancer. Hh signaling was initially described in Drosophila, and much of what we know today about mammalian Hh signaling was directly guided by discoveries in the fly. Indeed, Hh signaling is a wonderful example of the use of non-vertebrate model organisms to make basic discoveries that lead to new disease treatment. The first pharmaceutical to treat hyperactive Hh signaling in Basal Cell Carcinoma was released in 2012, approximately 30 years after the isolation of Hh mutants in Drosophila. The study of Hh signaling has been greatly facilitated by the imaginal wing disc, a tissue with terrific experimental advantages. Studies using the wing disc have led to an understanding of Hh ligand processing, packaging into particles for transmission, secretion, reception, signal transduction, target gene activation, and tissue patterning. Here we describe the imaginal wing disc, how Hh patterns this tissue, and provide methods to use wing discs to study Hh signaling in Drosophila. The tools and approaches we highlight form the cornerstone of research efforts in many laboratories that use Drosophila to study Hh signaling, and are essential for ongoing discoveries.


Subject(s)
Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Hedgehog Proteins/genetics , Signal Transduction , Animals , Molecular Biology/methods , Wings, Animal/growth & development , Wings, Animal/metabolism
8.
Genetics ; 195(1): 127-46, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23821596

ABSTRACT

Dynamic regulation of chromosome structure and organization is critical for fundamental cellular processes such as gene expression and chromosome segregation. Condensins are conserved chromosome-associated proteins that regulate a variety of chromosome dynamics, including axial shortening, lateral compaction, and homolog pairing. However, how the in vivo activities of condensins are regulated and how functional interactors target condensins to chromatin are not well understood. To better understand how Drosophila melanogaster condensin is regulated, we performed a yeast two-hybrid screen and identified the chromo-barrel domain protein Mrg15 to interact with the Cap-H2 condensin subunit. Genetic interactions demonstrate that Mrg15 function is required for Cap-H2-mediated unpairing of polytene chromosomes in ovarian nurse cells and salivary gland cells. In diploid tissues, transvection assays demonstrate that Mrg15 inhibits transvection at Ubx and cooperates with Cap-H2 to antagonize transvection at yellow. In cultured cells, we show that levels of chromatin-bound Cap-H2 protein are partially dependent on Mrg15 and that Cap-H2-mediated homolog unpairing is suppressed by RNA interference depletion of Mrg15. Thus, maintenance of interphase chromosome compaction and homolog pairing status requires both Mrg15 and Cap-H2. We propose a model where the Mrg15 and Cap-H2 protein-protein interaction may serve to recruit Cap-H2 to chromatin and facilitates compaction of interphase chromatin.


Subject(s)
Adenosine Triphosphatases/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Pairing , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila/metabolism , Multiprotein Complexes/metabolism , Polytene Chromosomes/metabolism , Adenosine Triphosphatases/genetics , Animals , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , Drosophila/genetics , Drosophila Proteins/genetics , Epigenesis, Genetic , Homeodomain Proteins/genetics , Interphase , Multiprotein Complexes/genetics , Polytene Chromosomes/chemistry , Protein Binding , Transcription Factors/genetics
9.
J Cell Biol ; 201(1): 49-63, 2013 Apr 01.
Article in English | MEDLINE | ID: mdl-23530065

ABSTRACT

Condensin complexes play vital roles in chromosome condensation during mitosis and meiosis. Condensin II uniquely localizes to chromatin throughout the cell cycle and, in addition to its mitotic duties, modulates chromosome organization and gene expression during interphase. Mitotic condensin activity is regulated by phosphorylation, but mechanisms that regulate condensin II during interphase are unclear. Here, we report that condensin II is inactivated when its subunit Cap-H2 is targeted for degradation by the SCF(Slimb) ubiquitin ligase complex and that disruption of this process dramatically changed interphase chromatin organization. Inhibition of SCF(Slimb) function reorganized interphase chromosomes into dense, compact domains and disrupted homologue pairing in both cultured Drosophila cells and in vivo, but these effects were rescued by condensin II inactivation. Furthermore, Cap-H2 stabilization distorted nuclear envelopes and dispersed Cid/CENP-A on interphase chromosomes. Therefore, SCF(Slimb)-mediated down-regulation of condensin II is required to maintain proper organization and morphology of the interphase nucleus.


Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Multiprotein Complexes/metabolism , Nuclear Envelope/metabolism , Proteolysis , Ubiquitin-Protein Ligases/metabolism , Adenosine Triphosphatases/genetics , Animals , Cell Cycle Proteins/genetics , Cell Line , Centromere Protein A , Chromatin/genetics , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , Down-Regulation/physiology , Drosophila Proteins/genetics , Drosophila melanogaster , Histones/genetics , Histones/metabolism , Interphase/physiology , Multiprotein Complexes/genetics , Nuclear Envelope/genetics , Phosphorylation/physiology , Ubiquitin-Protein Ligases/genetics
10.
Genetics ; 194(1): 101-15, 2013 May.
Article in English | MEDLINE | ID: mdl-23436180

ABSTRACT

Nucleostemin 3 (NS3) is an evolutionarily conserved protein with profound roles in cell growth and viability. Here we analyze cell-autonomous and non-cell-autonomous growth control roles of NS3 in Drosophila and demonstrate its GTPase activity using genetic and biochemical assays. Two null alleles of ns3, and RNAi, demonstrate the necessity of NS3 for cell autonomous growth. A hypomorphic allele highlights the hypersensitivity of neurons to lowered NS3 function. We propose that NS3 is the functional ortholog of yeast and human Lsg1, which promotes release of the nuclear export adapter from the large ribosomal subunit. Release of the adapter and its recycling to the nucleus are essential for sustained production of ribosomes. The ribosome biogenesis role of NS3 is essential for proper rates of translation in all tissues and is necessary for functions of growth-promoting neurons.


Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , GTP-Binding Proteins/metabolism , Ribosomes/metabolism , Alleles , Amino Acid Sequence , Animals , Cell Nucleus/metabolism , Cell Survival , Dopamine/metabolism , Drosophila Proteins/chemistry , Drosophila melanogaster/cytology , GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/chemistry , Genetic Loci/genetics , Humans , Larva/cytology , Larva/growth & development , Molecular Sequence Data , Mutation/genetics , Phenotype , Protein Structure, Tertiary , RNA Interference , Recombinant Fusion Proteins/metabolism , Ribosomal Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Sequence Homology, Amino Acid
11.
PLoS Genet ; 8(8): e1002873, 2012.
Article in English | MEDLINE | ID: mdl-22956908

ABSTRACT

The eukaryotic nucleus is both spatially and functionally partitioned. This organization contributes to the maintenance, expression, and transmission of genetic information. Though our ability to probe the physical structure of the genome within the nucleus has improved substantially in recent years, relatively little is known about the factors that regulate its organization or the mechanisms through which specific organizational states are achieved. Here, we show that Drosophila melanogaster Condensin II induces axial compaction of interphase chromosomes, globally disrupts interchromosomal interactions, and promotes the dispersal of peri-centric heterochromatin. These Condensin II activities compartmentalize the nucleus into discrete chromosome territories and indicate commonalities in the mechanisms that regulate the spatial structure of the genome during mitosis and interphase.


Subject(s)
Adenosine Triphosphatases/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Heterochromatin/genetics , Multiprotein Complexes/genetics , Polytene Chromosomes/genetics , Animals , Cell Compartmentation/genetics , Cell Nucleus/genetics , Cell Nucleus/metabolism , Centromere/genetics , Interphase/genetics , Mitosis , Polytene Chromosomes/metabolism
12.
Science ; 322(5906): 1384-7, 2008 Nov 28.
Article in English | MEDLINE | ID: mdl-19039137

ABSTRACT

Polytene chromosome structure is a characteristic of some polyploid cells where several to thousands of chromatids are closely associated with perfect alignment of homologous DNA sequences. Here, we show that Drosophila condensin II promotes disassembly of polytene structure into chromosomal components. Condensin II also negatively regulates transvection, a process whereby certain alleles are influenced transcriptionally via interallelic physical associations. We propose that condensin II restricts trans-chromosomal interactions that affect transcription through its ability to spatially separate aligned interphase chromosomes.


Subject(s)
Adenosine Triphosphatases/metabolism , Chromosomes/physiology , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Multiprotein Complexes/metabolism , Adenosine Triphosphatases/genetics , Alleles , Animals , Cell Cycle , Chromosomes/genetics , Chromosomes/ultrastructure , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila Proteins/physiology , Drosophila melanogaster/growth & development , Gene Expression Regulation , Genes, Insect , Homeodomain Proteins/genetics , Homeodomain Proteins/physiology , Interphase , Larva/genetics , Multiprotein Complexes/genetics , Mutant Proteins/metabolism , Pigmentation/genetics , Salivary Glands , Transcription Factors/genetics , Transcription Factors/physiology , Transcription, Genetic
13.
PLoS Genet ; 4(10): e1000228, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18927632

ABSTRACT

Several meiotic processes ensure faithful chromosome segregation to create haploid gametes. Errors to any one of these processes can lead to zygotic aneuploidy with the potential for developmental abnormalities. During prophase I of Drosophila male meiosis, each bivalent condenses and becomes sequestered into discrete chromosome territories. Here, we demonstrate that two predicted condensin II subunits, Cap-H2 and Cap-D3, are required to promote territory formation. In mutants of either subunit, territory formation fails and chromatin is dispersed throughout the nucleus. Anaphase I is also abnormal in Cap-H2 mutants as chromatin bridges are found between segregating heterologous and homologous chromosomes. Aneuploid sperm may be generated from these defects as they occur at an elevated frequency and are genotypically consistent with anaphase I segregation defects. We propose that condensin II-mediated prophase I territory formation prevents and/or resolves heterologous chromosomal associations to alleviate their potential interference in anaphase I segregation. Furthermore, condensin II-catalyzed prophase I chromosome condensation may be necessary to resolve associations between paired homologous chromosomes of each bivalent. These persistent chromosome associations likely consist of DNA entanglements, but may be more specific as anaphase I bridging was rescued by mutations in the homolog conjunction factor teflon. We propose that the consequence of condensin II mutations is a failure to resolve heterologous and homologous associations mediated by entangled DNA and/or homolog conjunction factors. Furthermore, persistence of homologous and heterologous interchromosomal associations lead to anaphase I chromatin bridging and the generation of aneuploid gametes.


Subject(s)
Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Chromosome Segregation/genetics , Chromosome Segregation/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Meiosis/genetics , Meiosis/physiology , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Adenosine Triphosphatases/chemistry , Alleles , Anaphase/genetics , Anaphase/physiology , Animals , Animals, Genetically Modified , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/chemistry , Drosophila Proteins/chemistry , Drosophila melanogaster/cytology , Female , Fertility/genetics , Fertility/physiology , Genes, Insect , Male , Models, Biological , Multiprotein Complexes/chemistry , Mutation , Nondisjunction, Genetic , Prophase , Protein Subunits , Sex Chromosomes
14.
Chromosoma ; 116(2): 197-214, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17219175

ABSTRACT

We have used gene amplification in Drosophila follicle cells as a model of metazoan DNA replication to address whether changes in histone modifications are associated with replication origin activation. We observe that replication initiation is associated with distinct histone modifications. Acetylated lysines K5, K8, and K12 on histone H4 and K14 on histone H3 are specifically enriched during replication initiation at the amplification origins. Strikingly, H4 acetylation persists at an amplification origin well after replication forks have progressed significantly outward from the origin, indicating that H4 acetylation is associated with origin regulation and not histone deposition at the replication forks. Origin recognition complex subunit 2 (orc2) mutants with severe amplification defects do not abolish H4 acetylation, whereas the dup/cdt1 mutant delays the appearance of acetylation foci, and mutants in rbf result in temporal persistence. These data indicate that core histone acetylation is associated with origin activity. Furthermore, follicle cells undergoing gene amplification exhibit high levels of histone H1 phosphorylation. The patterns of H1 phosphorylation provide insights into cell cycle states during amplification, as H1 kinase activity in follicle cells is responsive to high Cyclin E activity, and it can be abolished by overexpressing the retinoblastoma homolog, Rbf, that represses Cyclin E. These data suggest that amplification origins are able to initiate when the cells are in a late S-phase, when the genome is normally not licensed for replication.


Subject(s)
Cell Cycle/physiology , DNA Replication/physiology , Gene Amplification/physiology , Histones/metabolism , Origin Recognition Complex/metabolism , Acetylation , Animals , Bromodeoxyuridine , Chromatin Immunoprecipitation , Cyclin E/metabolism , Drosophila , Drosophila Proteins/metabolism , Female , Fluorescent Antibody Technique , Nucleic Acid Amplification Techniques , Ovarian Follicle/metabolism , Phosphorylation , Replication Origin/physiology , Retinoblastoma Protein/metabolism , Transcription Factors/metabolism
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