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.     

Comparative Analysis of DNA Replication Timing Reveals Conserved Large-Scale Chromosomal Architecture

Recent evidence suggests that the timing of DNA replication is coordinated across megabase-scale domains in metazoan genomes, yet the importance of this aspect of genome organization is unclear. Here we show that replication timing is remarkably conserved between human and mouse, uncovering large regions that may have been governed by similar replication dynamics since these species have diverged. This conservation is both tissue-specific and independent of the genomic G+C content conservation. Moreover, we show that time of replication is globally conserved despite numerous large-scale genome rearrangements. We systematically identify rearrangement fusion points and demonstrate that replication time can be locally diverged at these loci. Conversely, rearrangements are shown to be correlated with early replication and physical chromosomal proximity. These results suggest that large chromosomal domains of coordinated replication are shuffled by evolution while conserving the large-scale nuclear architecture of the genome.     

Replication in Drosophila chromosomes

The temporal order of replication of specific sites in polytene chromosomes from salivary glands and gastric caeca of Drosophila nasuta larvae was compared using ³H-thymidine autoradiography. Labelling of different cytological regions in segments of chromosome 2R (section 47 A to 49 C) and chromosome 3 (section 80 A to 82 C) was examined in detail in nuclei showing late S-period labelling (2 D and 1D types) in both cell types. The different labelling sites (22 on the 2R segment and 38 on the chromosome 3 segment) are cytologically similar in the two cell types. However, there are profound differences in the labelling frequencies of certain sites in polytene nuclei from salivary glands and gastric caeca during the late S-phase. This suggests that even though a comparable number of chromosomal replicating units operates in the two polytene cell types, the temporal order of completion of replication differs.     

Replication in Drosophila chromosomes

DNA fibre autoradiography of highly polytenized nuclei in salivary glands of Drosophila nasuta larvae reveals two distinct types of active replicons. Type I replicons are longer (mean size=64 m), have a very high rate of fork migration (average rate=0.95 m/min) and generally occur in large arrays often extending over several thousand m. In contrast, the type II replicons are smaller (mean size= 20 m), slow replicating (average rate=0.07 m/min) and occur in short arrays containing only a few closely spaced active replicons. Evidence is presented that type I replicons are active in the early S and type II in the late S. Observations on autoradiographic labelling of partially lysed polytene chromosomes provide evidence for a lack of temporal and spatial agreement in the activation of origin points in homologous regions of the lateral polytene strands; these observations also suggest local variations in levels of polyteny within a chromosome. On the basis of this and other available information on replication in polytene chromosomes the possible roles of the two replicon types in the generation of the different ³H-thymidine labelling patterns of polytene chromosomes are discussed.We take pleasure in dedicating this paper to our inspiring teacher Prof. S.P. Ray Chaudhuri on his completing 75 years of fruitful life     

Replication in Drosophila chromosomes

Prolongation of larval life in Drosophila melanogaster, by growing wild type larvae at lower temperature, or in animals carrying the X-linked mutation giant is known to result in a greater proportion of nuclei in salivary glands showing the highest level of polyteny. We have examined by autoradiography the patterns of ³H-thymidine incorporation during 10 min or 1 min pulses in salivary gland polytene chromosomes of older giant larvae and of wild type late third instar larvae of D. melanogaster grown since hatching either at 24 ° C or at 10 ° C. The various patterns of labelling and their relative frequencies are generally similar in glands from the warm-(24 ° C) or cold (10 ° C)-reared wild type larvae, except the interband (IB) labelling patterns which are very frequent in the later group but rare in the former. The IB type labelled nuclei in cold-reared wild type larvae show labelling ranging from only a few puffs/interbands labelled to nearly all puffs/interbands labelled. In warm-reared wild type larvae, very low labelled IB patterns are not seen. In older giant larvae, the ³H-thymidine labelling patterns are in most respects similar to those seen in cold-reared wild type larvae. In 1 min pulsed preparations from all larvae, the IB patterns are relatively more frequent than in corresponding 10 min pulsed preparations. No nuclei with the continuous (2C or 3C) type of labelling pattern, with all bands and interbands/puffs labelled, were seen in 1 min pulsed preparations from cold-reared wild type or in giant larvae, and only a few nuclei in 1 min pulsed preparations from warm-reared wild type larvae exhibited the 2C labelling pattern. Analysis of silver grain density on specific late replicating sites in late discontinuous (1D) type labelled nuclei suggests that the rate of DNA synthesis per chromosomal site is not different at the two developmental temperatures. It is suggested that correlated with the prolongation of larval life under cold-rearing conditions or in giant larvae, the polytene replication cycles are also prolonged. It is further suggested that the polytene S-period in these larvae is longer due to a considerable asynchrony in the initiation and termination of replication of different sites during a replication cycle.     

Replication of chromosomal DNA in diploid Drosophila melanogaster cells cultured in vitro

Replication rate and replicon sizes in chromosomal DNA of in vitro cultured diploid D. melanogaster cells were determined using autoradiography of ³H-thymidine labeled DNA. Synthesis of DNA in euchromatic and heterochromatic regions of Drosophila diploid cells occurs at different periods of the S phase which lasts 10 h. During the first 4 h the synthesis is observed only in euchromatic regions. The heterochromatic synthesis starts shortly before the synthesis in euchromatic regions is completed and lasts for 6 h until the end of the S phase. The cells were synchronized by 5fluorodeoxyuridine which blocked the diploid cell DNA synthesis. Synthesis was found to start simultaneously in most euchromatic replicons. In the majority of the replicons the synthesis started at a single point and proceeded bidirectionally. The average rate of DNA synthesis per fork was 12.5 m/h (38 kb). The mean distance between the middle points of adjacent labeled regions was 70 m (210 kb). The size of most replicons ranged from 40 to 120 m. — These estimates do not apply to the heterochromatic portions of the D. melanogaster genome since the measurements have been carried out on DNA preparations obtained during the first 2 h of the S phase. — On the average, a replicon can consist of 7 chromomeres since the size of a replicon in diploid cell chromosomal DNA and DNA length of a polytene chromomere average 210 and 30 kb, respectively.     

Replication in Drosophila chromosomes

It is widely known that the bulk of the pericentromeric heterochromatin (-heterochromatin) does not replicate during polytenization in Drosophila. However, a recent DNA-Feulgen cytophotometric study (Dennhöfer 1982a) has claimed equal polytenization of all heterochromatin regions. To re-examine this issue, the amount of Hoechst 33258-bright heterochromatin in non-polytene and polytene nuclei in salivary glands and Malpighian tubules of late third instar larvae of D. nasuta has been compared by cytofluorometry. Since the amount of Hoechst 33258-bright heterochromatin is similar in non-polytene and polytene nuclei in spite of the latter having an enormously high euchromatin DNA content, it is concluded that the -heterochromatin does not replicate during polytenization. The present results further indicate that in the polytene nuclei of Malpighian tubules the -heterochromatin remains at the 2C level whereas in salivary gland polytene nuclei it varies between the 2C and 4C levels.I would like to dedicate this paper to the memory of E. Heitz to commemorate 50 years of - and -heterochromatin     

Machine Learning Modeling of Protein-intrinsic Features Predicts Tractability of Targeted Protein Degradation

Targeted protein degradation(TPD) has rapidly emerged as a therapeutic modality to eliminate previously undruggable proteins by repurposing the cell’s endogenous protein degradation machinery. However, the susceptibility of proteins for targeting by TPD approaches, termed‘‘degradability”, is largely unknown. Here, we developed a machine learning model, model-free analysis of protein degradability(MAPD), to predict degradability from features intrinsic to protein targets. MAPD shows accurate perf...     

Chromatin structure of Drosophila nasutoides satellites as probed by cross-linking DNA in situ

Chromatin structure can be probed by cross-linking DNA in situ using trioxsalen and irradiation with UV light. Presumably DNA within a nucleosome is protected from cross-linking so that this region appears as a single-strand loop in the electron microscope under a condition in which single-strands and double-strands are distinguished. Unprotected regions appear as duplex due to cross-linking.We have used this approach to investigate the structure of chromatins containing satellite DNAs of Drosophila nasutoides. We have previously shown that D. nasutoides has an unusually large autosome pair which is almost entirely heterochromatic. Its nuclear DNA reveals four major satellite components amounting up to 60% of the total genome. All of them are localized in this large heterochromatic chromosome. We wish to ask whether chromatins containing different satellite sequences have different arrangements of nucleosomes. Our results from cross-linking experiments show that all DNA components including main band DNA have different patterns of protected and unprotected regions: (a) The length distributions of protected regions show multiple peaks with the smallest unit lengths being 200 nucleotides for main band DNA, 180 for satellites I, II and III, and 160 for satellite IV. (b) The amounts of unprotected regions, presumably internucleosome DNA, vary from 16% for main band DNA to 60% for satellite IV, suggesting that satellite chromatins have fewer nucleosomes per given length of chromatin than main band DNA chromatin. The spacings between nucleosomes appear to be random in satellite chromatins.     

Replication of DNA in larval salivary glands of Drosophila after in vivo synchronization

The replication of DNA in the giant chromosomes in different cells of Drosophila larval salivary glands is asynchronous. A method of in vivo synchronization of the nuclei has been successfully devised by a 5-fluorodeoxyuridine (FdU) block-release-thymidine chase technique, and the patterns of replication sequences have been examined by ³H-thymidine autoradiography. When the larvae of Drosophila melanogaster are fed on FdU for 48 h, and the block is released thereafter, most cells are found in mid-replication phase (termed 3C). When the larvae are subjected to a chase in normal Drosophila medium (or sucrose), a series of cells arrive at 3C phase about every 8 h. When they are chased in sucrose containing thymidine, the number of cells in 3C phase rises to 70%, and then drops rapidly to 1–2% of all labelled cells. The terminal phases (3D, 2D and 1D) reach a peak between 4–8 h. At 12–14 h of chase the 3D-1D peaks decline and a third peak consisting mostly of the initial phases (DD-1C) is found at 14–16 h. The replication of DNA in polytene chromosomes of Drosophila thus seems to proceed in a regular sequence of DD-3C-1D.Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

Identification of Genomic Regions Required for DNA Replication during Drosophila Embryogenesis

A collection of Drosophila deficiency stocks was examined by bromodeoxyuridine (BrdU) labeling of embryos to analyze the DNA replication patterns in late embryogenesis. This permitted us to screen 34% of the genome for genes that when absent in homozygous deficiencies affect the cell cycle or DNA replication. We found three genomic intervals that when deleted result in cessation of DNA replication in the embryo, 39D2-3;E2-F1, 51E and 75C5-7;F1. Embryos deleted for the 75C5-7;F1 region stop DNA replication at the time in embryogenesis when a G(1) phase is added to the mitotic cell cycle and the larval tissues begin to become polytene. Thus, this interval may contain a gene controlling these cell cycle transitions. DNA replication arrests earlier in embryos homozygous for deletions for the other two regions. Analysis of the effects of deletions in the 39D2-3;E2-F1 region on DNA replication showed that the block to DNA replication correlates with deletion of the histone genes. We were able to identify a single, lethal complementation group in 51E, l(2)51Ec, that is responsible for the cessation of replication observed in this interval. Deficiencies that removed one of the Drosophila cdc2 genes and the cyclin A gene had no effect on replication during embryogenesis. Additionally, our analysis identified a gene, pimples, that is required for the proper completion of mitosis in the post-blastoderm divisions of the embryo.     

The Spatiotemporal Program of DNA Replication Is Associated with Specific Combinations of Chromatin Marks in Human Cells

The duplication of mammalian genomes is under the control of a spatiotemporal program that orchestrates the positioning and the timing of firing of replication origins. The molecular mechanisms coordinating the activation of about predicted origins remain poorly understood, partly due to the intrinsic rarity of replication bubbles, making it difficult to purify short nascent strands (SNS). The precise identification of origins based on the high-throughput sequencing of SNS constitutes a new methodological challenge. We propose a new statistical method with a controlled resolution, adapted to the detection of replication origins from SNS data. We detected an average of 80,000 replication origins in different cell lines. To evaluate the consistency between different protocols, we compared SNS detections with bubble trapping detections. This comparison demonstrated a good agreement between genome-wide methods, with 65% of SNS-detected origins validated by bubble trapping, and 44% of bubble trapping origins validated by SNS origins, when compared at the same resolution. We investigated the interplay between the spatial and the temporal programs of replication at fine scales. We show that most of the origins detected in regions replicated in early S phase are shared by all the cell lines investigated whereas cell-type-specific origins tend to be replicated in late S phase. We shed a new light on the key role of CpG islands, by showing that 80% of the origins associated with CGIs are constitutive. Our results further show that at least 76% of CGIs are origins of replication. The analysis of associations with chromatin marks at different timing of cell division revealed new potential epigenetic regulators driving the spatiotemporal activity of replication origins. We highlight the potential role of H4K20me1 and H3K27me3, the coupling of which is correlated with increased efficiency of replication origins, clearly identifying those marks as potential key regulators of replication origins.