Genome-Wide Maps of Mononucleosomes and Dinucleosomes Containing Hyperacetylated Histones of Aspergillus fumigatus

It is suggested that histone modifications and/or histone variants influence the nucleosomal DNA length. We sequenced both ends of mononucleosomal and dinucleosomal DNA fragments of the filamentous fungus Aspergillus fumigatus, after treatment with the histone deacetylase inhibitor trichostatin A (TSA). After mapping the DNA fragments to the genome, we identified >7 million mononucleosome positions and >7 million dinucleosome positions. We showed that the distributions of the lengths of the mononucleosomal DNA fragments after 15-min and 30-min treatments with micrococcal nuclease (MNase) showed a single peak at 168 nt and 160 nt, respectively. The distributions of the lengths of the dinucleosomal DNA fragments after 15-min- and 30-min-treatment with MNase showed a single peak at 321 nt and 306 nt, respectively. The nucleosomal DNA fragments obtained from the TSA-treated cells were significantly longer than those obtained from the untreated cells. On the other hand, most of the genes did not undergo any change after treatment. Between the TSA-treated and untreated cells, only 77 genes had ≥2-fold change in expression levels. In addition, our results showed that the locations where mononucleosomes were frequently detected were conserved between the TSA-treated cells and untreated cells in the gene promoters (lower density of the nucleosomes). However, these locations were less conserved in the bodies (higher density of the nucleosomes) of genes with ≥2-fold changes. Our findings indicate that TSA influences the nucleosome positions, especially of the regions with high density of the nucleosomes by elongation of the nucleosomal DNA. However, most of the nucleosome positions are conserved in the gene promoters, even after treatment with TSA, because of the low density of nucleosomes in the gene promoters.     

Structure-based Analysis of DNA Sequence Patterns Guiding Nucleosome Positioning in vitro

Abstract

Recent studies of genome-wide nucleosomal organization suggest that the DNA sequence is one of the major determinants of nucleosome positioning. Although the search for underlying patterns encoded in nucleosomal DNA has been going on for about 30 years, our knowledge of these patterns still remains limited. Based on our evaluations of DNA deformation energy, we developed new scoring functions to predict nucleosome positioning. There are three principal differences between our approach and earlier studies: (i) we assume that the length of nucleosomal DNA varies from 146 to 147 bp; (ii) we consider the anisotropic flexibility of pyrimidine-purine (YR) dimeric steps in the context of their neighbors (e.g., YYRR versus RYRY); (iii) we postulate that alternating AT-rich and GC-rich motifs reflect sequence-dependent interactions between histone arginines and DNA in the minor groove. Using these functions, we analyzed 20 nucleosome positions mapped in vitro at single nucleotide resolution (including clones 601, 603, 605, the pGUB plasmid, chicken β-globin and three 5S rDNA genes). We predicted 15 of the 20 positions with 1-bp precision, and two positions with 2-bp precision. The predicted position of the ‘601’ nucleosome (i.e., the optimum of the computed score) deviates from the experimentally determined unique position by no more than 1 bp—an accuracy exceeding that of earlier predictions.

Our analysis reveals a clear heterogeneity of the nucleosomal sequences which can be divided into two groups based on the positioning ‘rules’ they follow. The sequences of one group are enriched by highly deformable YR/YYRR motifs at the minor-groove bending sites SHL ±3.5 and ±5.5, which is similar to the α-satellite sequence used in most crystallized nucleosomes. Apparently, the positioning of these nucleosomes is determined by the interactions between histones H2A/H2B and the terminal parts of nucleosomal DNA. In the other group (that includes the ‘601’ clone) the same YR/YYRR motifs occur predominantly at the sites SHL ±1.5. The interaction between the H3/H4 tetramer and the central part of the nucleosomal DNA is likely to be responsible for the positioning of nucleosomes of this group, and the DNA trajectory in these nucleosomes may differ in detail from the published structures.

Thus, from the stereochemical perspective, the in vitro nucleosomes studied here follow either an X-ray-like pattern (with strong deformations in the terminal parts of nucleosomal DNA), or an alternative pattern (with the deformations occurring predominantly in the central part of the nucleosomal DNA). The results presented here may be useful for genome-wide classification of nucleosomes, linking together structural and thermodynamic characteristics of nucleosomes with the underlying DNA sequence patterns guiding their positions.     

Phylogenetic analysis of DNA length mutations in a repetitive region of the Hawaiiandrosophila yolk protein geneYp2

Nucleotide sequence analysis has demonstrated that interspecific size variation in the YP2 yolk protein among HawaiianDrosophila is due to in-frame insertions and deletions in two repetitive segments of the coding region of the Yp2 gene. Sequence comparisons of the complex repetitive region close to the 5′ end of this gene across 34 endemic Hawaiian taxa revealed five length morphs, spanning a length difference of 21 nucleotides (nt). A phylogenetic character reconstruction of the length mutations on an independently derived molecular phylogeny showed clade-specific length variants arising from six ancient events: two identical insertions of 6 nt, and four deletions, one of 6 nt, one of 12 nt, and two identical but independent deletions of 15 nt. These mutations can be attributed to replication slippage with nontandem trinucleotide repeats playing a major role in the slipped-strand mispairing. Geographic analysis suggests that the 15 nt deletion which distinguishes theplanitibia subgroup from thecyrtoloma subgroup occurred on Oahu about 3 million years ago. The homoplasies observed caution against relying too heavily on nucleotide insertions/deletions for phylogenetic inference. In contrast to the extensive repeat polymorphisms within otherDrosophila and the human species, the more complex 5′Yp2 repetitive region analyzed here appears to lack polymorphism among HawaiianDrosophila, perhaps due to founder effects, low population sizes, and hitchhiking effects of selection on the immediately adjacent 5′ region. Correspondence to: M.P. Kambysellis     

Molecular cloning and expression analysis of <Emphasis Type="Italic">Bmrbp1</Emphasis>, the <Emphasis Type="Italic">Bombyx mori</Emphasis> homologue of the <Emphasis Type="Italic">Drosophila</Emphasis> gene <Emphasis Type="Italic">rbp1</Emphasis>

RBP1 is an important splicing factor involved in alternative splicing of the pre-mRNA of Drosophila sex-determining gene dsx. In this work, the Bombyx mori homologue of the rbp1 gene, Bmrbp1, was cloned. The pre-mRNA of Bmrbp1 gene is alternatively spliced to produce four mature mRNAs, named Bmrbp1-PA, Bmrbp1-PB, Bmrbp1-PC and Bmrbp1-PD, with nucleotide lengths of 799 nt, 1,316 nt, 894 nt and 724 nt, coding for 142 aa, 159 aa, 91 aa and 117 aa, respectively. BmRBP1-PA and BmRBP1-PD contain a N terminal RNA recognization motif (RRM) and a C terminal arginine/serine-rich domain, while BmRBP1-PB and BmRBP1-PC only share a RRM. Amino acid sequence alignments showed that BmRBP1 is conserved with its homologues in other insects and with other SR family proteins. The RT-PCR showed that Bmrbp1-PA was strongly expressed in all examined tissues and development stages, but Bmrbp1-PB was weakly expressed in these tissues and stages. The expression of both Bmrbp1-PA and Bmrbp1-PB showed no obvious sex difference. While the Bmrbp1-PC and Bmrbp1-PD were beyond detection by RT-PCR very likely due to their tissue/stage specificity. These results suggested that Bmrbp1 should be a member of SR family splicing factors, whether it is involved in the sex-specific splicing of Bmdsx pre-mRNA needs further research.     

Speculations on the subject of alcohol dehydrogenase and its properties in Drosophila and other flies

The alcohol dehydrogenases (ADH) and their genes (Adh) of Drosophila have been much studied by population and evolutionary biologists. I attempt to put some of these studies into a broad adaptionist perspective, suggesting the co-evolution of this enzyme with the fleshy fruits of angiosperms and fermenting yeasts. I suggest that these events occurred at about the K/T boundary (65 million years ago) and that the typical Drosophila (as exemplified by D. melanogaster) evolved from flies unable to use fermenting substrates as breeding sites. I also hint that the ADH enzymes of other flies (e.g., the tephritid fruit flies) may have evolved independently of those of Drosophila, but from a common ancestral gene. BioEssays 20:949–954, 1998. © 1998 John Wiley & Sons, Inc.     

Histone hyperacetylation facilitates chromatin remodelling in a Drosophila embryo cell-free system

We found that Drosophila embryo extract contains a protein activity (or activities) that can destabilize nucleosomes, resulting in increased sensitivity to DNase I, release of nucleosomal supercoiling, high levels of conformational flexibility of DNA and more diffuse micrococcal nuclease digestion patterns. Incorporation of histone H1 did not significantly affect this nucleosome remodelling. Remodelling occurs more efficiently in hyperacetylated chromatin. It was shown previously that hyperacetylated chromatin, reconstituted in a Drosophila embryo cell-free system, exhibits increased DNase I sensitivity and a high degree of conformational flexibility of DNA. The present data suggest that the more diffuse structure of acetylated chromatin is a result of chromatin remodelling by protein activities in the Drosophila embryo extract. Received: 4 November 1998 / Accepted: 10 May 1999     

Theory of DNA melting curves

Exact algorithms for the calculation of melting curves of heterogeneous DNA with N base pairs apparently require computer time proportional to N². However, it is shown that a decomposition of the loop entropy factor into a sum of I exponential functions (1) gives an extremely accurate approximation to the loop entropy factor for small values of I and (2) makes the computer time for the exact algorithms proportional to I·N. In effect, exact results for melting curves and lengths of helix or coil stretches are obtained with computer time comparable to that required for the Frank-Kamenetskii approximation. The remarkable accuracy of the latter for the fraction of helical content (errors of 0.01–0.05) is confirmed, but appreciably larger errors are found for the lengths of helix or coil stretches (typical errors of 30–100%).     

Pharmacogenetic Regulation of Acetylcholinesterase Activity in Drosophila Reveals the Regulatory Mechanisms of AChE Inhibitors in Synaptic Plasticity

We conducted experiments in Drosophila to investigate the consequences of altered acetylcholinesterase (AChE) activity in the nervous system. In ace hypomorphic mutant larvae, the amount of ace mRNA and the activity of AChE both in vivo and in vitro were significantly reduced compared with those of controls. Reduced Ace in Drosophila larvae resulted in significant down-regulation of branch length and the number of boutons in Type 1 glutamatergic neuromuscular junctions (NMJs). These defects in ace hypomorphic mutant larvae were suppressed when Musca domestica AChE was transgenically expressed. Because AChE inhibitors are utilized for medications for Alzheimer’s disease, we investigated whether pharmacological inhibition of AChE activity induced any synaptic defects. We found that controls exposed to a sublethal dose of DDVP phenocopied the synaptic structural defects of the ace hypomorphic mutant. These results suggest that down-regulation of AChE activity, regardless of whether it is due to genetic or pharmacological manipulations, results in altered synaptic architecture. Our study suggests that exposure to AChE inhibitors for 6–12 months may induce altered synaptic architectures in human brains with Alzheimer’s diseases, similar to those reported here. These changes may underlie or contribute to the loss of efficacy of AChE inhibitors after prolonged treatment.

     

Case–Case Comparison of Candida auris Versus Other Candida Species Bloodstream Infections: Results of an Outbreak Investigation in Colombia

Background

Candida auris is an emerging multidrug-resistant yeast that causes outbreaks in healthcare settings around the world. In 2016, clinicians and public health officials identified patients with C. auris bloodstream infections (BSI) in Colombian healthcare facilities. To evaluate potential risk factors and outcomes for these infections, we investigated epidemiologic and clinical features of patients with C. auris and other Candida species BSI.

Methods

We performed a retrospective case-case investigation in four Colombian acute care hospitals, defining a case as Candida spp. isolated from blood culture during January 2015–September 2016. C. auris BSI cases were compared to other Candida species BSI cases. Odds ratio (OR), estimated using logistic regression, was used to assess the association between risk factors and outcomes.

Results

We analyzed 90 patients with BSI, including 40 with C. auris and 50 with other Candida species. All had been admitted to the intensive care unit (ICU). No significant demographic differences existed between the two groups. The following variables were independently associated with C. auris BSI:?≥?15 days of pre-infection ICU stay (OR: 5.62, CI: 2.04–15.5), evidence of severe sepsis (OR: 3.70, CI 1.19–11.48), and diabetes mellitus (OR 5.69, CI 1.01–31.9).

Conclusion

Patients with C. auris BSI had longer lengths of ICU stay than those with other candidemias, suggesting that infections are acquired during hospitalization. This is different from other Candida infections, which are usually thought to result from autoinfection with host flora.

     

Synthesis of Dumbbell-Shaped Circular DMA-Molecules

Abstract

The syntheses of two dumbbell-shaped circular DMA-Molecules of chain lengths 140 and 150 nt are described. They are formed by enzymic ligation of chemically synthesized oligonucleotides containing self-complementary 5′ protruding ends.     

Different requirements for l(1)giant in two embryonic domains of Drosophila melanogaster

L(1)giant is a zygotic lethal mutation which affects the embryonic development of both the labial/thoracic segments and a subset of posterior abdominal segments. Using antibodies specific for proteins encoded by several Drosophila genes to identify the compartmental origin of the defects, we show that the requirement of giant activity is different in these two embryonic domains. Anteriorly, the posterior compartment of the labial segment is missing at the blastoderm stage. Posteriorly, cells are specifically deleted by cell death within the anterior compartments of abdominal segments 5–7 during germ band elongation. In mature embryos, posterior compartment structures of the peripheral nervous system of A5–7 are fused. In addition to a different pattern of defect in the two parts of the embryo, the kind of action appears different. Anteriorly, giant resembles a gap mutation in that a particular region is missing from the blastoderm fate map, whereas in the abdominal domain, giant affects the development of anterior compartment-specific structures.     

Drosophila centrosomes are unable to trigger parthenogenetic development of Xenopus eggs

Centrosomes are powerful and exclusive parthenogenetic agents in the Xenopus egg. We have previously shown that heterologous centrosomes from various vertebrate species were able to promote egg cleavage in Xenopus and that human centrosome activity was associated with an insoluble proteinacious structure that is not significantly simpler than the native centrosome. In this work, we have investigated the parthenogenetic capacity of more evolutionary distant centrosomes. We show that centrosomes devoid of centrioles, such as SPBs isolated from Saccharomyces cerevisiae, do not form asters of microtubules in cytoplasmic extracts from Xenopus eggs, and are inactive in the parthenogenetic test. We further show that Drosophila centrosomes which possess a typical centriole architecture, and are quite active to nucleate microtubules in Xenopus cytoplasmic extracts, are unable to trigger egg cleavage. This was observed both with centrosomes isolated from Drosophila syncytial embryos and nucleus-centrosome complexes from the Drosophila Kc23 cell line. We demonstrate that this inability could not be restored after pre-incubation of Drosophila centrosomes in the egg cytoplasm before injection. We conclude that the parthenogenetic activity of a centrosome is not directly linked to its capacity to nucleate microtubules from the egg tubulin, and that the evolutionary conserved nine-fold symmetrical structure of the centriole cannot be considered as sufficient for triggering procentriole assembly.     

Morphologische Variabilität der chromosomalen Funktionsstrukturen in den Spermatocytenkernen von Drosophila-Arten

Zusammenfassung In der Gattung Drosophila treten in den Zellkernen von primären Spermatocyten chromosomale Funktionsstrukturen auf, die im Prinzip wie die lateralen Schleifenpaare von Lampenbürstenchromosomen organisiert sind. Die Form der Schleifen ist bei jeder Art in artspezifischer Weise abgewandelt. Die morphologische Variabilität der Spermatocytenstrukturen von 54 Drosophila-Arten wird beschrieben. Das genetische Material in den Schleifen spielt möglicherweise eine entscheidende Rolle bei der Vorfertigung, Stabilisierung und programmierten Verpackung von Genprodukten, die erst in späteren Stadien während der Spermiohistogenese in den Zellen verbraucht werden.

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In the genus Drosophila the nuclei of primary spermatocytes contain special chromosomal functional structures which are organized as the pairs of lateral loops in lampbrush chromosomes. In each species the loops have their own species specific morphology. The morphological variability of the spermatocyte structures in 54 Drosophila species is described. Although the nuclei of spermatids are synthetically inactive, the cells are able to synthesize proteins which are essential for the differentiation of sperm. Therefore, special mechanisms are necessary for the preformation, stabilization, and programmed packaging of gene products during the spermatocyte stage. This may be the function of the spermatocyte loops.

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Verfasser dankt der Deutschen Forschungsgemeinschaft für eine Reisebeihilfe. Die Untersuchungen werden jetzt ebenfalls mit Unterstützung durch die Deutsche Forschungsgemeinschaft fortgesetzt. Mein besonderer Dank gilt Herrn Prof. Dr. W. S. Stone und seinen Mitarbeitern von der Genetics Foundation an der Universität von Texas in Austin. Die Zeichnungen wurden von Herrn E. Freiberg angefertigt.

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Herrn Prof. Dr. W. E. Ankel, meinem verehrten Lehrer, zum 70. Geburtstag gewidmet.     

CAF-1 is required for efficient replication of euchromatic DNA in <Emphasis Type="Italic">Drosophila</Emphasis> larval endocycling cells

The endocycle constitutes an effective strategy for cell growth during development. In contrast to the mitotic cycle, it consists of multiple S-phases with no intervening mitosis and lacks a checkpoint ensuring the replication of the entire genome. Here, we report an essential requirement of chromatin assembly factor-1 (CAF-1) for Drosophila larval endocycles. This complex promotes histone H3–H4 deposition onto newly synthesised DNA in vitro. In metazoans, the depletion of its large subunit leads to the rapid accumulation of cells in S-phase. However, whether this slower S-phase progression results from the activation of cell cycle checkpoints or whether it reflects a more direct requirement of CAF-1 for efficient replication in vivo is still debated. Here, we show that, strikingly, Drosophila larval endocycling cells depleted for the CAF-1 large subunit exhibit normal dynamics of progression through endocycles, although accumulating defects, such as perturbation of nucleosomal organisation, reduction of the replication efficiency of euchromatic DNA and accumulation of DNA damage. Given that the endocycle lacks a checkpoint ensuring the replication of the entire genome, the biological context of Drosophila larval development offered a unique opportunity to highlight the requirement of CAF-1 for chromatin organisation and efficient replication processes in vivo, independently of checkpoint activation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The dynamics of the motions of Drosophila melanogaster laboratory populations correlates with the parameters of space weather

Analysis of the motions of Drosophila melanogaster laboratory populations and their relationship to the heliogeophysical parameters (the radio-wave flux at the wavelength of 10.7 cm (F10.7), Wolf numbers (W), the planetary K _p-index, an indicator of geomagnetic activity) was carried out. A significant correlation between the changes in the characteristics of solar activity (F10.7, W) and the dynamics of the motions of Drosophila was detected. The association strongly depends on the sexual structure of the Drosophila populations, which follows from the difference in the distributions of this association.

     

Ihog proteins contribute to integrin-mediated focal adhesions

Integrin expression forms focal adhesions, but how this process is physiologically regulated is unclear. Ihog proteins are evolutionarily conserved, playing roles in Hedgehog signaling and serving as trans-homophilic adhesion molecules to mediate cell-cell interactions. Whether these proteins are also engaged in other cell adhesion processes remains unknown. Here, we report that Drosophila Ihog proteins function in the integrin-mediated adhesions. Removal of Ihog proteins causes blister and spheroidal muscle in wings and embryos, respectively. We demonstrate that Ihog proteins interact with integrin via the extracellular portion and that their removal perturbs integrin distribution. Finally, we show that Boc, a mammalian Ihog protein, rescues the embryonic defects caused by removing its Drosophila homologs. We thus propose that Ihog proteins contribute to integrin-mediated focal adhesions.

     

Up-regulation of stress-inducible genes in tobacco and <Emphasis Type="Italic">Arabidopsis</Emphasis> cells in response to abiotic stresses and ABA treatment correlates with dynamic changes in histone H3 and H4 modifications

Animal cells react to mitogenic or stress stimuli by rapid up-regulation of immediate-early (IE) genes and a parallel increase in characteristic modifications of core histones: chromatin changes, collectively termed the nucleosomal response. With regard to plants little is known about the accompanying changes at the chromatin level. We have used tobacco BY-2 and Arabidopsis T87 cell lines to study the nucleosomal response of plant cells to high salinity, cold and exogenous abscisic acid (ABA). When in quiescent stage, both tobacco and Arabidopsis cells show the typical nucleosomal response to high salinity and cold stress, manifested by rapid transient up-regulation of histone H3 Ser-10 phosphorylation, immediately followed by transient up-regulation of H3 phosphoacetylation and histone H4 acetylation. For each of the studied stresses the observed nucleosomal response was strictly correlated with the induction of stress-type specific genes. The dynamics of histone modifications in BY-2 cells in response to exogenous ABA exhibited a more complex pattern than that evoked by the two abiotic stresses, probably due to superposition of the primary and secondary effects of ABA. A rapid increase in H3 Ser-10 phosphorylation was also observed in whole leaves subjected to high salinity; however, the rate of change in this modification was much slower than in cultured cells. Together, these results indicate that the quiescent BY-2 and T87 cell lines show a typical nucleosomal response to abiotic stresses and ABA treatment and may represent suitable models for the study of chromatin-mediated mechanisms of stress tolerance in plants.     

Regulation of intestinal stem cells in mammals and Drosophila

The digestive systems in mammals and Drosophila are quite different in terms of their complexity and organization, but their biological functions are similar. The Drosophila midgut is a functional equivalent of the mouse small intestine. Adult intestinal stem cells (ISCs) have been identified in both the mouse small intestine and Drosophila midgut. The anatomy and cell renewal in the Drosophila midgut are similar to those in the mouse small intestine: the intestinal epithelium in both systems is a tube composed of epithelial cells with absorptive and secretory functions; the Notch signaling controls absorptive versus secretory fate decisions in the intestinal epithelium; cell renewal in both systems starts from stem cells in the basal cell layer, and the differentiated cells then move toward the lumen. However, it is clear that the stem cells in the two systems are regulated in different ways. In this review, we will compare cell renewal and stem cell regulation in the two systems. J. Cell. Physiol. 222:33–37, 2010. © 2009 Wiley‐Liss, Inc.