Identification and Chronological Analysis of Genomic Signatures in Influenza A Viruses

An increase in the availability of data on the influenza A viruses (IAV) has enabled the identification of the potential determinants of IAV host specificity using computational approaches. In this study, we proposed an alternative approach, based on the adjusted Rand index (ARI), for the evaluation of genomic signatures of IAVs and their ability to distinguish hosts they infected. Our experiments showed that the host-specific signatures identified using the ARI were more characteristic of their hosts than those identified using previous measures. Our results provided updates on the host-specific genomic signatures in the internal proteins of the IAV based on the sequence data as of February 2013 in the National Center for Biotechnology Information (NCBI). Unlike other approaches for signature recognition, our approach considered not only the ability of signatures to distinguish hosts (according to the ARI), but also the chronological relationships among proteins. We identified novel signatures that could be mapped to known functional domains, and introduced a chronological analysis to investigate the changes in host-specific genomic signatures over time. Our chronological analytical approach provided results on the adaptive variability of signatures, which correlated with previous studies’ findings, and indicated prospective adaptation trends that warrant further investigation.     

Isolation and Phylogenetic Analysis of Novel Viruses Infecting the Phytoplankton Phaeocystis globosa (Prymnesiophyceae)

Viruses infecting the harmful bloom-causing alga Phaeocystis globosa (Prymnesiophyceae) were readily isolated from Dutch coastal waters (southern North Sea) in 2000 and 2001. Our data show a large increase in the abundance of putative P. globosa viruses during blooms of P. globosa, suggesting that viruses are an important source of mortality for this alga. In order to examine genetic relatedness among viruses infecting P. globosa and other phytoplankton, DNA polymerase gene (pol) fragments were amplified and the inferred amino acid sequences were phylogenetically analyzed. The results demonstrated that viruses infecting P. globosa formed a closely related monophyletic group within the family Phycodnaviridae, with at least 96.9% similarity to each other. The sequences grouped most closely with others from viruses that infect the prymnesiophyte algae Chrysochromulina brevifilum and Chrysochromulina strobilus. Whether the P. globosa viruses belong to the genus Prymnesiovirus or form a separate group needs further study. Our data suggest that, like their phytoplankton hosts, the Chrysochromulina and Phaeocystis viruses share a common ancestor and that these prymnesioviruses and their algal host have coevolved.     

Discovery of Two Novel Viruses Expands the Diversity of Single-Stranded DNA and Single-Stranded RNA Viruses Infecting a Cosmopolitan Marine Diatom

Recent studies have suggested that diatom viruses are an important factor affecting diatom population dynamics, which in turn are important in considering marine primary productivity. The marine planktonic diatom Chaetoceros tenuissimus Meunier is a cosmopolitan species and often causes blooms off the western coast of Japan. To date, two viruses, C. tenuissimus DNA virus (CtenDNAV) type I and CtenRNAV type I, have been identified that potentially affect C. tenuissimus population dynamics in the natural environment. In this study, we successfully isolated and characterized two additional novel viruses (CtenDNAV type II and CtenRNAV type II). This paper reports the basic characteristics of these new viruses isolated from surface water or sediment from the Hiroshima Bay, Japan. The physiological and morphological characteristics of the two new viruses were similar to those of the previously isolated viruses. However, the amino acid sequences of the structural proteins of CtenDNAV type II and CtenRNAV type II were clearly distinct from those of both type I viruses, with identity scores of 38.3% and 27.6%, respectively. Our results suggest that at least four genetically distinct viruses sharing the same diatom host are present in western Japan and affect the population dynamics of C. tenuissimus. Moreover, the result that CtenRNAV type II lysed multiple diatom species indicates that RNA viruses may affect various diatom populations in the natural environment.     

Editor's choice: Large,Male Germ Cell-Specific Hypomethylated DNA Domains With Unique Genomic and Epigenomic Features on the Mouse X Chromosome

To understand the epigenetic regulation required for germ cell-specific gene expression in the mouse, we analysed DNA methylation profiles of developing germ cells using a microarray-based assay adapted for a small number of cells. The analysis revealed differentially methylated sites between cell types tested. Here, we focused on a group of genomic sequences hypomethylated specifically in germline cells as candidate regions involved in the epigenetic regulation of germline gene expression. These hypomethylated sequences tend to be clustered, forming large (10 kb to ∼9 Mb) genomic domains, particularly on the X chromosome of male germ cells. Most of these regions, designated here as large hypomethylated domains (LoDs), correspond to segmentally duplicated regions that contain gene families showing germ cell- or testis-specific expression, including cancer testis antigen genes. We found an inverse correlation between DNA methylation level and expression of genes in these domains. Most LoDs appear to be enriched with H3 lysine 9 dimethylation, usually regarded as a repressive histone modification, although some LoD genes can be expressed in male germ cells. It thus appears that such a unique epigenomic state associated with the LoDs may constitute a basis for the specific expression of genes contained in these genomic domains.     

Synthesis and characterization of a d(ApG) platinated nonanucleotide duplex.

The nonamer 5'd(CTCAGCCTC) 3' 1 has been reacted with cis-diamminediaquaplatinum(II) in water at pH 4.2. The major reaction product was shown by enzymatic digestion and 1H NMR to be the d(ApG)cis-Pt(NH3)2 chelate [cis-Pt(NH3)2[d(CTCAGCCTC)-N7(4),N7(5)]] 1-Pt. When mixed with its complementary strand 2, 1-Pt forms a B DNA type duplex 3-Pt with a Tm of 35 degrees C (versus 58 degrees C for the unplatinated duplex). The NMR study of the exchangeable protons of 3-Pt revealed that the helix distortion is localized on the CA*G*-CTG moiety (the asterisks indicating the platinum chelation sites) with a strong perturbation of the A*(4)T(15) base pair related to a large tilt of A*(4).     

Entry and Disassembly of Large DNA Viruses: Electron Microscopy Leads the Way

Nucleocytoplasmic large DNA viruses are a steadily growing group of viruses that infect a wide range of hosts and are characterized by large particle dimensions and genome sizes. Understanding how they enter into the host cell and deliver their genome in the cytoplasm is therefore particularly intriguing. Here, we review the current knowledge on the entry of two of the best-characterized nucleocytoplasmic large DNA viruses: the poxvirus Vaccinia virus (VACV) and the giant virus Mimivirus. While previous studies on VACV had proposed both direct fusion at the plasma membrane and endocytosis as entry routes, more recent biochemical and morphological data argue for macropinocytosis as well. Notably, direct imaging by electron microscopy (EM) also supported the existence of parallel ways of entry for VACV. Instead, all the giant viruses studied so far only enter cells by phagocytosis as observed by EM, and we discuss the mechanisms for opening of the particle, fusion of the viral and phagosomal membranes and genome delivery via a unique portal, specific for each giant virus. VACV core uncoating, in contrast, remains a morphologically ill-defined process. We argue that correlated light and electron microscopy methods are required to study VACV entry and uncoating in a direct and systematic manner. Such EM studies should also address whether entry of single particles and viral aggregates is different and thus provide an explanation for the different modes of entry described in the literature.     

The Clinical Physiology of Water Metabolism: Part III: The Water Depletion (Hyperosmolar) and Water Excess (Hyposmolar) Syndromes

Hyperosmolality occurs when there are defects in the two major homeostatic mechanisms required for water balance—thirst and arginine vasopressin (AVP) release. In this situation hypotonic fluids are lost in substantial quantities causing depletion of both intracellular and extracellular fluid compartments. Patients with essential hypernatremia have defective osmotically stimulated AVP release and thirst but may have intact mechanisms for AVP release following hypovolemia. Hyperosmolality can also be seen in circumstances in which impermeable solutes are present in excessive quantities in extracellular fluid. Under these conditions there is cellular dehydration and the serum sodium may actually be reduced by water drawn out of cells along an osmotic gradient.Hyposmolality and hyponatremia may be seen in a variety of clinical conditions. Salt depletion, states in which edema occurs and the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) may all produce severe dilution of body fluids resulting in serious neurologic disturbances. The differential diagnosis of these states is greatly facilitated by careful clinical assessment of extracellular fluid volume and by determination of urine sodium concentration. Treatment of the hyposmolar syndromes is contingent on the pathophysiology of the underlying disorder; hyponatremia due to salt depletion is treated with infusions of isotonic saline whereas mild hyponatremia in cirrhosis and ascites is best treated with water restriction. Severe symptomatic hyponatremia due to SIADH is treated with hypertonic saline therapy, sometimes in association with intravenous administration of furosemide. Less severe, chronic cases may be treated with dichlormethyltetracycline which blocks the action of AVP on the collecting duct.     

Characterization of a DNA damage-recognition protein from mammalian cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the anticancer drug cisplatin

A factor has been identified in extracts from human HeLa and hamster V79 cells that retards the electrophoretic mobility of several DNA restriction fragments modified with the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin). Binding of the factor to cisplatin-modified DNA was sensitive to pretreatment with proteinase K, establishing that the factor is a protein. Gel mobility shifts were observed with probes containing as few as seven Pt atoms per kilobase of duplex DNA. By competition experiments the dissociation constant, Kd, of the protein from cisplatin-modified DNA was estimated to be (1-20) X 10(-10) M. Protein binding is selective for DNA modified with cisplatin, [Pt(en)Cl2] (en, ethylenediamine), and [Pt(dach)Cl2] (dach, 1,2-diaminocyclohexane) but not with chemotherapeutically inactive trans-diamminedichloroplatinum(II) or monofunctionally coordinating [Pt(dien)Cl]Cl (dien, diethylenetriamine) complexes. The protein also does not bind to DNA containing UV-induced photoproducts. The protein binds specifically to 1,2-intrastrand d(GpG) and d(ApG) cross-links formed by cisplatin, as determined by gel mobility shifts with synthetic 110-bp duplex oligonucleotides; these modified oligomers contained five equally spaced adducts of either cis-[Pt(NH3)2d(GpG) or cis-[Pt(NH3)2d(ApG)]. Oligonucleotides containing the specific adducts cis-[Pt(NH3)2d(GpTpG)], trans-[Pt(NH3)2d(GpTpG)], or cis-[Pt(NH3)2(N3-cytosine)d(G)] were not recognized by the protein. The apparent molecular weight of the protein is 91,000, as determined by sucrose gradient centrifugation of a preparation partially purified by ammonium sulfate fractionation. Binding of the protein to platinum-modified DNA does not require cofactors but is sensitive to treatment with 5 mM MnCl2, CdCl2, CoCl2, or ZnCl2 and with 1 mM HgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

从烟草上分离的中国番茄曲叶病毒及其卫星DNA全基因组结构特征

本研究从具有典型曲叶病症状的广西靖西烟草病植株上分离到病毒分离物JX-2,全基因组序列测定结果表明,JX-2 DNA-A 全长2 738个核苷酸,共编码6个开放阅读框架(open reading frames,ORFs),其中病毒链编码AV1 (CP)和AV2两个ORFs,互补链编码AC1、AC2、AC3和AC4 共4个ORFs.BLAST结果表明,JX-2 DNA-A与中国番茄曲叶病毒(Tomato leaf curl China virus,ToLCCNV)各分离物的相似性在93.0%~99.7%之间,其中与ToLCCNV广西番茄分离物ToLCCNV-G32的相似性最高,达99.7%,而与其它双生病毒的同源性均在88.0%以下,表明JX-2是ToLCCNV的一个分离物.基于JX-2和已报道的双生病毒属代表种DNA-A全基因组核苷酸序列构建的系统进化树显示,JX-2与ToLCCNV-G32分离物的亲缘关系最近,并与ToLCCNV其它分离物形成一个分支,而与其它10种双生病毒的亲缘关系均相对较远.利用双生病毒卫星DNAβ的特异性引物β01/β02在JX-2样品中扩增到DNAβ分子(JX-2β),全长为1 341个核苷酸,其互补链编码1个ORF (即βC1),并包含一个富含A序列和一个卫星病毒保守序列.序列分析表明,JX-2β与ToLCCNV伴随的DNAβ的相似性在91.0%~96.1%之间,其中与ToLCCNV-G61DNAβ和ToLCCNV-G18 DNAβ的相似性最高(96.1%),与其它卫星DNAβ的相似性均低于61.8%.基于JX-2β全基因组核苷酸序列构建的系统进化关系树显示,JX-2β与ToLCCNV G61分离物伴随的DNAβ亲缘关系最近,并形成一个独立的分支,再与ToLCCNV 其余两个分离物伴随的DNAβ形成一个较大的分支.这是首次报道从烟草中分离到的中国番茄曲叶病毒及其伴随卫星DNA分子的全基因组结构特征.     

Quick Identification and Localization of CpG Islands in Large Genomic Fragments by Partial Digestion with Hpa II and Hha I

More than 50% of mammalian genes are associated with CpG islandsand thus they serve as a good gene marker. We have devised asimple method to scan large pieces of native or cloned genomicDNA for CpG islands. The method is based on the presence ofmultiple Hpa II and Hha I sites in CpG islands, at a frequency30 times higher than in the rest of the genome. The steps includecomplete digestion of DNA with a rare-cutting restriction endonuclease(to produce large fragments with defined ends), partial digestionwith Hpa II and Hha I, and subsequent Southern hybridizationwith an end probe. This identifies a CpG island as a clusterof sub-bands and, based on their electrophoretic mobility, onecan immediately locate the island relative to the ends. Formany vectors, universal probes flanking the cloning site areavailable, enabling the simultaneous analysis of a large numberof samples. We demonstrated the usefulness of the method byanalyzing known CpG islands in native genomic DNA and lambda,cosmid and P1 clones, and by isolating two novel transcribedislands from anonymous cosmid clones. Our method is quick, inexpensive,and can detect CpG islands with few or even no rare-cutter sites.     

Phages Infecting Vibrio vulnificus Are Abundant and Diverse in Oysters (Crassostrea virginica) Collected from the Gulf of Mexico

Phages infecting Vibrio vulnificus were abundant (>10⁴ phages g of oyster tissue⁻¹) throughout the year in oysters (Crassostrea virginica) collected from estuaries adjacent to the Gulf of Mexico (Apalachicola Bay, Fla.; Mobile Bay, Ala.; and Black Bay, La.). Estimates of abundance ranged from 10¹ to 10⁵ phages g of oyster tissue⁻¹ and were dependent on the bacterial strain used to assay the sample. V. vulnificus was near or below detection limits (<0.3 cell g⁻¹) from January through March and was most abundant (10³ to 10⁴ cells g⁻¹) during the summer and fall, when phage abundances also tended to be greatest. The phages isolated were specific to strains of V. vulnificus, except for one isolate that caused lysis in a few strains of V. parahaemolyticus. Based on morphological evidence obtained by transmission electron microscopy, the isolates belonged to the Podoviridae, Styloviridae, and Myoviridae, three families of double-stranded DNA phages. One newly described morphotype belonging to the Podoviridae appears to be ubiquitous in Gulf Coast oysters. Isolates of this morphotype have an elongated capsid (mean, 258 nm; standard deviation, 4 nm; n = 35), with some isolates having a relatively broad host range among strains of V. vulnificus. Results from this study indicate that a morphologically diverse group of phages which infect V. vulnificus is abundant and widely distributed in oysters from estuaries bordering the northeastern Gulf of Mexico.     

Unique mutations in mitochondrial DNA of senescence-accelerated mouse (SAM) strains

Mitochondrial DNA (mtDNA) is exclusively inherited maternally and hence could offer a good method for tracing the lineage of mouse strains. We examined the mtDNA sequence of senescence-accelerated mouse (SAM) strains as well as other laboratory strains of inbred mice to deduce the ancestral strain of SAM. Four unique mutations were identified at bases 2256, 10,847, 11,181, and 13,053 in SAM strains. The mutations were not found in other mouse strains including AKR/J, one of the parental strains of SAM. Comparison of the mtDNA sequences also led to the consensus mtDNA sequence of laboratory strains of inbred mice. The seven laboratory strains of common inbred mice showed polymorphisms at base 9348, thymine repeat from base 9818, and adenine repeat from base 9821, and could be classified into five types by combination of the differences. Although we could not identify mouse strains with the same type of mtDNA as SAM in this study, the polymorphisms would provide a promising clue to ascertain the ancestral strain(s) of SAM. The polymorphism in mtDNA could be used to ascertain the genealogy of other mouse strains as well.     

Interaction of cis- and trans-RuCl2(DMSO)4 with the nucleotides GpA, d(GpA), ApG, d(ApG) and d(CCTGGTCC): high-field NMR characterization of the reaction products

 Both cis- and trans-RuCl₂(DMSO)₄ (cis-Ru and trans-Ru) react with ApG, GpA, d(ApG) and d(GpA) to yield products with bifunctional metal coordination of the bases. For each dinucleotide one major product and several minor species are formed. This is in contrast to previous results on analogous reactions between trans-Ru and d(GpG) where a substantial amount of an intermediate species was found. The rates of reaction between dinucleotides and cis-Ru are approximately 20-fold slower than for trans-Ru. The compounds formed with the two isomers exhibit identical proton NMR spectra, suggesting the same coordination mode for ruthenium in the final product. The two purine bases are coordinated to ruthenium through N7 in a head-to-head conformation with the glycosidic angles being in the anti range. Coupling constants indicate a relatively pure 3′-endo conformation for the 5′-sugar and mainly 2′-endo for the 3′-sugar. The similar bifunctional binding mode of cis- and trans-Ru(II) with dinucleotides as evident from the NMR spectra are in contrast to the different mode of interaction suggested earlier for cis- and trans-Ru complexes with DNA. trans-Ru interacts with the deoxyoctanucleotide d(CCTGGTCC), giving two main products during the first 2 h of incubation time. Four H8 guanine resonances are shifted downfield, characteristic of N7 metal coordination. The products are not analyzed in detail, but it is suggested that the structures may be described as two chiral G(N7/N7) chelates. Received: 20 August 1998 / Accepted: 20 January 1999     

An Evolutionary View of the Arms Race between Protein Kinase R and Large DNA Viruses

To establish productive infections, viruses must counteract numerous cellular defenses that are poised to recognize viruses as nonself and to activate antiviral pathways. The opposing goals of host and viral factors lead to evolutionary arms races that can be illuminated by evolutionary and computational methods and tested in experimental models. Here we illustrate how this perspective has been contributing to our understanding of the interactions of the protein kinase R pathway with large DNA viruses.     

Formation and structure of reaction products of cis-PtCl2(NH3)2 with d(ApG) and/or d(GpA) in di-, tri- and penta-nucleotides. Preference for GpA chelation over ApG chelation

The reaction products of cis-PtCl2(NH)3)2 with several deoxyribonucleotides containing d(ApG) and/or d(GpA) have been studied. The various reaction products were separated by high-performance liquid chromatography and characterized by means of absorbance at 254 nm in combination with atomic absorption spectroscopy and 300-MHz 1H-NMR (pH dependence of the non-exchangeable base-protons, T1 relaxation time determinations). For the larger fragments the results from these techniques were confirmed by enzymatic degradation studies of the platinated fragments. The smallest of the investigated nucleotides, d(ApG) and d(GpA), both formed a variety of different platinum chelates. In the reaction with d(ApG) 15% cis-Pt(NH3)2-[d(ApG)N1(1),N7(2)] and 78% cis-Pt(NH3)2[d(ApG)N7(1),N7(2)] were found, 4% of the reacted material consisted of a 1 mol Pt/2 mol dinucleotide product, and 3% of an unidentified 1:1 product. From the main product two rotamers were found to occur: at room temperature, 81% anti,anti and 19% anti,syn product is present. With d(GpA) about equal amounts of N1,N7 and N7,N7 products were found; for both products the anti,anti and anti,syn conformations were found, respectively. Upon reaction of cis-PtCl2(NH3)2 with d(pApG) and d(pGpA) only the N7,N7 products were found; at room temperature and pH greater than 1.5 these products were present in anti,anti conformation. However, for the d(pApG)-platinum chelate at -20 degrees C a small amount (less than 5%) of a second product could be observed in NMR. For the d(pGpA)-platinum chelate a second N7,N7-coordinated product was observed when the pH of the NMR sample was lowered to 1.1 (at this pH the free 5'-phosphate group is protonated). With the larger fragments d(ApGpA), d(pApGpA) and d(TpApGpApT) the intra-molecular competition between the formation of the d(ApG) or the d(GpA) chelates could be studied. Using these nucleotides no N1-coordinated products or rotamers were observed. In the case of d(ApGpA) and d(TpApGpApT) the d(GpA) chelate (67% and 75% respectively) was favoured over the d(ApG) chelate, while with d(pApGpA) about equal amounts of both chelates were formed.     

Tumor Cell Migration and Invasion Are Enhanced by Depletion of Rap1 GTPase-activating Protein (Rap1GAP)

The functional significance of the widespread down-regulation of Rap1 GTPase-activating protein (Rap1GAP), a negative regulator of Rap activity, in human tumors is unknown. Here we show that human colon cancer cells depleted of Rap1GAP are endowed with more aggressive migratory and invasive properties. Silencing Rap1GAP enhanced the migration of confluent and single cells. In the latter, migration distance, velocity, and directionality were increased. Enhanced migration was a consequence of increased endogenous Rap activity as silencing Rap expression selectively abolished the migration of Rap1GAP-depleted cells. ROCK-mediated cell contractility was suppressed in Rap1GAP-depleted cells, which exhibited a spindle-shaped morphology and abundant membrane protrusions. Tumor cells can switch between Rho/ROCK-mediated contractility-based migration and Rac1-mediated mesenchymal motility. Strikingly, the migration of Rap1GAP-depleted, but not control cells required Rac1 activity, suggesting that loss of Rap1GAP alters migratory mechanisms. Inhibition of Rac1 activity restored membrane blebbing and increased ROCK activity in Rap1GAP-depleted cells, suggesting that Rac1 contributes to the suppression of contractility. Collectively, these findings identify Rap1GAP as a critical regulator of aggressive tumor cell behavior and suggest that the level of Rap1GAP expression influences the migratory mechanisms that are operative in tumor cells.