Inactivation of Bacteriophages by Thiol Reducing Agents

Adaptation of the vaccinia virus expression system to HeLa S3 suspension bioreactor culture for the production of recombinant protein was conducted. Evaluation of hollow fiber perfusion of suspension culture demonstrated its potential for increased cell density prior to infection. The hollow fiber was also used for medium manipulations prior to infection. Two process parameters, multiplicity of infection (MOI) and temperature during the protein production phase, were evaluated to determine their effect on expression of the reporter protein, enhanced green fluorescent protein (EGFP). An MOI of 1.0 was sufficient for infection and led to the highest level of intracellular EGFP expression. Reducing the temperature to 34 °C during the protein production phase increased production of the protein two-fold compared to 37 °C in spinner flask culture. Scaling up the process to a 1.5-liter bioreactor with hollow fiber perfusion led to an overall production level of 9.9 μg EGFP/10⁶ infected cells, or 27 mg EGFP per liter.     

Inactivation of Poliovirus 1 and F-Specific RNA Phages and Degradation of Their Genomes by UV Irradiation at 254 Nanometers

Several models (animal caliciviruses, poliovirus 1 [PV1], and F-specific RNA bacteriophages) are usually used to predict inactivation of nonculturable viruses. For the same UV fluence, viral inactivation observed in the literature varies from 0 to 5 logs according to the models and the methods (infectivity versus molecular biology). The lack of knowledge concerning the mechanisms of inactivation due to UV prevents us from selecting the best model. In this context, determining if viral genome degradation may explain the loss of infectivity under UV radiation becomes essential. Thus, four virus models (PV1 and three F-specific RNA phages: MS2, GA, and Qβ) were exposed to UV radiation from 0 to 150 mJ · cm⁻². PV1 is the least-resistant virus, while MS2 and GA phages are the most resistant, with phage Qβ having an intermediate sensitivity; respectively, 6-log, 2.3-log, 2.5-log, and 4-log decreases for 50 mJ · cm⁻². In parallel, analysis of RNA degradation demonstrated that this phenomenon depends on the fragment size for PV1 as well as for MS2. Long fragments (above 2,000 bases) for PV1 and MS2 fell rapidly to the background level (>1.3-log decrease) for 20 mJ · cm⁻² and 60 mJ · cm^{− 2}, respectively. Nevertheless, the size of the viral RNA is not the only factor affecting UV-induced RNA degradation, since viral RNA was more rapidly degraded in PV1 than in the MS2 phage with a similar size. Finally, extrapolation of inactivation and UV-induced RNA degradation kinetics highlights that genome degradation could fully explain UV-induced viral inactivation.     

Human Origin Recognition Complex Binds Preferentially to G-quadruplex-preferable RNA and Single-stranded DNA

Origin recognition complex (ORC), consisting of six subunits ORC1–6, is known to bind to replication origins and function in the initiation of DNA replication in eukaryotic cells. In contrast to the fact that Saccharomyces cerevisiae ORC recognizes the replication origin in a sequence-specific manner, metazoan ORC has not exhibited strict sequence-specificity for DNA binding. Here we report that human ORC binds preferentially to G-quadruplex (G4)-preferable G-rich RNA or single-stranded DNA (ssDNA). We mapped the G-rich RNA-binding domain in the ORC1 subunit, in a region adjacent to its ATPase domain. This domain itself has an ability to preferentially recognize G4-preferable sequences of ssDNA. Furthermore, we found, by structure modeling, that the G-rich RNA-binding domain is similar to the N-terminal portion of AdoMet_MTase domain of mammalian DNA methyltransferase 1. Therefore, in contrast with the binding to double-stranded DNA, human ORC has an apparent sequence preference with respect to its RNA/ssDNA binding. Interestingly, this specificity coincides with the common signature present in most of the human replication origins. We expect that our findings provide new insights into the regulations of function and chromatin binding of metazoan ORCs.     

Inactivation of the ATP-dependent DNase of Escherichia coli After Infection with Double-Stranded DNA Phages

The ATP-dependent DNase activity of Escherichia coli disappeared or was markedly reduced after infection with double-stranded DNA phages, T2, T3, T4, T5, T6, T7, lambda, phi80, and P1, but not with the single-stranded DNA phage f1, or the RNA phage Qbeta. This DNase activity was not reduced when chloramphenicol was added prior to phage infection.     

Strong Binding of Single-stranded DNA by Stem-loop Oligonucleotides

Abstract

We report that oligodeoxynucleotides which form stem-loop hairpin structures and which have pyrimidine-rich loops can form strong complexes with complementary single-stranded DNA sequences. Stem-loop oligonucleotides were constructed with a 25-nt T-rich loop and with variable Watson-Crick stems. The complexes of these oligomers with the sequence dA_gwere studied by thermal denaturation. Evidence is presented that the complexes are one-to-one, bimolecular complexes in which the pyrimidine loop bases comprise the outer strands in a pyr · pur · pyr triplex, in effect chelating the purine strand in the center of the loop. Melting temperatures for the loop complexes are shown to be up to 29 °C higher than Watson- Crick duplex of the same length. It is shown that the presence of a stem increases stability of the triplex relative to an analogous oligomer without a stem. The effect of stem length on the stability of such a complex is examined. Such hairpin oligomers represent a new approach to the sequence-specific binding of single-stranded RNA and DNA. In addition, the finding raises the possibility that such a complex may exist in natural RNA folded sequences.     

Grouping of RNA Phages by a Millipore Filtration Method

Twenty-five strains of RNA phages were tested for their filtration and elution patterns (F-E patterns) by a millipore filtration method. These strains, including MS2, f2 and R17, were separated in three groups in this aspect. We named these groups as group I, II and III. According to this grouping, MS2, f2 and R17 belonged to group I and Qβ belonged to group III. Furthermore, it was shown that group III was further divided in two sub-groups (IIIa and IIIb) by this method. Grouping based on the F-E patterns was in extremely good accordance with the grouping based on the serological properties. This grouping was also supported by the results of chemical and physical analyses of these RNA phages, that is, RNA phages which belonged to the same group had several common properties. Basic Information on the millipore filtration method was also presented in this paper.     

Single-stranded DNA binding and methylation by EcoP1I DNA methyltransferase

EcoP1I methyltransferase (M.EcoP1I) belongs to the type III restriction-modification system encoded by prophage P1 that infects Escherichia coli. Binding of M.EcoP1I to double-stranded DNA and single-stranded DNA has been characterized. Binding to both single- and double-stranded DNA could be competed out by unlabeled single-stranded DNA. Metal ions did not influence DNA binding. Interestingly, M.EcoP1I was able to methylate single-stranded DNA. Kinetic parameters were determined for single- and double-stranded DNA methylation. This feature of the enzyme probably functions in protecting the phage genome from restriction by type III restriction enzymes and thus could be considered as an anti-restriction system. This study describing in vitro methylation of single-stranded DNA by the type III methyltransferase EcoP1I allows understanding of the mechanism of action of these enzymes and also their role in the biology of single-stranded phages.     

Separation of Single-stranded DNA,Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth^1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes ³. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes ^4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates ¹⁰. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community’s genetic signature. Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s¹¹. By exploiting the charge interaction between the positively-charged Ca²⁺ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing ¹². Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.Download video file.^{(86M, mov)}     

Mechanism of Inactivation of Bacteriophage J1 by Ascorbic Acid

The mechanism of inactivation of a double-stranded DNA phage, phage J1 of Lactobacillus casei, by ascorbic acid was investigated.

Bubbling air, oxidizing agents and transition metal ions enhanced the rate of inactivation of the phage by ascorbic acid. In contrast, bubbling nitrogen gas, other reducing agents and radical scavengers prevented the inactivation. The results indicated that the inactivating effect of ascorbic acid was oxygen dependent and caused by free radicals formed during the autoxidation of ascorbic acid.

The target of ascorbic acid in the phage particle was not the tail protein but DNA. Ascorbic acid caused single-strand scissions in phage DNA, as exhibited by alkaline sucrose density gradient centrifugation analysis, and caused a slight decrease in the viscosity of DNA.     

Subgroups in Group III of RNA Phages

In our previous studies, RNA phage strains were separated into 3 major groups on the basis of filtration efficiency through Millipore filters. In the present study, the strains of group III were shown to be further divided into 3 subgroups: (a) Qβ, NH, SG; (b) VK, SO; (c) ST. This subgrouping was found to be compatible with the serological grouping and pronase sensitivity with the exception of strain NM. Strain NM was classified in subgroup (a) by the Millipore filtration and in subgroup (b) by the other two methods.     

Human DNA Exonuclease TREX1 Is Also an Exoribonuclease That Acts on Single-stranded RNA

3′ repair exonuclease 1 (TREX1) is a known DNA exonuclease involved in autoimmune disorders and the antiviral response. In this work, we show that TREX1 is also a RNA exonuclease. Purified TREX1 displays robust exoribonuclease activity that degrades single-stranded, but not double-stranded, RNA. TREX1-D200N, an Aicardi-Goutieres syndrome disease-causing mutant, is defective in degrading RNA. TREX1 activity is strongly inhibited by a stretch of pyrimidine residues as is a bacterial homolog, RNase T. Kinetic measurements indicate that the apparent K_m of TREX1 for RNA is higher than that for DNA. Like RNase T, human TREX1 is active in degrading native tRNA substrates. Previously reported TREX1 crystal structures have revealed that the substrate binding sites are open enough to accommodate the extra hydroxyl group in RNA, further supporting our conclusion that TREX1 acts on RNA. These findings indicate that its RNase activity needs to be taken into account when evaluating the physiological role of TREX1.     

Regulation of Single-stranded DNA Binding by the C Termini of Escherichia coli Single-stranded DNA-binding (SSB) Protein

The homotetrameric Escherichia coli single-stranded DNA-binding (SSB) protein plays a central role in DNA replication, repair, and recombination. In addition to its essential activity of binding to transiently formed single-stranded (ss) DNA, SSB also binds an array of partner proteins and recruits them to their sites of action using its four intrinsically disordered C-terminal tails. Here we show that the binding of ssDNA to SSB is inhibited by the SSB C-terminal tails, specifically by the last 8 highly acidic amino acids that comprise the binding site for its multiple partner proteins. We examined the energetics of ssDNA binding to short oligodeoxynucleotides and find that at moderate salt concentration, removal of the acidic C-terminal ends increases the intrinsic affinity for ssDNA and enhances the negative cooperativity between ssDNA binding sites, indicating that the C termini exert an inhibitory effect on ssDNA binding. This inhibitory effect decreases as the salt concentration increases. Binding of ssDNA to approximately half of the SSB subunits relieves the inhibitory effect for all of the subunits. The inhibition by the C termini is due primarily to a less favorable entropy change upon ssDNA binding. These observations explain why ssDNA binding to SSB enhances the affinity of SSB for its partner proteins and suggest that the C termini of SSB may interact, at least transiently, with its ssDNA binding sites. This inhibition and its relief by ssDNA binding suggest a mechanism that enhances the ability of SSB to selectively recruit its partner proteins to sites on DNA.     

Thermal Inactivation of Aeromonas hydrophila As Affected by Sodium Chloride and Ascorbic Acid

The combined effects of sodium chloride (0, 1.0, 1.5, and 3.0%) and ascorbic acid (0, 1.0, and 2.0 mmol/liter) with mild heat (46°C) on the survival of Aeromonas hydrophila were evaluated. Because of the nonlinear nature of the survivor curves obtained, several equations yielding an R² (coefficient of multiple determination) of 1 were tested. The equation that most closely fit the curvature of the observed data set was a hyperbolic function. Equation coefficients were combined to obtain a so-called death value. This value (46.67% explained variance) was calculated by extracting the larger eigenvalue and the relative eigenvector from the correlation matrix of the coefficients. the effects of the experimental factors on the death value were described by a quadratic response surface model. Results revealed that the death value was not influenced by the presence of ascorbic acid. However, increased mortality resulted from the interaction between sodium chloride and ascorbic acid.