Nucleotide sequences of the 5' termini of phi X174 mRNAs synthesized in vitro

When using X174 RFI DNA as a template, in vitro, E. coli RNA polymerase synthesizes four major purine triphosphate-containing 5′ end sequences. RNase A digests of α³²P labeled RNA were further digested with spleen exonuclease to remove the bulk of the oligonucleotides with 5′ hydroxyls and then chromatographed on DEAE cellulose to resolve the remaining 5′ terminal oligonucleotides. By application of standard separation and sequence techniques, the major 5′ end sequences were shown to be: pppApUp(Cp), pppApApApUp(Cp), pppApApApApUp(Cp), and pppGpApUp(Gp).     

An Active Immune Defense with a Minimal CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA and without the Cas6 Protein

The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3′ handle are still active in triggering an interference reaction. The complete 3′ handle could be removed without loss of activity. However, manipulations of the 5′ handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference.     

Structure/Function Analysis of PARP-1 in Oxidative and Nitrosative Stress-Induced Monomeric ADPR Formation

Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2^nd messenger monomeric adenosine diphosphate-ribose (mADPR). Previous studies have delineated specific roles for several of PARP-1′s structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1′s BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2^nd messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.     

Complementary Roles for Exonuclease 1 and Flap Endonuclease 1 in Maintenance of Triplet Repeats

Trinucleotide repeats can form stable secondary structures that promote genomic instability. To determine how such structures are resolved, we have defined biochemical activities of the related RAD2 family nucleases, FEN1 (Flap endonuclease 1) and EXO1 (exonuclease 1), on substrates that recapitulate intermediates in DNA replication. Here, we show that, consistent with its function in lagging strand replication, human (h) FEN1 could cleave 5′-flaps bearing structures formed by CTG or CGG repeats, although less efficiently than unstructured flaps. hEXO1 did not exhibit endonuclease activity on 5′-flaps bearing structures formed by CTG or CGG repeats, although it could excise these substrates. Neither hFEN1 nor hEXO1 was affected by the stem-loops formed by CTG repeats interrupting duplex regions adjacent to 5′-flaps, but both enzymes were inhibited by G4 structures formed by CGG repeats in analogous positions. Hydroxyl radical footprinting showed that hFEN1 binding caused hypersensitivity near the flap/duplex junction, whereas hEXO1 binding caused hypersensitivity very close to the 5′-end, correlating with the predominance of hFEN1 endonucleolytic activity versus hEXO1 exonucleolytic activity on 5′-flap substrates. These results show that FEN1 and EXO1 can eliminate structures formed by trinucleotide repeats in the course of replication, relying on endonucleolytic and exonucleolytic activities, respectively. These results also suggest that unresolved G4 DNA may prevent key steps in normal post-replicative DNA processing.     

Identification of a 5′-Deoxyadenosine Deaminase in Methanocaldococcus jannaschii and Its Possible Role in Recycling the Radical S-Adenosylmethionine Enzyme Reaction Product 5′-Deoxyadenosine

We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5′-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes the conversion of 5′-deoxyadenosine to 5′-deoxyinosine as its major product but will also deaminate 5′-methylthioadenosine, S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5′-deoxyadenosine deaminase (DadD). The K_m for 5′-deoxyadenosine was found to be 14.0 ± 1.2 μM with a k_cat/K_m of 9.1 × 10⁹ M⁻¹ s⁻¹. Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5′-deoxyadenosine. Since 5′-dA has been demonstrated to be an inhibitor of radical SAM enzymes; a pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5′-deoxyadenosine, whereupon the 5′-deoxyribose moiety of 5′-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens.     

Structure of the Legionella Effector,lpg1496, Suggests a Role in Nucleotide Metabolism

Pathogenic Gram-negative bacteria use specialized secretion systems that translocate bacterial proteins, termed effectors, directly into host cells where they interact with host proteins and biochemical processes for the benefit of the pathogen. lpg1496 is a previously uncharacterized effector of Legionella pneumophila, the causative agent of Legionnaires disease. Here, we crystallized three nucleotide binding domains from lpg1496. The C-terminal domain, which is conserved among the SidE family of effectors, is formed of two largely α-helical lobes with a nucleotide binding cleft. A structural homology search has shown similarity to phosphodiesterases involved in cleavage of cyclic nucleotides. We have also crystallized a novel domain that occurs twice in the N-terminal half of the protein that we term the KLAMP domain due to the presence of homologous domains in bacterial histidine kinase-like ATP binding region-containing proteins and S-adenosylmethionine-dependent methyltransferase proteins. Both KLAMP structures are very similar but selectively bind 3′,5′-cAMP and ADP. A co-crystal of the KLAMP1 domain with 3′,5′-cAMP reveals the contribution of Tyr-61 and Tyr-69 that produces π-stacking interactions with the adenine ring of the nucleotide. Our study provides the first structural insights into two novel nucleotide binding domains associated with bacterial virulence.     

Novel cis-Acting Element within the Capsid-Coding Region Enhances Flavivirus Viral-RNA Replication by Regulating Genome Cyclization

cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5′ cyclization sequence (5′CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5′CS, and the presence of DCS-PK facilitates the formation of 5′-3′ RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution.     

Functional Characterization of Core Components of the Bacillus subtilis Cyclic-Di-GMP Signaling Pathway

Bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is an intracellular second messenger that regulates adaptation processes, including biofilm formation, motility, and virulence in Gram-negative bacteria. In this study, we have characterized the core components of a c-di-GMP signaling pathway in the model Gram-positive bacterium Bacillus subtilis. Specifically, we have directly identified and characterized three active diguanylate cyclases, DgcP, DgcK, and DgcW (formerly YtrP, YhcK, and YkoW, respectively), one active c-di-GMP phosphodiesterase, PdeH (formerly YuxH), and a cyclic-diguanylate (c-di-GMP) receptor, DgrA (formerly YpfA). Furthermore, elevation of c-di-GMP levels in B. subtilis led to inhibition of swarming motility, whereas biofilm formation was unaffected. Our work establishes paradigms for Gram-positive c-di-GMP signaling, and we have shown that the concise signaling system identified in B. subtilis serves as a powerful heterologous host for the study of c-di-GMP enzymes from bacteria predicted to possess larger, more-complex signaling systems.     

Identification of Escherichia coli ygaQ and rpmG as novel mitomycin C resistance factors implicated in DNA repair

Using the ASKA (A Complete Set of Escherichia coli K-12 ORF Archive) library for genome-wide screening of E. coli proteins we identified that expression of ygaQ and rpmG promotes mitomycin C resistance (MMC^R). YgaQ mediated MMC^R was independent of homologous recombination involving RecA or RuvABC, but required UvrD. YgaQ is an uncharacterized protein homologous with α-amylases that we identified to have nuclease activity directed to ssDNA of 5′ flaps. Nuclease activity was inactivated by mutation of two amino acid motifs, which also abolished MMC^R. RpmG is frequently annotated as a bacterial ribosomal protein, although forms an operon with MutM glycosylase and a putative deubiquitinating (DUB) enzyme, YicR. RpmG associated MMC^R was dependent on MutM. MMC^R from RpmG resembles DNA repair phenotypes reported for ‘idiosyncratic ribosomal proteins’ in eukaryotes.     

N2-guanine specific transfer RNA methyltransferase II from rat liver

An enzyme was purified from rat liver and leukemic rat spleen which methylates guanosine residues in tRNA to N²-methylguanosine. By sequence analysis of bulk E. coli tRNA methylated with crude extracts it was shown that the enzyme is responsible for about 50% of total m²G formed invitro. The extent of methylation of a number of homogenous tRNA species was measured using the purified enzyme from both sources. Among tested E. coli tRNAs only tRNA^Arg, tRNA^Phe, and tRNA^Val yielded significantly more m²G than the bulk tRNA. The K_m for tRNA^Arg in the methylation reaction with enzymes from either tissue was 7.8 × 10⁻⁷ M as compared to the value 1 × 10⁻⁵ M obtained for the bulk tRNA. In a pancreatic RNase digest of bulk tRNA as well as of pure tRNA^Arg, tRNA^Phe, and tRNA^Val, A-m²G-Cp was found to be the only sequence methylated. Thus, the mammalian methyltransferase specifically recognizes the guanylate residue at position 10 from the 5′-end contained in a sequence (s⁴)U-A-G-Cp. Furthermore, there is no change between the enzyme from normal liver and leukemic spleen in the affinity for tRNA, the methylating capacity, and tRNA site and sequence recognition specificity.     

Double-stranded Endonuclease Activity in Bacillus halodurans Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas2 Protein

The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5′-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg²⁺ or Mn²⁺), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1–α1 loop.     

Novel RNA structural features of an alternatively splicing group II intron from Clostridium tetani

Group II introns are ribozymes in bacterial and organellar genomes that function as self-splicing introns and as retroelements. Previously, we reported that the group II intron C.te.I1 of Clostridium tetani alternatively splices in vivo to produce five distinct coding mRNAs. Accurate fusion of upstream and downstream reading frames requires a shifted 5′ splice site located 8 nt upstream of the usual 5′ GUGYG motif. This site is specified by the ribozyme through an altered intron/exon-binding site 1 (IBS1–EBS1) pairing. Here we use mutagenesis and self-splicing assays to investigate in more detail the significance of the structural features of the C.te.I1 ribozyme. The shifted 5′ splice site is shown to be affected by structures in addition to IBS1–EBS1, and unlike other group II introns, C.te.I1 appears to require a spacer between IBS1 and the GUGYG motif. In addition, the mechanism of 3′ exon recognition is modified from the ancestral IIB mechanism to a IIA-like mechanism that appears to be longer than the typical single base-pair interaction and may extend up to 4 bp. The novel ribozyme properties that have evolved for C.te.I1 illustrate the plasticity of group II introns in adapting new structural and catalytic properties that can be utilized to affect gene expression.     

Allelic Exchange in Actinomyces oris with mCherry Fluorescence Counterselection

Described here is a method for facile generation of markerless gene deletion mutants of Actinomyces oris. Homologous integration of a nonreplicative vector carrying a gene exchange cassette into the bacterial chromosome was selected for by using mCherry fluorescence and resistance to kanamycin. Completion of allelic replacement was counterselected for by using loss of fluorescence.Actinomyces oris (formerly Actinomyces naeslundii [3]) is Gram positive, facultatively anaerobic, and commonly found in the human oral cavity and plays a major role in the formation of oral biofilm or dental plaque. It is thought that adherence of A. oris to the tooth surface and its coaggregation with oral streptococci create an adhesive platform for subsequent colonization of bacteria in the plaque community (4). A. oris surface molecules such as fimbriae and pili have been shown previously to be required for the bacterial interactions with host tissues and other oral bacteria (7). However, the roles of fimbrial molecules or other surface proteins involved in these processes and their molecular assembly on the cell surface remain elusive. Lack of a facile gene disruption technology is the main reason for this obscurity.Conventional methods of genetic manipulation employing nonreplicative plasmids as delivery vectors in A. oris have been used to create gene disruption by allelic exchange, which allows insertion of a selectable marker (1, 8, 9). Often, this strategy generates polar mutations that affect downstream genes, and it is inadequate for multigene deletion because antibiotic markers for Actinomyces are scarce. To circumvent this problem, we successfully developed a method that utilizes a pUC19 derivative (namely, pHTT177) to generate a nonpolar, in-frame deletion of the sortase gene srtC2 (5). However, this system proved extremely laborious because the second homologous recombination (double-crossover) event leading to chromosomal excision and loss of the plasmid could not be efficiently selected for. Consequently, we explored fluorescence as a positive selection marker for A. oris, as described below.To generate a nonreplicative delivery vector for gene replacement with a counterselectable marker, we cloned the gene encoding the red fluorescent protein mCherry under the control of the constitutive promoter PrpsJ into pHTT177 by using EcoRI and NdeI sites (5) (Fig. ​(Fig.1).1). Initially, the mCherry sequence was amplified from plasmid pRSET-B-mCherry DNA (6) by using primers P1 (5′-GGCGGCTAGCATGGTGAGCAAGGGCGAGGAG-3′) and P2 (5′-GGCGCATATGCTACTACTTGTACAGCTCGTCCATG-3′), which contain NheI and NdeI sites (underlined), respectively. Primers P3 (5′-GGCGGAATTCCGCCCGAGCGCGGGGACCAGT-3′) and P4 (5′-GGCGGCTAGCGGCGCCTAACCTCTCTTGTACTTG-3′), containing EcoRI and NheI sites, were used to amplify the untranslated region of rpsJ from A. oris MG-1 chromosomal DNA (see gene identification no. ANA_0026 in the A. oris database at www.oralgen.lanl.gov). Both fragments were subcloned into pJRD215 at EcoRI and NdeI sites (2). The resulting vector, pCWU3, has a multiple-cloning site (MCS) containing EcoRI, SacI, KpnI, BamHI, XbaI, SalI, and HindIII sites for cloning purposes (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Construction of the nonreplicative delivery vector with red fluorescent mCherry protein as a counterselectable marker. The mCherry gene under the control of the A. oris rpsJ promoter was subcloned into the Escherichia coli/Actinomyces shuttle vector pJRD215 before being cloned into pHTT177, which is a derivative of pUC19. The resulting plasmid, pCWU3, has a kanamycin resistance cassette (kan^R) and an MCS containing EcoRI, SacI, KpnI, BamHI, XbaI, SalI, and HindIII sites.As a proof of concept, we utilized the vector pCWU3, created as described above, to generate an in-frame deletion of acaA (see gene identification no. ANA_0196 in the database at www.oralgen.lanl.gov), encoding a putative cell wall anchor protein (called Aca for actinomyces cell wall anchor). Primer sets P5/P6 (5′-GGCGGAATTCGCCGGAGGCGCCGTCGGGGAAG-3′/5′-GGCGGGTACCAGGATCTCCGTTAGACACGG-3′) and P7/P8 (5′-GGCGGGTACCCAGCGAGACTGCGACCAGCAG-3′/5′-GGCGTCTAGAGGTGGGCGTACTTCTGGTCCAT-3′) were used to amplify ∼1.0-kb sequences upstream and downstream, respectively, of acaA from A. oris MG-1 chromosomal DNA. The upstream DNA fragment was digested with EcoRI and KpnI, while the downstream fragment was digested with KpnI and XbaI (restriction enzyme sites are underlined); both fragments were ligated into pCWU3, which had been precut with EcoRI and XbaI. A. oris MG-1 was transformed with the resulting plasmid by electroporation (5), and kanamycin-resistant colonies representing integration of the plasmid into the bacterial chromosome were selected for their ability to grow on heart infusion (HI; Difco) agar plates supplemented with 50-μg ml⁻¹ kanamycin. These colonies were also examined for their fluorescence by using an Olympus XI71 inverted microscope equipped with a Hamamatsu charge-coupled device camera and a tetramethyl rhodamine isothiocyanate (TRITC) filter set (Fig. ​(Fig.22 A).Open in a separate windowFIG. 2.Analysis of a gene deletion in A. oris. (A) A. oris cells expressing mCherry under the control of the rpsJ promoter were viewed with an Olympus inverted microscope using a TRITC filter. (B and C) Selection of A. oris acaA deletion mutants was performed with a FluorChem Q imaging system (Alpha Innotech, CA). Fluorescent cells appeared green (pseudocolored) with the Cy3 setting, while nonfluorescent cells (potential mutants, indicated by the white arrow in panel C) were gray. An enlarged area (indicated by the white box in panel B) is shown in panel C. (D and E) Nonfluorescent bacteria were further examined for the absence of the acaA gene by PCR amplification (D) and Southern blot analysis (E). Bands of approximately 2.2 kb indicate acaA deletion, whereas bands of approximately 3.3 kb indicate a wild-type (WT) genotype (D). Samples from the parent strain MG-1 (WT) and size markers (M) are indicated, and the black arrow marks a 3.4-kb hybridized fragment.To select Actinomyces clones that had undergone the double-crossover event leading to chromosomal excision and loss of the plasmid, we inoculated a sample of bacteria carrying the integrated plasmid into HI broth overnight at 37°C. The bacterial culture was then serially passaged seven times with a 1:40 dilution in HI broth without antibiotics. Forty-microliter aliquots of the 10,000-fold-diluted final culture were plated onto HI agar plates. After 3 days of growth at 37°C, plates were screened for nonfluorescent colonies by using a FluorChem Q imaging system (Alpha Innotech, CA) with a Cy3 filter. The Cy3 filter was chosen because it produced brighter images than those produced by the Cy5 filter (data not shown) and the images were given with false green coloring (Fig. ​(Fig.2B).2B). Of approximately 16,000 colonies that were screened (a procedure taking less than 30 min), 11 showed no fluorescence (an example indicated by an arrow is shown in Fig. ​Fig.2C),2C), corresponding to a frequency of ∼7.0 × 10⁻⁴. This is consistent with the low frequency of homologous recombination in A. oris (5). Nonfluorescent colonies were also confirmed to be sensitive to kanamycin (data not shown), and their genomic DNA was extracted for PCR and Southern blot analyses. For PCR analysis, primers P5 and P8 were used. As shown in Fig. ​Fig.2D,2D, 8 of the 11 isolates generated amplicons of approximately 2.2 kb, which is indicative of acaA deletion, while the remaining 3 isolates generated the expected wild-type amplicons of approximately 3.3 kb. For further confirmation, DNA samples from three acaA mutants and the wild-type strain MG-1 were analyzed by Southern blotting using a 550-bp probe generated by primers 5′-AGTCTCCAACGCATCCGTCTC-3′ and 5′-GTGTCCCGAGACATTGGCCGTG-3′. Based on sequence analysis of acaA and surrounding genes, digestion by PstI will generate a 3.4-kb fragment for the wild type. As expected, the probe hybridized to the 3.4-kb DNA fragment, which was missing from the three mutant samples (Fig. ​(Fig.2E).2E). Thus, the lack of a hybridization signal for the three mutants further confirmed the absence of acaA in these mutants.In summary, we have developed a facile allelic exchange system for A. oris that reduces the laborious step of screening for a double-crossover event to less than 30 min. To our knowledge, this is the first report of an application that employs a fluorescent protein as a positive selectable marker for gene disruption in bacteria. Conceptually, this strategy can be applied for gene disruption in any system.     

Chemically Modified DNA Aptamers Bind Interleukin-6 with High Affinity and Inhibit Signaling by Blocking Its Interaction with Interleukin-6 Receptor

Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates immune and inflammatory responses, and its overproduction is a hallmark of inflammatory diseases. Inhibition of IL-6 signaling with the anti-IL-6 receptor antibody tocilizumab has provided some clinical benefit to patients; however, direct cytokine inhibition may be a more effective option. We used the systematic evolution of ligands by exponential enrichment (SELEX) process to discover slow off-rate modified aptamers (SOMAmers) with hydrophobic base modifications that inhibit IL-6 signaling in vitro. Two classes of IL-6 SOMAmers were isolated from modified DNA libraries containing 40 random positions and either 5-(N-benzylcarboxamide)-2′-deoxyuridine (Bn-dU) or 5-[N-(1-naphthylmethyl)carboxamide]-2′-deoxyuridine (Nap-dU) replacing dT. These modifications facilitate the high affinity binding interaction with IL-6 and provide resistance against degradation by serum endonucleases. Post-SELEX optimization of one Bn-dU and one Nap-dU SOMAmer led to improvements in IL-6 binding (10-fold) and inhibition activity (greater than 20-fold), resulting in lead SOMAmers with sub-nanomolar affinity (K_d = 0.2 nm) and potency (IC₅₀ = 0.2 nm). Although similar in inhibition properties, the two SOMAmers have unique sequences and different ortholog specificities. Furthermore, these SOMAmers were stable in human serum in vitro for more than 48 h. Both SOMAmers prevented IL-6 signaling by blocking the interaction of IL-6 with its receptor and inhibited the proliferation of tumor cells in vitro as effectively as tocilizumab. This new class of IL-6 inhibitor may be an effective therapeutic alternative for patients suffering from inflammatory diseases.     

2'-O-methyl ribothymidine: a component of rabbit liver lysine transfer RNA

One of the lysine transfer RNAs of rabbit liver is shown to contain 2′-O-methyl ribothymidine in place of ribothymidine. This represents the first demonstration of the presence of 2′-O-methyl ribothymidine in a nucleic acid.     

The molecular complexity of mu and pi symbiont DNA of Paramecium aurelia

The molecular size of mu and pi symbionts of Parameciumaurelia has been calculated from renaturation kinetic data. Observed values were 0.78 × 10⁹ daltons for mu particle DNA and 0.81 × 10⁹ daltons for pi particle DNA. Estimates of analytical complexity were 4.45 × 10⁹ and 5.05 × 10⁹ daltons respectively. Based on these data, mu and pi symbionts appear to possess multiple genomes and contain a minimum of 5 or 6 copies of each DNA sequence.