Increasing the length of poly-pyrimidine bulges broadens RNA conformational ensembles with minimal impact on stacking energetics.

Helical elements separated by bulges frequently undergo transitions between unstacked and coaxially stacked conformations during the folding and function of noncoding RNAs. Here, we examine the dynamic properties of poly-pyrimidine bulges of varying length (n = 1-4, 7) across a range of Mg2+ concentrations using HIV-1 TAR RNA as a model system and solution NMR spectroscopy. In the absence of Mg2+, helices linked by bulges with n ≥ 3 residues adopt predominantly unstacked conformations (stacked population <15%), whereas one-bulge and two-bulge motifs adopt predominantly stacked conformations (stacked population >74%). In the presence of 3 mM Mg2+, the helices predominantly coaxially stack (stacked population >84%), regardless of bulge length, and the midpoint for the Mg2+-dependent stacking transition is within threefold regardless of bulge length. In the absence of Mg2+, the difference between free energy of interhelical coaxial stacking across the bulge variants is estimated to be ∼2.9 kcal/mol, based on an NMR chemical shift mapping with stacking being more energetically disfavored for the longer bulges. This difference decreases to ∼0.4 kcal/mol in the presence of Mg2+ NMR RDCs and resonance intensity data show increased dynamics in the stacked state with increasing bulge length in the presence of Mg2+ We propose that Mg2+ helps to neutralize the growing electrostatic repulsion in the stacked state with increasing bulge length thereby increasing the number of coaxial conformations that are sampled. Energetically compensated interhelical stacking dynamics may help to maximize the conformational adaptability of RNA and allow a wide range of conformations to be optimally stabilized by proteins and ligands.

Distinct Dynamic Modes Enable the Engagement of Dissimilar Ligands in a Promiscuous Atypical RNA Recognition Motif.

Conformational dynamics play a critical role in ligand binding, often conferring divergent activities and specificities even in species with highly similar ground-state structures. Here, we employ time-resolved electrospray ionization hydrogen-deuterium exchange (TRESI-HDX) to characterize the changes in dynamics that accompany oligonucleotide binding in the atypical RNA recognition motif (RRM2) in the C-terminal domain (CTD) of human La protein. Using this approach, which is uniquely capable of probing changes in the structure and dynamics of weakly ordered regions of proteins, we reveal that binding of RRM2 to a model 23-mer single-stranded RNA and binding of RRM2 to structured IRES domain IV of the hepatitis C viral (HCV) RNA are driven by fundamentally different dynamic processes. In particular, binding of the single-stranded RNA induces helical "unwinding" in a region of the CTD previously hypothesized to play an important role in La and La-related protein-associated RNA remodeling, while the same region becomes less dynamic upon engagement with the double-stranded HCV RNA. Binding of double-stranded RNA also involves less penetration into the RRM2 binding pocket and more engagement with the unstructured C-terminus of the La CTD. The complementarity between TRESI-HDX and Δδ nuclear magnetic resonance measurements for ligand binding analysis is also explored.

3'-UTR poly(T/U) repeat of EWSR1 is altered in microsatellite unstable colorectal cancer with nearly perfect sensitivity.

Approximately 15% of colorectal cancers exhibit instability of short nucleotide repeat regions, microsatellites. These tumors display a unique clinicopathologic profile and the microsatellite instability status is increasingly used to guide clinical management as it is known to predict better prognosis as well as resistance to certain chemotherapeutics. A panel of five repeats determined by the National Cancer Institute, the Bethesda panel, is currently the standard for determining the microsatellite instability status in colorectal cancer. Recently, a quasimonomorphic mononucleotide repeat 16T/U at the 3' untranslated region of the Ewing sarcoma breakpoint region 1 gene was reported to show perfect sensitivity and specificity in detecting mismatch repair deficient colorectal, endometrial, and gastric cancers in two independent populations. To confirm this finding, we replicated the analysis in 213 microsatellite unstable colorectal cancers from two independent populations, 148 microsatellite stable colorectal cancers, and the respective normal samples by PCR and fragment analysis. The repeat showed nearly perfect sensitivity for microsatellite unstable colorectal cancer as it was altered in 212 of the 213 microsatellite unstable (99.5%) and none of the microsatellite stable colorectal tumors. This repeat thus represents the first potential single marker for detecting microsatellite instability.

Unified translation repression mechanism for microRNAs and upstream AUGs.

BACKGROUND: MicroRNAs (miRNAs) are endogenous small RNAs that modulate gene expression at the post-transcriptional level by binding complementary sites in the 3'-UTR. In a recent genome-wide study reporting a new miRNA target class (miBridge), we identified and validated interactions between 5'-UTRs and miRNAs. Separately, upstream AUGs (uAUGs) in 5'-UTRs are known to regulate genes translationally without affecting mRNA levels, one of the mechanisms for miRNA-mediated repression. RESULTS: Using sequence data from whole-genome cDNA alignments we identified 1418 uAUG sequences on the 5'-UTR that specifically interact with 3'-ends of conserved miRNAs. We computationally identified miRNAs that can target six genes through their uAUGs that were previously reported to suppress translation. We extended this meta-analysis by confirming expression of these miRNAs in cell-lines used in the uAUG studies. Similarly, seven members of the KLF family of genes containing uAUGs were computationally identified as interacting with several miRNAs. Using KLF9 as an example (whose protein expression is limited to brain tissue despite the mRNA being expressed ubiquitously), we show computationally that miRNAs expressed only in HeLa cells and not in neuroblastoma (N2A) cells can bind the uAUGs responsible for translation inhibition. Our computed results demonstrate that tissue- or cell-line specific repression of protein translation by uAUGs can be explained by the presence or absence of miRNAs that target these uAUG sequences. We propose that these uAUGs represent a subset of miRNA interaction sites on 5'-UTRs in miBridge, whereby a miRNA binding a uAUG hinders the progression of ribosome scanning the mRNA before it reaches the open reading frame (ORF). CONCLUSIONS: While both miRNAs and uAUGs are separately known to down-regulate protein expression, we show that they may be functionally related by identifying potential interactions through a sequence-specific binding mechanism. Using prior experimental evidence that shows uAUG effects on translation repression together with miRNA expression data specific to cell lines, we demonstrate through computational analysis that cell-specific down-regulation of protein expression (while maintaining mRNA levels) correlates well with the simultaneous presence of miRNA and target uAUG sequences in one cell type and not others, suggesting tissue-specific translation repression by miRNAs through uAUGs.

X-ray structure and activity of the yeast Pop2 protein: a nuclease subunit of the mRNA deadenylase complex.

In Saccharomyces cerevisiae, a large complex, known as the Ccr4-Not complex, containing two nucleases, is responsible for mRNA deadenylation. One of these nucleases is called Pop2 and has been identified by similarity with PARN, a human poly(A) nuclease. Here, we present the crystal structure of the nuclease domain of Pop2 at 2.3 A resolution. The domain has the fold of the DnaQ family and represents the first structure of an RNase from the DEDD superfamily. Despite the presence of two non-canonical residues in the active site, the domain displays RNase activity on a broad range of RNA substrates. Site-directed mutagenesis of active-site residues demonstrates the intrinsic ability of the Pop2 RNase D domain to digest RNA. This first structure of a nuclease involved in the 3'-5' deadenylation of mRNA in yeast provides information for the understanding of the mechanism by which the Ccr4-Not complex achieves its functions.

Failure to generate atheroprotective apolipoprotein AI phenotypes using synthetic RNA/DNA oligonucleotides (chimeraplasts).

BACKGROUND: Elevated plasma high-density lipoprotein (HDL), and its major constituent apolipoprotein AI (apoAI), are cardioprotective. Paradoxically, two natural variants of apoAI, termed apoAI(Milano) and apoAI(Paris), are associated with low HDL, but nevertheless provide remarkable protection against heart disease for heterozygous carriers and may even lead to longevity. Both variants arise from point mutations and have Arg(173) and Arg(151) to Cys substitutions, respectively, which allow disulphide-linked dimers to form. Potentially, synthetic RNA/DNA oligonucleotides (chimeraplasts) can permanently correct single point mutations in genomic DNA. Here, we use a variation of such targeted gene repair technology, 'gain-of-function chimeraplasty', and attempt to enhance the biological activity of apoAI by altering a single genomic base to generate the atheroprotective phenotypes, apoAI(Milano) and apoAI(Paris). METHODS: We targeted two cultured cell lines that secrete human apoAI, hepatoblastoma HepG2 cells and recombinant CHO-AI cells, using standard 68-mer chimeraplasts with polyethyleneimine (PEI) as carrier and then systematically varied several experimental conditions. As a positive control we targeted the dysfunctional APOE2 gene, which we have previously converted to wild-type APOE3. RESULTS: Conversion of wild-type apoAI to apoAI(Milano) proved refractory, with limited correction in CHO-AI cells only. However, a successful conversion to apoAI(Paris) was achieved, as demonstrated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and direct genomic sequencing. Unexpectedly, attempts with a new batch of 68-mer chimeraplast to enhance conversion, by using different delivery vehicles, including chemically modified PEI, failed to show a base change; nor could conversion be detected with an 80-mer or a 52-76-mer series. In contrast, when a co-culture of CHO-E2 and CHO-AI cells was co-targeted, a clear conversion of apoE2 to apoE3 was seen, whereas no apoAI(Paris) could be detected. When the individual chimeraplasts were analysed by denaturing electrophoresis only the active apoE2-to-E3 chimeraplast gave a sharp band. CONCLUSIONS: Our findings suggest that different batches of chimeraplasts have variable characteristics and that their quality may be a key factor for efficient targeting and/or base conversion. We conclude that, although an evolving technology with enormous potential, chimeraplast-directed gene repair remains problematical.

Comparison of fluorescently labeled oligonucleotide and polynucleotide probes for the detection of pelagic marine bacteria and archaea.

We compared the detection of bacteria and archaea in the coastal North Sea and at Monterey Bay, Calif., after fluorescence in situ hybridization (FISH) either with rRNA-targeted oligonucleotide probes monolabeled with the cyanin dye Cy3 (oligoFISH) or with fluorescein-labeled polyribonucleotide probes (polyFISH). During an annual cycle in German Bight surface waters, the percentages of bacteria visualized by polyFISH (annual mean, 77% of total counts) were significantly higher than those detected by oligoFISH (53%). The fraction of total bacteria visualized by oligoFISH declined during winter, whereas cell numbers determined by polyFISH remained constant throughout the year. Depth profiles from Monterey Bay showed large differences in the fraction of bacterial cells visualized by polyFISH and oligoFISH in the deeper water layers irrespective of the season. Image-analyzed microscopy indicated that the superior detection of cells by polyFISH with fluorescein-labeled probes in bacterioplankton samples was less a consequence of higher absolute fluorescence intensities but was rather related to quasi-linear bleaching dynamics and to a higher signal-to-background ratio. The relative abundances of archaea in North Sea and Monterey Bay spring samples as determined by oligoFISH were on average higher than those determined by polyFISH. However, simultaneous hybridizations with oligonucleotide probes for bacteria and archaea suggested that the oligoFISH probe ARCH915 unspecifically stained a population of bacteria. Using either FISH technique, blooms of archaea were observed in North Sea surface waters during the spring and summer months. Marine group II archaea (Euryarchaeota) reached >30% of total picoplankton abundances, as determined by polyFISH. We suggest that studies of pelagic microbial community structure using oligoFISH with monolabeled probes should focus on environments that yield detections > or =70% of total cell counts, e.g., coastal surface waters during spring and summer.

RNA-binding characteristics of the chloroplast S1-like ribosomal protein CS1.

The chloroplast ribosomal protein CS1, the homolog of the bacterial ribosomal protein S1, is believed to be involved in the process of ribosome binding to mRNA during translation. Since translation control is an important step in chloroplast gene expression, and in order to study initiation complex formation, we studied the RNA-binding properties of CS1 protein. We found that most of the CS1 protein in spinach chloroplast co-purified with the 30S ribosomal subunit. The relative binding affinity of RNA to CS1 was determined using the UV-crosslinking competition assay. CS1 protein binds the ribohomopolymer poly(U) with a relatively high binding affinity. Very low binding affinities were obtained for the other ribohomopolymers, poly(G), poly(A) and poly(C). In addition, no specific binding of CS1, either in the 30S complex or as a recombinant purified protein, was obtained to the 5'-untranslated region of the mRNA in comparison to the other parts. RNA-binding experiments, in which the N- and C-termini of the protein were analyzed, revealed that the RNA-binding site is located in the C-terminus half of the protein. These results suggest that CS1 does not direct the 30S complex to the initiation codon of the translation site by specific binding to the 5'-untranslated region. In bacteria, specific binding is derived by base pairing between 16S rRNA and the Shine-Dalagarno sequences. In the chloroplast, nuclear encoded and gene-specific translation factors may be involved in the determination of specific binding of the 30S subunit to the initiator codon.

Molecular interactions and metal binding in the theophylline-binding core of an RNA aptamer.

An RNA aptamer containing a 15-nt binding site shows high affinity and specificity for the bronchodilator theophylline. A variety of base modifications or 2' deoxyribose substitutions in binding-site residues were tested for theophyllinebinding affinity and the results were compared with the previously determined three-dimensional structure of the RNA-theophylline complex. The RNA-theophylline complex contains a U6-A28-U23 base triple, and disruption of this A28-U23 Hoogsteen-pair by a 7-deaza, 2'-deoxy A28 mutant reduces theophylline binding >45-fold at 25 degrees C. U24 is part of a U-turn in the core of the RNA, and disruption of this U-turn motif by a 2'-deoxy substitution of U24 also reduces theophylline binding by >90-fold. Several mutations outside the "conserved core" of the RNA aptamer showed reduced binding affinity, and these effects could be rationalized by comparison with the three-dimensional structure of the complex. Divalent ions are absolutely required for high-affinity theophylline binding. High-affinity binding was observed with 5 mM Mg2+, Mn2+, or Co2+ ions, whereas little or no significant binding was observed for other divalent or lanthanide ions. A metal-binding site in the core of the complex was revealed by paramagnetic Mn2+-induced broadening of specific RNA resonances in the NMR spectra. When caffeine is added to the aptamer in tenfold excess, the NMR spectra show no evidence for binding in the conserved core and instead the drug stacks on the terminal helix. The lack of interaction between caffeine and the theophylline-binding site emphasizes the extreme molecular discrimination of this RNA aptamer.

A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA.

All of the previously reported recombinant RNA-dependent RNA polymerases (RdRp), the NS5B enzymes, of hepatitis C virus (HCV) could function only in a primer-dependent and template-nonspecific manner, which is different from the expected properties of the functional viral enzymes in the cells. We have now expressed a recombinant NS5B that is able to synthesize a full-length HCV genome in a template-dependent and primer-independent manner. The kinetics of RNA synthesis showed that this RdRp can initiate RNA synthesis de novo and yield a full-length RNA product of genomic size (9.5 kb), indicating that it did not use the copy-back RNA as a primer. This RdRp was also able to accept heterologous viral RNA templates, including poly(A)- and non-poly(A)-tailed RNA, in a primer-independent manner, but the products in these cases were heterogeneous. The RdRp used some homopolymeric RNA templates only in the presence of a primer. By using the 3'-end 98 nucleotides (nt) of HCV RNA, which is conserved in all genotypes of HCV, as a template, a distinct RNA product was generated. Truncation of 21 nt from the 5' end or 45 nt from the 3' end of the 98-nt RNA abolished almost completely its ability to serve as a template. Inclusion of the 3'-end variable sequence region and the U-rich tract upstream of the X region in the template significantly enhanced RNA synthesis. The 3' end of minus-strand RNA of HCV genome also served as a template, and it required a minimum of 239 nt from the 3' end. These data defined the cis-acting sequences for HCV RNA synthesis at the 3' end of HCV RNA in both the plus and minus senses. This is the first recombinant HCV RdRp capable of copying the full-length HCV RNA in the primer-independent manner expected of the functional HCV RNA polymerase.

Biosynthesis of osteogenic growth peptide via alternative translational initiation at AUG85 of histone H4 mRNA.

The osteogenic growth peptide (OGP) is an extracellular mitogen identical to the histone H4 (H4) COOH-terminal residues 90-103, which regulates osteogenesis and hematopoiesis. By Northern analysis, OGP mRNA is indistinguishable from H4 mRNA. Indeed, cells transfected with a construct encoding [His102]H4 secreted the corresponding [His13]OGP. These results suggest production of OGP from H4 genes. Cells transfected with H4-chloramphenicol acetyltransferase (CAT) fusion genes expressed both "long" and "short" CAT proteins. The short CAT was retained following an ATG --> TTG mutation of the H4 ATG initiation codon, but not following mutation of the in-frame internal ATG85 codon, which, unlike ATG1, resides within a perfect context for translational initiation. These results suggest that a PreOGP is translated starting at AUG85. The translational initiation at AUG85 could be inhibited by optimizing the nucleotide sequence surrounding ATG1 to maximally support upstream translational initiation, thus implicating leaky ribosomal scanning in usage of the internal AUG. Conversion of the predicted PreOGP to OGP was shown in a cell lysate system using synthetic [His102]H4-(85-103) as substrate. Together, our results demonstrate that H4 gene expression diverges at the translational level into the simultaneous parallel production of both H4, a nuclear structural protein, and OGP, an extracellular regulatory peptide.

RNAs in the sera of Persian Gulf War veterans have segments homologous to chromosome 22q11.2.

Reverse transcriptase PCR (RT-PCR) was used for polyribonucleotide assays with sera from deployed Persian Gulf War veterans with the Gulf War Syndrome and a cohort of nonmilitary controls. Sera from veterans contained polyribonucleotides (amplicons) that were obtained by RT-PCR and that ranged in size from 200 to ca. 2,000 bp. Sera from controls did not contain amplicons larger than 450 bp. DNA sequences were derived from two amplicons unique to veterans. These amplicons, which were 414 and 759 nucleotides, were unrelated to each other or to any sequence in gene bank databases. The amplicons contained short segments that were homologous to regions of chromosome 22q11.2, an antigen-responsive hot spot for genetic rearrangements. Many of these short amplicon segments occurred near, between, or in chromosome 22q11.2 Alu sequences. These results suggest that genetic alterations in the 22q11.2 region, possibly induced by exposures to environmental genotoxins during the Persian Gulf War, may have played a role in the pathogenesis of the Gulf War Syndrome. However, the data did not exclude the possibility that other chromosomes also may have been involved. Nonetheless, the detection of polyribonucleotides such as those reported here may have application to the laboratory diagnosis of chronic diseases that have a multifactorial etiology.

Evidence for a repressive function of the long polyglutamine tract in the human androgen receptor: possible pathogenetic relevance for the (CAG)n-expanded neuronopathies.

We have reported that polyglutamine (polyGln)-expanded human androgen receptors (hAR) have reduced transactivational competence in transfected cells. We presumed that maximal hAR transactivation requires a normal-size polyGln tract. Here we report, however, that hAR transactivity and polyGln-tract length are related inversely: n = 0 > 12 > 20 > 40 > 50. Thus, a normal-size polyGln tract represses the transactivational competence of a polyGln-free hAR, and polyGln expansion increases that negative effect. This observation has pathogenetic implications for X-linked spinobular muscular atrophy (Kennedy syndrome), and possibly for the autosomal dominant central neuronopathies associated with (CAG)n expansion in the translated portion of four different genes.

[Biological actions and therapeutic perspectives of double stranded polyribonucleotides: a reappraisal]. / Actions biologiques et perspectives thérapeutiques des polyribonucléotides double-brins: une réévaluation.

Double-stranded polynucleotides, which are composed of two complementary homopolyribonucleotides containing no genetic information, are synthetic molecules capable of mimicking the action of natural double-stranded RNA or viral RNA on cells. Double-stranded polyribonucleotides act as an alarm system alerting the cell to the presence of an external aggression, e.g. a viral attack. In addition, polyribonucleotides have a more active function in that they trigger cell defense processes through activation of a family of genes, of which some encode cytokines, activation of cytoplasmic enzymes involved in antiviral mechanisms or signal transduction, and activation of nonspecific immune responses. Double-stranded polyribonucleotides containing one mismatched base pair per helix have been found to be especially interesting. The best known example is poly(I).poly(C12U), also called ampligen. Poly(I).poly(C12U) is capable, in experimental models, of limiting the development of viruses (including HIV), reducing tumor growth, eliminating metastases, and, according to one report, preventing steady declines in T-cell counts in HIV-positive patients. Therapeutic doses used in the USA as an experimental drug induced little toxicity. In vitro, poly(I).poly(C12U) acts synergistically with interferon, interleukin 2 or AZT, suggesting that these latter drugs may be effective in lower, less toxic doses when used in combination with poly(I).poly(C12U). The therapeutic activity of poly(I).poly(C12U) holds promise. More extensive prospective studies of this agent are warranted.

Attenuation of Mengo virus through genetic engineering of the 5' noncoding poly(C) tract.

The murine cardioviruses, such as the Mengo and encephalomyocarditis viruses, and the bovine aphthoviruses, such as foot-and-mouth disease virus, are distinguished among positive-strand RNA viruses by the presence of long homopolymeric poly(C) tracts within their 5' noncoding sequences. Although the specific lengths (60-350 bases) and sequence discontinuities (for example, uridine residues) that sometimes disrupt the homopolymer have served to characterize natural viral isolates, the biological function of the poly(C) region has never been clear. We now report that complementary DNA-mediated truncation of the Mengo virus poly(C) tract dramatically attenuates the pathogenicity of the virus in mice. Animals injected with viruses with short tracts not only survived inoculation of up to 50 micrograms live virus (10(11) plaque-forming units) but consistently produced high titres of neutralizing antibodies, which conferred long-term immunogenic protection from (normally) lethal virus challenge. We propose that analogous synthetic strains of foot and mouth disease virus could serve as the basis for new attenuated vaccines.

Cationic metal-specific structures adopted by the poly(dG) region and the direct repeats in the chicken adult beta A globin gene promoter.

Naturally occurring contiguous deoxyguanine residues and their surrounding sequences in the chicken adult beta A globin gene promoter were analyzed for their inherent potential to adopt non-B DNA structures in supercoiled plasmid DNA. In particular, cationic effects on structure were studied by treating the supercoiled plasmid DNA harboring the chicken adult beta A globin 5' flanking sequence with an unpaired DNA base-specific probe, chloroacetaldehyde in the presence of either Mg++, Cu++, Zn++, Ca++ or Co++ ions. The chloroacetaldehyde-reactive bases were mapped at a single base resolution by a chemical cleavage method that specifically cleaves DNA at the chloroacetaldehyde modified sites. These experiments revealed that while Mg++ and Ca++ ions induce a dG.dG.dC triple helix structure at the contiguous dG residues, Zn++, Cu++ and Co++ ions induce yet another structure at the direct repeats immediately 5' of the dG residues. When Mg++ and Zn++ ions are both present, Zn++ inhibits the dG.dG.dC triplex at the contiguous dG residues and induces a particular non-B DNA structure at the adjacent direct repeats. The specific induction of non-B DNA structures by metal ions at the two adjacent sequences within the promoter region may be of biological significance.

Cloning and synthesis of infectious cardiovirus RNAs containing short, discrete poly(C) tracts.

Mengovirus RNA transcripts with 5' noncoding poly(C) tracts of C8, C12, and C13UC10 have been synthesized in vitro from cDNA clones and shown to be infectious to HeLa cells. A chimeric clone has also been constructed which links the 5' end from one mengovirus clone (299 nucleotides, containing C13UC10) to a 7,424-base fragment derived from the 3' end of encephalomyocarditis (EMC) virus. Progeny virus isolated after transfection with the clone-derived RNAs had the same poly(C) tracts, mengovirus-specific sequences, or EMC virus-specific sequences as the transcript from which it was derived. Although the cloned poly(C) tracts were considerably shorter than those found in viral RNA from mengovirus (C50UC10) or EMC virus (C115UCUC3UC10), the growth characteristics of the progeny viruses in HeLa cells were indistinguishable from those of the parental viruses, indicating the length of this tract does not play a significant restrictive role for cardiovirus infectivity in tissue culture.

Aminoglycoside-induced mistranslation in thermophilic archaebacteria.

The effect of selected aminoglycoside antibiotics on the translational accuracy of poly(U) programmed ribosomes derived from the thermophilic archaebacteria Thermoplasma acidophilum, Sulfolobus solfataricus, Thermococcus celer and Desulfurococcus mobilis has been determined. Under optimum temperature and ionic conditions for polyphenylalanine synthesis, the four species investigated are found to be markedly diverse in their response to the miscoding-inducing action of aminoglycoside antibiotics. T. acidophilum is sensitive to all of the compounds tested except streptomycin; S. solfataricus responds to paromomycin and to hygromycin B; T. celer is only affected by neomycin, and D. mobilis is refractory to all drugs. The only feature shared by the four species under study, and by all archaebacteria so far investigated, is their complete insensitivity to streptomycin. The structural and phylogenetic implications of the remarkable diversity encountered among archaebacterial ribosomes in their susceptibility to aminoglycosides are discussed.

Heterogeneity of the polyribocytidilic acid tract in aphthovirus: changes in the size of the poly(C) of viruses recovered from persistently infected cattle.

A sample of aphthovirus type C3 strain Resende carrying two polyribocytidilic acid [poly(C)] tracts was cloned in tissue culture. One clone with a poly(C)-rich tract of about 145 nucleotides long (clone 3B) and another with a poly(C)-rich tract of about 230 nucleotides long (clone 12) and a mixture of both were injected intralingually into three steers. Samples from all three animals were recovered during the acute phase of the disease, from the blood and from the feet, and at various days after inoculation from the oesophageal-pharyngeal (OP) fluids. Analysis of the viral RNAs of the positive samples by means of RNase T1 maps on one- and two-dimensional gels showed (1) changes in the electrophoretic mobility of the poly(C)-rich tracts of viruses recovered from the OP fluids at various times after infection; (2) selection of virus populations with poly(C)-rich tracts of increased size; (3) later on, changes in the patterns of oligonucleotides of persistent viruses. These variations may lead to the production of new strains with altered biological properties that may contribute to the maintenance and spread of these viruses in the field.

Synthesis and functional activity of translation initiation regions in mRNA. 20-base polyribonucleotides from the replicase gene of phage MS2 and fr.

Three 20-base polyribonucleotides, AAACAUGAGGAAUACCCAUG (I), AAACAUGAGGAAAACCCAUG (II), AAACAUGAAGAAUACCCAUG (III), corresponding to the minimal initiation region for the replicase gene of phage MS2 and fr or having some differences were synthesized using enzymatic methods. The template activity of the synthesized polynucleotides in initiation and their capacity to bind phage coat protein were studied under conditions optimal for native mRNA. Polynucleotides I and II exhibit template activity comparable to that of the native phage RNA fragments. Polynucleotide III with the destroyed SD sequence dit not manifest any functional activity either as template or in binding to MS2 phage coat protein.