DNA partitions into triplets under tension in the presence of organic cations, with sequence evolutionary age predicting the stability of the triplet phase.

Using atomistic simulations, we show the formation of stable triplet structure when particular GC-rich DNA duplexes are extended in solution over a timescale of hundreds of nanoseconds, in the presence of organic salt. We present planar-stacked triplet disproportionated DNA (Σ DNA) as a possible solution phase of the double helix under tension, subject to sequence and the presence of stabilising co-factors. Considering the partitioning of the duplexes into triplets of base pairs as the first step of operation of recombinase enzymes like RecA, we emphasise the structure-function relationship in Σ DNA. We supplement atomistic calculations with thermodynamic arguments to show that codons for 'phase 1' amino acids (those appearing early in evolution) are more likely than a lower entropy GC-rich sequence to form triplets under tension. We further observe that the four amino acids supposed (in the 'GADV world' hypothesis) to constitute the minimal set to produce functional globular proteins have the strongest triplet-forming propensity within the phase 1 set, showing a series of decreasing triplet propensity with evolutionary newness. The weak form of our observation provides a physical mechanism to minimise read frame and recombination alignment errors in the early evolution of the genetic code.

Ataluren para o tratamento da distrofia muscular de Duchenne / Ataluren for the treatment of Duchenne muscular dystrophy

TECNOLOGIA:TranslarnaTM (ataluren). INDICAÇÃO NA BULA: TranslarnaTM é indicado para o tratamento da distrofia muscular de Duchenne resultante de uma mutação nonsense (DMDmn) no gene distrofina, em pacientes com capacidade de marcha e com idade igual ou superior a cinco anos. A eficácia não foi demonstrada em doentes sem capacidade de marcha(6) . PERGUNTA: o ataluren é eficaz e seguro para o tratamento da distrofia muscular de Duchenne? EVIDÊNCIAS: foi analisado um ensaio clínico randomizado que avaliou o ataluren em pacientes com distrofia muscular de Duchenne, comparado com placebo. A diferença na variação média da distância de caminhada de seis minutos entre o ataluren e o placebo, o desfecho primário avaliado, não foi estatisticamente significante entre todos os indivíduos randomizados. Os resultados foram favoráveis ao ataluren quanto a alteração na função muscular proximal e a alteração na força exercida durante a flexão e a extensão do joelho, comparado com o placebo. O ataluren foi geralmente bem tolerado. Não houve descontinuações do estudo devido a eventos adversos e, em sua maioria, os eventos adversos foram leves ou moderados. O ensaio clínico selecionado foi patrocinado pela empresa titular da autorização de introdução do ataluren no mercado. CONCLUSÕES: a distrofia muscular de Duchenne não tem cura e o seu tratamento se baseia na terapia sintomática. O ataluren é recomendado para o tratamento da distrofia muscular de Duchenne em pessoas com idade igual ou superior a cinco anos, que conservam a capacidade da marcha. Considerando a incerteza dos benefícios clínicos relevantes do ataluren na prática clínica, há que se buscar uma relação custo/benefício que seja favorável para o seu financiamento. O ataluren demonstrou ser bem tolerado, apresentando eventos adversos leves ou moderado, em sua maioria. O ataluren não tem registro de comercialização no Brasil.(AU)

A semi-synthetic organism with an expanded genetic alphabet.

Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A-T and G-C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM), which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism's genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA containing the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid containing d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.

A structural basis for streptomycin-induced misreading of the genetic code.

During protein synthesis, the ribosome selects aminoacyl-transfer RNAs with anticodons matching the messenger RNA codon present in the A site of the small ribosomal subunit. The aminoglycoside antibiotic streptomycin disrupts decoding by binding close to the site of codon recognition. Here we use X-ray crystallography to define the impact of streptomycin on the decoding site of the Thermus thermophilus 30S ribosomal subunit in complexes with cognate or near-cognate anticodon stem-loop analogues and messenger RNA. Our crystal structures display a significant local distortion of 16S ribosomal RNA induced by streptomycin, including the crucial bases A1492 and A1493 that participate directly in codon recognition. Consistent with kinetic data, we observe that streptomycin stabilizes the near-cognate anticodon stem-loop analogue complex, while destabilizing the cognate anticodon stem-loop analogue complex. These data reveal how streptomycin disrupts the recognition of cognate anticodon stem-loop analogues and yet improves recognition of a near-cognate anticodon stem-loop analogue.

The use of synthetic oligonucleotides as protein inhibitors and anticode drugs in cancer therapy: accomplishments and limitations.

The function of gene products can be altered at many levels, including the mutation of gene sequence and the change in steady state levels of mRNA and/or protein by various mechanisms. The cumulative malfunction of specific gene products underlies many pathological conditions such as the multi-step and multi-cause acquisition of cancer. Here we discuss two oligonucleotide-based strategies in which these compounds target defective gene products acting either as antiprotein or anticode agents. The SELEX technique (systematic evolution of ligands by exponential enrichment) is an antiprotein approach in which nuclease-resistant DNA or RNA aptamers are selected by their ability to bind their protein targets with high affinity and specificity of the same range as antibodies. Such inhibitors were previously evolved against a great variety of targets, including receptors, growth factors and adhesion molecules implicated in the genesis of some kinds of cancer. Moreover, some results have already been obtained in animal models. The antigene technology interferes with earlier steps in the information flow leading from gene to protein. In this approach selective gene silencing is provided by the formation of stable and specific complexes between triplex forming molecules and their DNA targets. The feasibility of this strategy as well as a molecular mechanism for the action of antigene oligonucleotides has been demonstrated in cellular systems and in vivo. The use of oligonucleotide drugs (of either the antiprotein or the anticode type) as a viable approach to cancer therapy is limited by some common problems that will be discussed.

[Differences in the gene structure of the matrix protein and hemagglutinin in remantadine-resistant and -sensitive variants of the influenza virus type A FPV]. / Razlichiia v strukture genov matrikskogo belka i gemaggliutinina ustoichivogo i chuvstvitel'nogo k remantadinu variantov virusa grippa tipa FPV.

The oligonucleotide mapping technique and RNA-RNA hybridization revealed structural differences between the HA- and M-protein genes of two variants of influenza A FPV, remantadine-sensitive and remantadine-resistant ones. A quantitative estimation of the structural divergence of the two influenza FPV pairs of genes was carried out. The possible functional role of the differences in the structure of the HA- and M-protein genes of the two FPV variants is discussed.

Altered 40 S ribosomal subunits in omnipotent suppressors of yeast.

The five suppressors SUP35, SUP43, SUP44, SUP45 and SUP46, each mapping at a different chromosomal locus in the yeast Saccharomyces cerevisiae, suppress a wide range of mutations, including representatives of all three types of nonsense mutations, UAA, UAG and UGA. We have demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosomes from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin. Furthermore, ribosomal subunit mixing experiments established that the 40 S ribosomal subunit, and this subunit only, is responsible for the higher levels of misreading. Thus, the gene products of SUP35, SUP44, SUP45 and SUP46 are components of the 40 S subunit or are enzymes that modify the subunit. In addition, a protein from the 40 S subunit of the SUP35 suppressor has an altered electrophoretic mobility; this protein is distinct from the altered protein previously uncovered in the 40 S subunit of the SUP46 suppressor. In contrast to the ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46, the ribosomes from the SUP43 suppressor do not significantly misread polyuridylate templates in vitro, suggesting that this locus may not encode a ribosomal component or that the misreading is highly specific.

[Characteristics of the derepressed mutant of the F-like plasmid pAP18-1 coding for tetracycline resistance in Escherichia coli]. / Kharakteristika derepressirovannogo mutanta F-podobnoi plazmidy pAP18-1, kodiruiushchei rezistentnost' k tetratsiklinu u Escherichia coli.

A transfer function derepressed mutant of the F-like plasmid pAP18-1 (Tc, ColV) was induced with the help of N-methyl-N'-nitro-N-nitrozoguanidine. The mutant plasmid pAP18-1drd belongs to the FVII incompatibility group of the F-like plasmids. The plasmid pAP18-1drd is characterized by the loss of the capacity for inhibiting the tra-genes functions of the Flac plasmid and is sensitive to the Tra-function inhibitors of the reference plasmids of the FinV and FinW groups.

[Identification of the number of mutant copies of the genetic transfer factor AP42]. / Identifikatsiia mutantov kopiinosti faktora geneticheskogo perenosa pAP42.

The copy number mutant of genetic transfer factor pAP42 (incFIX) was induced by the treatment of E. coli AP115 cells carrying the plasmid by N-methyl-N-nitro-N-nitrosoguanidine. The copy number mutant pAP42::Tn1cop1 is characterized by the increased copy number. The cells carrying the copy number mutant have a higher ampicillin resistance and higher beta-lactamase activity. Mutant plasmid pAP42: :Tn1cop1 is incompatible with plasmid pAP42 and compatible with plasmids of the other inc-groups of F-like plasmids.

Streptomycin-induced, third-position misreading of the genetic code.

Streptomycin was used to increase the frequency of errors in protein synthesis in vivo. In the system under study two misreading errors were observed. Both involved the erroneous insertion of lysine at asparagine codons, because of misreading of a pyrimidine as a purine at the 3' position of the codon. Streptomycin increased the errors at the two codons AAU and AAC to the same extent, thereby maintaining the error ratio found for basal level mistranslation.

Epigenetic carcinogens: problems with identification and risk estimation.

The mechanisms of carcinogenesis are just beginning to be understood. There is recent interest in the broad classification of carcinogens into two categories based upon their mechanism of action: those that interact with DNA via a genetic mechanism are termed genetic carcinogens; and those that do not directly interact with DNA, but may cause changes in DNA tertiary structure or methylation patterns, and are termed epigenetic carcinogens. Present knowledge is inadequate to justify separate risk assessment methods for genetic vs. epigenetic carcinogens. Quantitative estimates of carcinogenic risk are currently best made using non-threshold models.

Effect of codon shortening and the antibiotics viomycin and sparsomycin upon the behaviour of bound aminoacyl-tRNA. Decoding at the ribosomal A site.

70 S ribosomes were programmed with initiator tRNA and messenger oligonucleotides AUG(U)n and AUG(C)n, where n = 1, 2 or 3. The binding of the ternary complexes [Phe-tRNA X EF-Tu X GTP] and [Pro-tRNA X EF-Tu X GTP] to the programmed ribosomes was studied. If codon-anticodon interaction is restricted to only one basepair, the ternary complex leaves the ribosome before GTP hydrolysis. Two basepairs allow hydrolysis of GTP, but the aminoacyl-tRNA dissociates and is recycled, resulting in wastage of GTP. Three basepairs result in apparently stable binding of aminoacyl-tRNA to the ribosome. The antibiotic sparsomycin weakens the binding by an amount roughly equivalent to one messenger base, while viomycin has the reverse effect.

Concurrent transfer and recombination between plasmids encoding for heat-stable enterotoxin and drug resistance in porcine enterotoxigenic Escherichia coli.

The frequency of and genetic mechanisms for simultaneous transfer of genes encoding for tetracycline and sulpha-streptomycin resistance, heat-labile (LT) and heat-stable (ST-mouse) enterotoxin production in porcine enterotoxigenic Escherichia coli to Escherichia coli K12 were investigated. Seven E. coli strains of 0-group 149 were studied by conjugation and transformation experiments. All strains transferred tetracycline-resistance plasmids at a high frequency. No interaction was observed between these plasmids and those encoding for LT production. However, most tetracycline-resistant recipient cells were ST-mouse+ following recombination events between plasmids encoding for colicin B and ST-mouse production and plasmids encoding for tetracycline resistance. Alternatively, when selecting for sulpha or streptomycin resistance a majority of the transconjugants were also ST-mouse+, as plasmids coding for sulpha and streptomycin were mobilized by the colicin B and ST-mouse encoding plasmid. Since the simultaneous transfer of genes encoding for drug resistance, colicin B and ST-mouse production are common events in vitro, they might also occur frequently in vivo during antibiotic selective pressure.

[Hereditary and environmental factors in the causation of neoplasia based on studies in Xiphophorus (author's transl)]. / Erb- und Umweltfaktoren im Ursachengefüge des neoplastischen Wachstums nach Studien an Xiphophorus.

The genetic information for neoplastic transformation is inherited as a normal part of the genome in all individuals of Xiphophorus. Neoplasia, however, was found only in hybrids between members of different populations and local races. It can be classified in (a) a large group that is triggered by mutagens, (b) a large group that is triggered by mutagens, (b) a large group triggered by promoters, (c) a small group that develops, "spontaneously", and (d) a small group that is inherited according to Mendelian Laws. The process leading to susceptibility for neoplasia is represented by the disintegration of coadapted gene systems that normally protect the fish from neoplasia. Hybridization is the most effective process that leads to disintegration of the protection gene-systems. Environmental mutagens and promoters (i.e. carcinogens) may complete disintegration and thus may trigger neoplasia. The phenomenon of introducing susceptibility to neoplasia by means of hybridization has been observed in a large variety of plants and animals (Table 2). While we have no data on the relation between hybridization and cancer in human beings comparable to those in plants and animals, we put the question whether the many facts on tumor incidence in humans, that do not agree with the concept of the primacy of environmental factors in carcinogenesis may be explained by interpopulational and interracial hybridization in preceding generations. Based on our studies on Xiphophorus we suppose that environmental factors represent only the peak of an iceberg in the multistep process of the causation of neoplasia. The most important steps leading to neoplasia, i.e. those that bring out susceptibility, are supposed to be hidden in our ancestry.

Reverse transcriptase inhibitors and chemically induced bladder tumors in mice.

Recent immunologic and microbiologic evidence suggests that urothelial tumors may be caused by "C" type oncogenic viruses. Such viruses may exert their oncogenic potential in responce to stimulation by known chemical carcinogens. By means of a unique enzyme, reverse transcriptase, these viruses are able to incorporate genetic information into that of the host, and can thereby be transmitted vertically from generation to generation. An evaluation of the specific antiviral agents dimethylbenzyldemethl-rifampicin and streptovaricin-comples, which inhibit the enzyme reverse transcriptase, revealed no depay in the induction of bladder tumors by the chemical carcinogen, 2-formylamino-4-(5-nitro-2-furyl) thiazole (FANFT) in C3H mice. This observation suggests that the reproduction and release of virus may not be essential in the malignant transformation of bladder epithelial cells, but does not preclude the possiblity that inherited viral genetic information may be involved in the oncogenesis of bladder tumors.

[Genetic control of the ethanol effect on the human EEG]. / Genetische Determiniertheit der Alkohol-Wirkung auf das menschliche EEG

26 identical and 26 fraternal twin pairs were subjected to 1,2 ml/kg alcohol loading under standardized conditions; furthermore, 13 non-twin persons were repeatedly subjected to the same procedure in order to test the intraindividual variability. The alcohol effect was pursued by repeated EEG recordings. The EEGs of identical twins proved to react identically to alcohol loading, whereas the EEGs of fraternal twins became more dissimilar during the course of the experiment. The identical reaction of the brain wave pattern in monozygotic twins cannot be attributed to more similar blood alcohol concentrations; instead, this finding is a pharmacogenetic phenomenon of the central nervous system.

Structure-activity relationships among negamycin analogs.

Various negamycin analogs were examined for (1) miscoding activity and (2) inhibition of the termination of protein synthesis. Since properties (1) and (2) do not correlate for the investigated compounds they may depend on different structural features of negamycin analogs. The results of biochemical and antimicrobial studies indicate that (a) the natural configuration of the carbon atom carrying the beta-amino group is essential, (b) the delta-hydroxyl group is unnecessary, and (c) the acylation of the epsilon-amino group causes loss of activity.