Descripción: Splitting and apparent splicing of ribosomal RNA, both previously unknown in vertebrates, were found in rodents of the genus Ctenomys. Instead of being formed by a single molecule of 4.4 kb, 28S rRNA is split in two molecules of 2.6 and 1.8 kb. A hidden break, mapping within a 106 bp 'intron' located in the D6 divergent region, is expressed in mature ribosomes of liver, lung, heart and spleen, as well as in primary fibroblast cultures. Testis-specific processing eliminates the intron and concomitantly the break site, producing non-split 28S rRNA molecules exclusively in this organ. The intron is flanked by two 9 bp direct repeats, revealing the acquisition by insertion of a novel rRNA processing strategy in the evolution of higher organisms.

Descripción: In the present work, we study the effect of translational-rotational hydrodynamic coupling on the stationary electric linear dichroism of DNA fragments. The theoretical resolution of the problem has, so far, been dealt with analytic methods valid only in the limit of low electric fields. In this work, we apply numerical methods that allow us to study the problem and also consider electric fields of arbitrary strength. We use the bent rod molecules model to describe DNA fragments with physical properties characterized by their electric charge, electric polarizability tensor, rotational diffusion tensor, and translation-rotation coupling diffusion tensor. The necessary orientational distribution function to calculate electric dichroism is obtained by solving the Fokker-Planck equation through the finite difference method. We analyze the different contributions due to electric polarizability and translational- rotational coupling to the electric dichroism. © 2011 American Institute of Physics.

Descripción: Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5′ and 3′ ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy reveled that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5′ and 3′ CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5′ and 3′ untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5′ and 3′ UAR (upstream AUG region). In order to investigate the functional role of 5′-3′ UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5′ or 3′ UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Descripción: Inhibition of bacterial gene expression by RNase P-directed cleavage is a promising strategy for the development of antibiotics and pharmacological agents that prevent expression of antibiotic resistance. The rise in multiresistant bacteria harboring AAC(6′)-Ib has seriously limited the effectiveness of amikacin and other aminoglycosides. We have recently shown that recombinant plasmids coding for external guide sequences (EGS), short antisense oligoribonucleotides (ORN) that elicit RNase P-mediated cleavage of a target mRNA, induce inhibition of expression of aac(6′)-Ib and concomitantly induce a significant decrease in the levels of resistance to amikacin. However, since ORN are rapidly degraded by nucleases, development of a viable RNase P-based antisense technology requires the design of nuclease resistant RNA analog EGSs. We have assayed a variety of ORN analogs of which selected LNA/DNA co-oligomers elicited RNase P-mediated cleavage of mRNA in vitro. Although we found an ideal configuration of LNA/DNA residues, there seems not to be a correlation between number of LNA substitutions and level of activity. Exogenous administration of as low as 50 nM of an LNA/DNA co-oligomer to the hyperpermeable E. coli AS19 harboring the aac(6′)-Ib inhibited growth in the presence of amikacin. Our experiments strongly suggest an RNase P-mediated mechanism in the observed antisense effect.

Descripción: The mechanisms of RNA replication of plus-strand RNA viruses are still unclear. Here, we identified the first promoter element for RNA synthesis described in a flavivirus. Using dengue virus as a model, we found that the viral RdRp discriminates the viral RNA by specific recognition of a 5′ element named SLA. We demonstrated that RNA-RNA interactions between 5′ and 3′ end sequences of the viral genome enhance dengue virus RNA synthesis only in the presence of an intact SLA. We propose a novel mechanism for minus-strand RNA synthesis in which the viral polymerase binds SLA at the 5′ end of the genome and reaches the site of initiation at the 3′ end via long-range RNA-RNA interactions. These findings provide an explanation for the strict requirement of dengue virus genome cyclization during viral replication. © 2006 by Cold Spring Harbor Laboratory Press.

Descripción: Background: Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative strategies to high-throughput sequencing projects for the identification of novel genes differentially expressed in sunflower as a source of organ-specific genetic markers that can be functionally associated to important traits. Results: Differential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4). The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60%) did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences putatively related to responses to important agronomic traits and key regulatory and physiological genes. Conclusions: The application of suppressed subtracted hybridization technology not only enabled the cost effective isolation of differentially expressed sequences but it also allowed the identification of novel sequences in sunflower from a relative small number of analyzed sequences when compared to major sequencing projects. © 2003 Fernández et al; licensee BioMed Central Ltd.

Descripción: The energetic contributions of individual DNA-contacting side chains to specific DNA recognition in the human papillomavirus 16 E2C-DNA complex is small (less than 1.0 kcal mol-1), independent of the physical and chemical nature of the interaction, and is strictly additive. The sum of the individual contributions differs 1.0 kcal mol-1 from the binding energy of the wild-type protein. This difference corresponds to the contribution from the deformability of the DNA, known as "indirect readout." Thus, we can dissect the energetic contribution to DNA binding into 90% direct and 10% indirect readout components. The lack of high energy interactions indicates the absence of "hot spots," such as those found in protein-protein interfaces. These results are compatible with a highly dynamic and "wet" protein-DNA interface, yet highly specific and tight, where individual interactions are constantly being formed and broken. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: Nucleotide variation was studied in a 1.1 kb section of the coding region of an Esterase gene (Est-A) that maps in the center of the segments rearranged by polymorphic inversions in the cactophilic Drosophila buzzatii. We examine 30 homozygous second-chromosome lines differing in gene arrangement and three D. koepferae isofemale lines as outgroups. Our data show that Est-A is a highly polymorphic gene at both synonymous and replacement sites. Significant departures from homogeneity in the distribution of the ratio of silent polymorphism to divergence predicted by the neutral theory reveals a local excess of silent polymorphism. This is consistent with the presence of two apparent narrow peaks of elevated silent polymorphism surrounding nonconservative amino acid substitutions. These polymorphisms as well as others at synonymous and nonsynonymous sites are shared with D. koepferae. We suggest that the presence of shared nucleotide polymorphisms is probably due to interspecific gene flow and/or balancing selection acting on replacement variants and/or to a decreased probability of loss of ancestral polymorphisms caused by linkage to an adaptive inversion polymorphism. Recurrent mutation and persistence of neutral ancestral polymorphisms cannot, however, be ruled out. The analysis of the distribution of nucleotide variation among the three chromosomal arrangements sampled reveals that derived arrangements (J and JZ3) are less polymorphic than the ancestral ST, and that the widely distributed ST and J arrangements are genetically differentiated. However, a significant number of polymorphisms are shared between arrangements, suggesting frequent exchange either from gene conversion or from double crossovers in heterokaryotypes. Finally, our present results in combination with data of sequence variation at the breakpoints of inversion J suggest that this old gene arrangement has risen in frequency in relatively recent times.

Descripción: Background: A partial deficiency in Protoporphyrinogen oxidase (PPOX) produces the mixed disorder Variegate Porphyria (VP), the second acute porphyria more frequent in Argentina. Identification of patients with an overt VP is absolutely important because treatment depends on an accurate diagnosis but more critical is the identification of asymptomatic relatives to avoid acute attacks which may progress to death. Methods: We have studied at molecular level 18 new Argentinean patients biochemically diagnosed as VP. PPOX gene was amplified in one or in twelve PCR reactions. All coding exons, flanking intronic and promoter regions were manual or automatically sequenced. For RT-PCR studies RNA was retrotranscripted, amplified and sequenced. PPOX activity in those families carrying a new and uncharacterized mutation was performed. Results: All affected individuals harboured mutations in heterozygous state. Nine novel mutations and 3 already reported mutations were identified. Six of the novel mutations were single nucleotide substitutions, 2 were small deletions and one a small insertion. Three single nucleotide substitutions and the insertion were at exon-intron boundaries. Two of the single nucleotide substitutions, c.471G>A and c.807G>A and the insertion (c.388+3insT) were close to the splice donor sites in exons 5, 7 and intron 4 respectively. The other single nucleotide substitution was a transversion in the last base of intron 7, g.3912G>C (c.808-1G>C) so altering the consensus acceptor splice site. However, only in the first case the abnormal band showing the skipping of exon 5 was detected. The other single nucleotide substitutions were transversions: c.101A>T, c.995G>C and c.670 T>G that result in p.E34V, p.G332A and W224G aminoacid substitutions in exons 3, 10 and 7 respectively. Activity measurements indicate that these mutations reduced about 50% PPOX activity and also that they co-segregate with this reduced activity value. Two frameshift mutations, c.133delT and c.925delA, were detected in exons 3 and 9 respectively. The first leads to an early termination signal 22 codons downstream (p.S45fsX67) and the second leads to a stop codon 5 codons downstream (p.I309fsX314). One reported mutation was a missense mutation (p.G232R) and 2 were frameshift mutations: c.1082insC and 1043insT. The last mutation was detected in six new apparently unrelated Argentinean families. Conclusion: Molecular analysis in available family members revealed 14 individuals who were silent carriers of VP. Molecular techniques represent the most accurate approach to identify unaffected carriers and to provide accurate genetic counselling for asymptomatic individuals. The initial screening includes the insertion search. © 2008 Rossetti et al; licensee BioMed Central Ltd.

Descripción: EGS (external guide sequence) technology is a promising approach to designing new antibiotics. EGSs are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. The ftsZ mRNA secondary structure was modeled and EGSs complementary to two regions with high probability of being suitable targets were designed. In vitro reactions showed that EGSs targeting these regions bound ftsZ mRNA and elicited RNase P-mediated cleavage of ftsZ mRNA. A recombinant plasmid, pEGSb1, coding for an EGS that targets region "b" under the control of the T7 promoter was generated. Upon introduction of this plasmid into Escherichia coli BL21(DE3)(pLysS) the transformant strain formed filaments when expression of the EGS was induced. Concomitantly, E. coli harboring pEGSb1 showed a modest but significant inhibition of growth when synthesis of the EGSb1 was induced. Our results indicate that EGS technology could be a viable strategy to generate new antimicrobials targeting ftsZ. © 2012 Sala et al.

Descripción: Background: Premature ovarian failure (POF) is characterized by hypergonadotropic amenorrhoea before the age of 40. Inhibin α-subunit (INHα) gene is proposed as a candidate gene due to its role in negative feedback control of FSH. Methods: Polymorphism -16 C>T of INHα gene was studied in 61 POF patients and 82 controls above 40 years old (C > 40). Substitution 769G>A was studied in 59 POF patients, 76 C > 40 and 73 controls below 40 years old (C < 40). Results: No significant difference in risk of POF development for -16T allele was found when comparing idiopathic POF (I-POF) with C > 40 (Odds ratio = 1.46; 95% confidence interval = 0.63-3.19). Implication of -16C>T polymorphism in serum inhibin levels was analysed in 46 controls, and no significant differences (P > 0.05) were found between CC and CT + TT genotype groups when comparing either mid-follicular phase Pro-αC and inhibin B values or mid-luteal phase Pro-αC and inhibin A values. Heterozygosity for substitution 769G>A was found in 1 of 59 POF woman, 2 of 76 C > 40 and 6 of 73 C < 40. Presence of this su bstitution in a relevant number of control subjects is herein described for the first time. Conclusion: Our results indicate that -16C>T and 769G>A variants in INHα gene may not be associated to POF disease. © 2006 Oxford University Press.

Descripción: The major cAMP receptors in eukaryotes are the regulatory (R) subunits of PKA, an allosteric enzyme conserved in fungi through mammals. While mammals have four R-subunit genes, Saccharomyces cerevisiae has only one, Bcy1. To achieve a molecular understanding of PKA activation in yeast and to explore the evolution of cyclic-nucleotide binding (CNB) domains, we solved the structure of cAMP-bound Bcy1(168-416). Surprisingly, the relative orientation of the two CNB domains in Bcy1 is very different from mammalian R-subunits. This quaternary structure is defined primarily by a fungi-specific sequence in the hinge between the αB/αC helices of the CNB-A domain. The unique interface between the two CNB domains in Bcy1 defines the allosteric mechanism for cooperative activation of PKA by cAMP. Some interface motifs are isoform-specific while others, although conserved, play surprisingly different roles in each R-subunit. Phylogenetic analysis shows that structural differences in Bcy1 are shared by fungi of the subphylum Saccharomycotina. © 2010 Elsevier Ltd.

Descripción: Even though substantial progress has been made to elucidate the physiological and environmental factors underpinning differences in body size, little is known about its genetic architecture. Furthermore, all animal species bear a specific relationship between the size of each organ and overall body size, so different body size traits should be investigated as well as their sexual dimorphism that may have an important impact on the evolution of body size. We have surveyed 191 co-isogenic lines of Drosophila melanogaster, each one of them homozygous for a single P-element insertion, and assessed the effects of mutations on different body size traits compared to the P-element-free co-isogenic control. Nearly 60% of the lines showed significant differences with respect to the control for these traits in one or both sexes and almost 35% showed trait- and sex-specific effects. Candidate gene mutations frequently increased body size in males and decreased it in females. Among the 92 genes identified, most are involved in development and/or metabolic processes and their molecular functions principally include protein-binding and nucleic acid-binding activities. Although several genes showed pleiotropic effects in relation to body size, few of them were involved in the expression of all traits in one or both sexes. These genes seem to be important for different aspects related to the general functioning of the organism. In general, our results indicate that the genetic architecture of body size traits involves a large fraction of the genome and is largely sex and trait specific. © 2009 Macmillan Publishers Limited All rights reserved.

Descripción: In order to assess the relationship between the genus Kluyvera and other members of the family Enterobacteriaceae, the 16S rRNA genes of type strains of the recognized Kluyvera species, Kluyvera georgiana, Kluyvera cochleae, Kluyvera ascorbata and Kluyvera cryocrescens, were sequenced. A comparative phylogenetic analysis based on these 16S rRNA gene sequences and those available for strains belonging to several genera of the family Enterobacteriaceae showed that members of the genus Kluyvera form a cluster that contains all the known Kluyvera species. However, the type strain of Enterobacter intermedius (ATCC 33110 T ) was included within this cluster in a very close relationship with the type strain of K. cochleae (ATCC 51609 T ). In addition to the phylogenetic evidence, biochemical and DNA-DNA hybridization analyses of species within this cluster indicated that the type strain of E. intermedius is in fact a member of the genus Kluyvera and, within it, of the species Kluyvera cochleae. Therefore, following the current rules for bacterial nomenclature and classification, the transfer of E. intermedius to the genus Kluyvera as Kluyvera intermedia comb. nov. is proposed (type strain, ATCC 33110 T =CIP 79.27 T =LMG 2785 T =CCUG 14183 T ). Biochemical analysis of four E. intermedius strains and one K. cochleae strain independent of the respective type strains further indicated that E. intermedius and K. cochleae represent the same species and are therefore heterotypic synonyms. Nomenclatural priority goes to the oldest legitimate epithet. Consequently, Kluyvera cochleae Müller et al. 1996 is a later synonym of Kluyvera intermedia (Izard et al. 1980) Pavan et al. 2005. © 2005 IUMS.