A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5′ sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

Putative pyrophosphate phosphofructose 1-kinase genes identified in sugarcane may be getting energy from pyrophosphate

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) has been detected in several types of plant cells, but the gene has not been reported in sugar cane. Using Citrux paradixi PPi-PFK gene (AF095520 and AF095521) sequences to search the sugar cane EST database, we have identified both the α and β subunits of this enzyme. The deduced amino acid sequences showed 76 and 80% similarity with the corresponding α and β subunits of C. paradisi. A high degree of similarity was also observed among the PFK β subunits when the alignment of the sugar cane sequences was compared to those of Ricinus communis and Solanum tuberosum, it appears that α and β are two distinct subunits; they were found at different concentrations in several sugar cane tissues. It remains to be determined if the different gene expression levels have some physiological importance and how they affect sucrose synthesis, export, and storage in vacuoles. A comparison between the amino acid sequences of β PFKs from a variety of organisms allowed us to identify the two critical Asp residues typical of this enzyme's activity site and the other binding sites; these residues are tightly conserved in all members of this protein family. Apparently, there are catalytic residues on the β subunit of the pyrophosphate-dependent enzyme.

Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences

The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. In silico expression analysis suggests that some of these transcripts are expressed at low levels and in a restricted set of tissues.

The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

Background: Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. Methods. To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. Results: In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role in YFV replication. Conclusions: Although the precise function of eIF3L on interactions with viral proteins is not entirely understood, these results indicate an interaction of eIF3L with YF NS5 and that eIF3L overexpression facilitates translation, which has potential implications for virus replication. © 2013 Morais et al.; licensee BioMed Central Ltd.

Fast assembly of non-thiolated DNA on gold surface at lower pH

In a typical protocol for attaching DNA to a gold electrode, thiolated DNA is incubated with the electrode at neutral pH overnight. Here we report fast adsorption of non-thiolated DNA oligomers on gold electrodes at acidic pH (i.e., pH ~3.0). The peak-to-peak potential difference and the redox peak currents in typical cyclic voltammetry of [Fe(CN)6]3- are investigated to monitor the attachment. Compared with incubation at neutral pH, the lower pH can significantly promote the adsorption processes, enabling efficient adsorption even in 30min. The adsorption rate is DNA concentration-dependent, while the ionic strength shows no influence. Moreover, the adsorption is base-discriminative, with a preferred order of A>C≫G, T, which is attributed to the protonation of A and C at low pH and their higher binding affinity to gold surface. The immobilized DNA is functional and can hybridize with its complementary DNA but not a random DNA. This work is promising to provide a useful time-saving strategy for DNA assembly on gold electrodes, allowing fast fabrication of DNA-based biosensors and devices. © 2013 Elsevier Inc.

Construction and preliminary characterization of a river buffalo bacterial artificial chromosome library

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq (R))

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Telomere fragment induced amnion cell senescence: a contributor to parturition?

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Optimization of an amperometric biosensor for the detection of hepatitis C virus using fractional factorial designs

Fractional factorial design and factorial with center point design were applied to the development of an amperometric biosensor for the detection of the hepatitis C virus. Biomolecules were immobilized by adsorption on graphite electrodes modified with siloxane-poly(propyleneoxide) hybrid matrix prepared using the sol-gel method. Several parameters were optimized, such as the streptavidin concentration at 0.01 mg mL(-1) and 1.0% bovine serum albumin, the incubation time of the electrodes in the complementary DNA solution for 30 minutes and a 1: 1500 dilution of the avidin-peroxidase conjugate, among others. The application of chemometric studies has been efficient, since the best conditions have been established with a restricted number of experiments, indicating the influence of different factors on the system.

Avaliação de populações de possíveis rizobactérias em solos sob espécies florestais

Embora estudos recentes relatem a utilização de RCPC (Rizobactérias Promotoras de Crescimento de Plantas) no Brasil, raríssimos trabalhos avaliam a presença natural dessas espécies bacterianas no solo. O objetivo deste trabalho foi avaliar a ocorrência de RPCP em duas amostras de solo sob diferentes tipos de manejo, através da construção e do seqüenciamento de bibliotecas de DNA metagenômico. Utilizaram-se oligonucleotídeos específicos para amplificação da região hipervariável do espaço intergênico dos genes ribossomais 16S-23S de DNA extraído de diferentes solos, sob Eucalyptus sp. e sob mata. Os fragmentos obtidos foram inseridos em vetor e clonados. As bibliotecas geraram 495 clones, que foram seqüenciados e identificados através de comparações realizadas pelo software Blast. O solo sob Eucalyptus sp. apresentou maior número de RPCP do que sob mata. Os filos Actinobacteria e Proteobacteria eram maiores no solo sob Eucalyptus sp., estando o filo Firmicutes ausente no solo sob mata. Somente oito espécies diferentes de RPCP foram detectadas: Bacillus subtilis, Bacillus megaterium, Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium sp., Frankia sp., Pseudomonas fluorescens e Pseudomonas gladioli. O trabalho forneceu valiosos dados sobre a presença de RPCP em solos com espécies florestais e sua possível utilização em reflorestamentos, assim como para o melhor conhecimento desses microrganismos nos solos do Brasil.

Molecular characterization of hemoglobin alpha-D chains from Geochelone carbonaria and Geochelone denticulata land turtles

In order to help elucidate the evolution of alpha-globins, the complete cDNA and amino acid sequences of Geochelone carbonaria and Geochelone denticulata land turtles alpha-D chains have been described. In G. carbonaria, the cDNA is 539 bp with ATG start codon located at position 46, TGA stop codon at position 469 and AATAAA polyadenylation signal at position 520. In G. denticulata, the cDNA is 536 bp with ATG start codon located at position 46, TGA stop codon at position 469 and AATAAA polyadenylation signal at position 517. Both cDNAs codify 141 amino acid residues, differing from each other in only four amino acid residues. When comparing with human Hb alpha-chain, alterations in important regions can be noted: alpha110 Ala-Gly, alpha114 Pro-Gly, alpha117 Phe-Tyr and alpha122 His-Gln. There is a high homology between the amino acids of these turtles when compared with chicken alpha-D chains, progressively decreasing when compared with human, crocodile, snake, frog and fish alpha-chains. Phylogenetic analysis of alpha-D chains shows that those of turtles are closer to those of birds than to snakes and lizards. (C) 2002 Elsevier B.V. All rights reserved.

Cloning and expression of the cDNA encoding rat granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) acts on precursor hematopoietic cells to control the production and maintenance of neutrophils. Recombinant G-CSF (re-G-CSF)is used clinically to treat patients with neutropenia and has greatly reduced the infection risk associated with bone marrow transplantation. Cyclic hematopoiesis, a stem cell defect characterized by severe recurrent neutropenia, occurs in man and grey collie dogs, and can be treated by administration of re-G-CSF. Availability of the rat G-CSF cDNA would benefit the use of rats as models of gene therapy for the treatment of cyclic hematopoiesis. In preliminary rat experiments, retroviral-mediated expression of canine G-CSF caused neutralizing antibody formation which precluded long-term increases in neutrophil counts. To overcome this problem we cloned the rat G-CSF cDNA from RNA isolated from skin fibroblasts. The rat G-CSF sequence shared a high degree of identity in both the coding and non-coding regions with both the murine G-CSF (85%) and human G-CSF (74%). The signal peptides of murine and human G-CSF both contained 30 amino acids (aa), whereas the deduced signal sequence for rat G-CSF possessed 21 aa. A retrovirus encoding the rat G-CSF cDNA synthesized bioactive G-CSF from transduced vascular smooth muscle cells.

Growth hormone mRNA expression in the pituitary of Bos indicus and Bos taurus x Bos indicus crossbred young bulls treated with recombinant bovine somatotropin

The effects of breed and of recombinant bovine somatotropin (rbST) treatment on growth hormone gene expression were studied in young bulls. The experiment was completely randomized in a [2 × 2]-factorial arrangement, using two levels of rbst (0 or 250 mg/animal/14 days), and two breed groups (Nelore and Simmental x Nelore crossbred). A CDNA encoding Bos indicus growth hormone was cloned and sequenced for use as a probe in Northern and dot blot analyses. Compared to the Bos taurus structural gene, the Bos indicus CDNA was found to begin 21 bases downstream from the transcription initiation site and had only two discrepancies (C to T at position 144-His and T to C at position 354-Phe), without changes in the polypeptide sequence. However, two amino acid substitutions were found for Bubalus spp., which belong to the same tribe. The rbst treatment did not change any of the characteristics evaluated (body and pituitary gland weights, growth hormone MRNA expression level). Crossbred animals had significantly higher body weight and heavier pituitaries than Nelore cattle. Pituitary weight was proportional to body weight in both breed groups. Growth hormone MRNA expression in the pituitary was similar (P>0.075) for both breed and hormonal treatment groups, but was 31.9% higher in the pure Nelore group, suggesting that growth hormone gene transcription regulation differs among these breeds.

Cloning and characterization of the cDNA for the Brazilian Cratomorphus distinctus larval firefly luciferase: Similarities with European Lampyris noctiluca and Asiatic Pyrocoelia luciferases

Studies on firefly (Lampyridae) luciferases have focused on nearctic species of Photinus and Photuris and Euroasiatic species of Lampyris, Luciola, Hotaria, and Pyrocoelia. Despite accounting for the greatest diversity of fireflies in the world, no molecular studies have been carried out on the highly diverse genera from the neotropical region. Here we report the luciferase cDNA cloning for the larva of the Brazilian firefly Cratomorphus distinctus. The cDNA has 1978 bp and codes for a 547-residue-long polypeptide. Noteworthy, sequence comparison as well as functional properties show the highest degree of similarity with Lampyris noctiluca (93%) and Pyrocoelia spp. (91%) luciferases, suggesting a close phylogenetic relationship despite the geographical distance separating these species. The bioluminescence emission spectrum peaks at 550 nm and, as expected, is sensitive to pH, shifting to 605 nm at pH 6. The kinetic properties of the recombinant luciferase were similar to those of other firefly luciferases. © 2004 Elsevier Inc. All rights reserved.