cDNA sequence and molecular modeling of a nerve growth factor from Bothrops jararacussu venomous gland

The complete nucleotide sequence of a nerve growth factor precursor from Bothrops jararacussu snake (Bj-NGF) was determined by DNA sequencing of a clone from cDNA library prepared from the poly(A) + RNA of the venom gland of B.jararacussu. cDNA encoding Bj-NGF precursor contained 723 bp in length, which encoded a prepro-NGF molecule with 241 amino acid residues. The mature Bj-NGF molecule was composed of I 18 amino acid residues with theoretical pI and molecular weight of 8.31 and 13,537, respectively. Its amino acid sequence showed 97%, 96%, 93%, 86%, 78%, 74%, 76%, 76% and 55% sequential similarities with NGFs from Crotalus durissus terrificus, Agkistrodon halys pallas, Daboia (Vipera) russelli russelli, Bungarus multicinctus, Naja sp., mouse, human, bovine and cat, respectively. Phylogenetic analyses based on the amino acid sequences of 15 NGFs separate the Elapidae family (Naja and Bungarus) from those Crotalidae snakes (Bothrops, Crotalus and Agkistrodon). The three-dimensional structure of mature Bj-NGF was modeled based on the crystal structure of the human NGF. The model reveals that the core of NGF, formed by a pair of P-sheets, is highly conserved and the major mutations are both at the three beta-hairpin loops and at the reverse turn. (C) 2002 Societe francaise de biochimie et biologic moleculaire/Editions scientifiques et medicales Elsevier SAS. All rights reserved.

5S rDNA chromosomal mapping and COI sequence analysis reveal differentiation among distinct populations of a characid fish Serrapinnus notomelas

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

Completion of the Nucleotide Sequence of a Brazilian Isolate of Southern bean mosaic virus

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

Complete nucleotide sequence, genomic organization and phylogenetic analysis of Citrus leprosis virus cytoplasmic type

The complete nucleotide sequence of the genomic RNA 1 (8745 nt) and RNA 2 (4986 nt) of Citrus leprosis virus cytoplasmic type (CiLV-C) was determined using cloned cDNA. RNA 1 contains two open reading frames (ORFs), which correspond to 286 and 29 kDa proteins. The 286 kDa protein is a polyprotein putatively involved in virus replication, which contains four conserved domains: methyltransferase, protease, helicase and polymerase. RNA 2 contains four ORFs corresponding to 15, 61, 32 and 24 kDa proteins, respectively. The 32 kDa protein is apparently involved in cell-to-cell movement of the virus, but none of the other putative proteins exhibit any conserved domain. The 5' regions of the two genomic RNAs contain a 'cap' structure and poly(A) tails were identified in the 3'-terminals. Sequence analyses and searches for structural and non-structural protein similarities revealed conserved domains with members of the genera Furovirus, Bromovirus, Tobravirus and Tobamovirus, although phylogenetic analyses strongly suggest that CiLV-C is a member of a distinct, novel virus genus and family, and definitely demonstrate that it does not belong to the family Rhabdoviridae, as previously proposed. Based on these results it was proposed that Citrus leprosis virus be considered as the type member of a new genus of viruses, Cilevirus.

Tamoxifen down-regulates CaMKII messenger RNA levels in normal human breast tissue

Tamoxifen was proven to reduce the incidence of breast cancer by 49% in women at increased risk of the disease in the Breast Cancer Prevention Trial. In order to identify potential candidates to explain the preventive effect induced by tamoxifen on breast cancer, normal breast tissue obtained from 42 fibroadenoma patients, randomly assigned to receive placebo or tamoxifen, was analyzed by the reverse Northern blot and RT-PCR techniques. The cDNA fragments used on Northern blot membranes were generated by the Human Cancer Genome Project funded by the Ludwig Institute for Cancer Research and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil). Total RNA was obtained from normal breast tissue from patients with clinical, cytological and ultrasound diagnosis of fibroadenoma. After a 50-day treatment with tamoxifen (10 or 20 mg/day) or placebo, normal breast tissue adjacent to the tumor was collected during lumpectomy with local anesthesia. One differentially expressed gene, Calcium/calmodulin-dependent protein kinase II (CaMKII), was found to be down-regulated during TAM treatment. CaMKII is an ubiquitous serine/threonine protein kinase that has been implicated in the diverse effects of hormones utilizing Ca2+ as a second messenger as well as in c-fos activation. These results indicate that the down-regulation of CaMKII induced by TAM might represent alternative or additional mechanisms of the action of this drug on cell cycle control and response to hormones in normal human breast tissue.

Mitochondrial genome sequence of the Brazilian luminescent click beetle Pyrophorus divergens (Coleoptera: Elateridae): Mitochondrial genes utility to investigate the evolutionary history of Coleoptera and its bioluminescence

The Coleoptera order is the richest group among Metazoa, but its phylogenetics remains incompletely understood. Among Coleoptera, bioluminescence is found within the Elateroidea, but the evolution of this character remains a mystery. Mitochondrial DNA has been used extensively to reconstruct phylogenetic relationships, however, the evolution of a single gene does not always correspond to the species evolutionary history and the molecular marker choice is a key step in this type of analysis. To create a solid basis to better understand the evolutionary history of Coleoptera and its bioluminescence, we sequenced and comparatively analyzed the mitochondrial genome of the Brazilian luminescent click beetle Pyrophorus divergens (Coleoptera: Elateridae). © 2007 Elsevier B.V. All rights reserved.

Clonagem, expressão e purificação de domínios da proteína AtRLI2 (RNase L Inhibitor), um supressor endógeno de silenciamento por RNA de Arabidopis thaliana, visando estudos estruturais

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Modelo cinético estocástico para a transcrição considerando colisões entre as moléculas de RNA polimerase

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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)

Identities among actin-encoding cDNAs of the Nile tilapia (Oreochromis niloticus) and other eukaryote species revealed by nucleotide and amino acid sequence analyses

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

Complete genome sequence and structural characterization of a novel iflavirus isolated from Opsiphanes invirae (Lepidoptera: Nymphalidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Genome Sequence of Exiguobacterium antarcticum B7, Isolated from a Biofilm in Ginger Lake, King George Island, Antarctica

Exiguobacterium antarcticum is a psychotropic bacterium isolated for the first time from microbial mats of Lake Fryxell in Antarctica. Many organisms of the genus Exiguobacterium are extremophiles and have properties of biotechnological interest, e. g., the capacity to adapt to cold, which make this genus a target for discovering new enzymes, such as lipases and proteases, in addition to improving our understanding of the mechanisms of adaptation and survival at low temperatures. This study presents the genome of E. antarcticum B7, isolated from a biofilm sample of Ginger Lake on King George Island, Antarctic peninsula.

Expression profiling and cross-species RNA interference (RNAi) of desiccation-induced transcripts in the anhydrobiotic nematode Aphelenchus avenae

Abstract Background Some organisms can survive extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living mycophagous nematode Aphelenchus avenae can be induced to enter anhydrobiosis by pre-exposure to moderate reductions in relative humidity (RH) prior to extreme desiccation. This preconditioning phase is thought to allow modification of the transcriptome by activation of genes required for desiccation tolerance. Results To identify such genes, a panel of expressed sequence tags (ESTs) enriched for sequences upregulated in A. avenae during preconditioning was created. A subset of 30 genes with significant matches in databases, together with a number of apparently novel sequences, were chosen for further study. Several of the recognisable genes are associated with water stress, encoding, for example, two new hydrophilic proteins related to the late embryogenesis abundant (LEA) protein family. Expression studies confirmed EST panel members to be upregulated by evaporative water loss, and the majority of genes was also induced by osmotic stress and cold, but rather fewer by heat. We attempted to use RNA interference (RNAi) to demonstrate the importance of this gene set for anhydrobiosis, but found A. avenae to be recalcitrant with the techniques used. Instead, therefore, we developed a cross-species RNAi procedure using A. avenae sequences in another anhydrobiotic nematode, Panagrolaimus superbus, which is amenable to gene silencing. Of 20 A. avenae ESTs screened, a significant reduction in survival of desiccation in treated P. superbus populations was observed with two sequences, one of which was novel, while the other encoded a glutathione peroxidase. To confirm a role for glutathione peroxidases in anhydrobiosis, RNAi with cognate sequences from P. superbus was performed and was also shown to reduce desiccation tolerance in this species. Conclusions This study has identified and characterised the expression profiles of members of the anhydrobiotic gene set in A. avenae. It also demonstrates the potential of RNAi for the analysis of anhydrobiosis and provides the first genetic data to underline the importance of effective antioxidant systems in metazoan desiccation tolerance.

Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA

Abstract Background The ability to manipulate the genetic networks underlying the physiological and behavioural repertoires of the adult honeybee worker (Apis mellifera) is likely to deepen our understanding of issues such as learning and memory generation, ageing, and the regulatory anatomy of social systems in proximate as well as evolutionary terms. Here we assess two methods for probing gene function by RNA interference (RNAi) in adult honeybees. Results The vitellogenin gene was chosen as target because its expression is unlikely to have a phenotypic effect until the adult stage in bees. This allowed us to introduce dsRNA in preblastoderm eggs without affecting gene function during development. Of workers reared from eggs injected with dsRNA derived from a 504 bp stretch of the vitellogenin coding sequence, 15% had strongly reduced levels of vitellogenin mRNA. When dsRNA was introduced by intra-abdominal injection in newly emerged bees, almost all individuals (96 %) showed the mutant phenotype. An RNA-fragment with an apparent size similar to the template dsRNA was still present in this group after 15 days. Conclusion Injection of dsRNA in eggs at the preblastoderm stage seems to allow disruption of gene function in all developmental stages. To dissect gene function in the adult stage, the intra-abdominal injection technique seems superior to egg injection as it gives a much higher penetrance, it is much simpler, and it makes it possible to address genes that are also expressed in the embryonic, larval or pupal stages.

Expression, purification and structural analysis of the Pyrococcus abyssi RNA binding protein PAB1135

Abstract Background The gene coding for the uncharacterized protein PAB1135 in the archaeon Pyrococcus abyssi is in the same operon as the ribonuclease P (RNase P) subunit Rpp30. Findings Here we report the expression, purification and structural analysis of PAB1135. We analyzed the interaction of PAB1135 with RNA and show that it binds efficiently double-stranded RNAs in a non-sequence specific manner. We also performed molecular modeling of the PAB1135 structure using the crystal structure of the protein Af2318 from Archaeoglobus fulgidus (2OGK) as the template. Conclusions Comparison of this model has lead to the identification of a region in PAB1135 that could be involved in recognizing double-stranded RNA.