Molecular and biochemical characterization of the Neurospora crassa glycogen synthase encoded by the gsn cDNA

Glycogen synthases catalyze the transfer of a glucosyl moiety from a nucleotide phosphosugar to a nascent glycogen chain via an alpha1-->4 linkage. Although many genes coding for glycogen synthases have been described, the enzymes from rabbit and yeast are the best characterized. The fungus Neurospora crassa accumulates glycogen during exponential growth, and mobilizes it at the onset of stationary phase, or when placed at high temperature or starved for carbon. Through a PCR methodology, the gsn cDNA coding for the N. crassa glycogen synthase was isolated, and the amino acid sequence of the protein was deduced. The product of the cDNA seems to be the only glycogen synthase present in N. crassa. Characterization of the gsn cDNA revealed that it codes for a 706-amino acids protein, which is very similar to mammalian and yeast glycogen synthases. Gene expression increased during exponential growth, reaching its maximal level at the end of the exponential growth phase, which is consistent with the pattern of glycogen synthase activity and glycogen level. Expression of the gsn is highly regulated at the transcriptional level. Under culture conditions that induce heat shock, conidiation, and carbon starvation, expression of the gsn gene was decreased, and glycogen synthase activity and glycogen content behaved similarly.

A molecular mechanism for Lys(49)-phospholipase A(2) activity based on ligand-induced conformational change

Agkistrodon contortrix laticinctus myotoxin is a Lys(49)- phospholipase A(2) (EC 3.1.1.4) isolated from the venom of the serpent A contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A contortrix laticinctus myotoxin involves the side chains of Phe(121) and Phe(124) and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118-125. Comparison with other Lys(49)-phospholipase A(2) myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca2+-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase active site and the C-terminal myotoxic site, justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.

Molecular characterisation and evolutionary relationships of a potyvirus infecting Crotalaria in Brazil - Brief report

The 3' terminal genomic region of a potyvirus causing mosaic disease in several Crotalaria species has been cloned and sequenced. Comparisons of the nucleotide and deduced amino acid (aa) sequences of the cloned cDNA with those from other potyviruses show that the Crotalaria-infecting virus (designated Crotalaria mosaic virus; CrMV) is closely related to Cowpea aphid-borne mosaic virus (CABMV). Maximum identity (95.4%) at the coat protein (CP) aa level was observed between CrMV and a Brazilian strain of CABMV. Phylogenetic analyses derived from the sequence alignments of the CP and 3' untranslated region confirmed the identification of CrMV as a strain of CABMV and the name CABMV-Cr is suggested.

SYSTEMATIC RELATIONSHIPS WITHIN CHIRODERMA (CHIROPTERA, PHYLLOSTOMIDAE) BASED ON CYTOCHROME-B SEQUENCE VARIATION

The Neotropical bat genus Chiroderma consists of five recognized species. This study uses DNA-sequence variation of the mitochondrial cytochrome b gene to infer the phylogenetic relationships within Chiroderma. Phylogenetic relationships deduced from these data by parsimony analyses resulted in the discovery of a single most-parsimonious tree with C. salvini diverging basal to the other four species of Chiroderma and sister-group relationships of C. villosum with C. improvisum and C. trinitatum with C. doriae. This is a relatively young group of species with approximate times of divergence ranging from 1.6 million years before present (mya) for the divergence of C. doriae from C. trinitatum to 4.6 mya for the divergence of C. salvini from the other four species of Chiroderma.

Isolation and sequence determination of peptides in the venom of the spider wasp (Cyphononyx dorsalis) guided by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry

Micro-scale (sub-pmol) isolation and sequence determination of three peptides from the venom of the solitary spider wasp Cyphononyx dorsalis is described. We isolated two novel peptides Cd-125 and Cd-146 and a known peptide Thr(6)-bradykinin from only two venom sacs of solitary spider wasp Cyphononyx dorsalis without bioassay-guided fractionation. but instead guided by MALDI-TOF MS. The MALDI-TOF MS analysis of each fraction showed the purity and molecular weight of the components, which led to the isolation of the peptides virtually without loss of sample amount. The sequences of the novel peptides Cd-125 (Asp-Thr-Ala-Arg-Leu-Lys-Trp-His) and Cd-146 (Ser-Glu-Thr-Gly-Asn-Thr-Val-Thr-Val-Lys-Gly-Phe-Ser-Pro-Leu-Arg) were determined by Edman degradation together with mass spectrometry. and finally corroborated by solid-phase synthesis. The known peptide Thr(6)-bradykinin (Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg) was identified by comparison with the synthetic authentic specimen. This is the first example for any kinins to be found in Pompilidae wasp venoms. The procedure reported here can be applicable to studies on many other components of solitary wasp venoms with limited sample availability. (C) 2001 Elsevier B.V. Ltd. All rights reserved.

Molecular phylogeny of the green lacewings (Neuroptera : Chrysopidae)

The first quantitative analysis of phylogenetic relationships of green lacewings (Chrysopidae) is presented based on DNA sequence data. A single nuclear and two mitochondrial genes are used in the analysis: carbomoylphosphate synthase (CPS) domain of carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) (i.e. rudimentary locus), large subunit ribosomal gene (16S) and cytochrome oxidase I (COI). This study represents the first use of the CAD gene to investigate phylogenetic relationships of the lacewings. DNA sequences for 33 chrysopid species from 18 genera, representing all subfamilies and tribes, were compared with outgroups sampled from families Hemerobiidae, Osmylidae and Polystoechotidae. Parsimony analyses of the combined data set recovered all of the previously established subfamilial and tribal groups as monophyletic clades (although relatively weakly supported) except Apochrysinae sensu lato. The enigmatic Nothancyla verreauxi Navas has historically been difficult to place in a subfamily group based on morphological characteristics; molecular data presented herein do not adequately resolve this problem.

Phylogeography of the Solanaceae-infecting Basidiomycota fungus Rhizoctonia solani AG-3 based on sequence analysis of two nuclear DNA loci

Background: the soil fungus Rhizoctonia solani anastomosis group 3 (AG-3) is an important pathogen of cultivated plants in the family Solanaceae. Isolates of R. solani AG-3 are taxonomically related based on the composition of cellular fatty acids, phylogenetic analysis of nuclear ribosomal DNA (rDNA) and beta-tubulin gene sequences, and somatic hyphal interactions. Despite the close genetic relationship among isolates of R. solani AG-3, field populations from potato and tobacco exhibit comparative differences in their disease biology, dispersal ecology, host specialization, genetic diversity and population structure. However, little information is available on how field populations of R. solani AG-3 on potato and tobacco are shaped by population genetic processes. In this study, two field populations of R. solani AG-3 from potato in North Carolina (NC) and the Northern USA; and two field populations from tobacco in NC and Southern Brazil were examined using sequence analysis of two cloned regions of nuclear DNA (pP42F and pP89).Results: Populations of R. solani AG-3 from potato were genetically diverse with a high frequency of heterozygosity, while limited or no genetic diversity was observed within the highly homozygous tobacco populations from NC and Brazil. Except for one isolate (TBR24), all NC and Brazilian isolates from tobacco shared the same alleles. No alleles were shared between potato and tobacco populations of R. solani AG-3, indicating no gene flow between them. To infer historical events that influenced current geographical patterns observed for populations of R. solani AG-3 from potato, we performed an analysis of molecular variance (AMOVA) and a nested clade analysis (NCA). Population differentiation was detected for locus pP89 (Phi(ST) = 0.257, significant at P < 0.05) but not for locus pP42F (Phi(ST) = 0.034, not significant). Results based on NCA of the pP89 locus suggest that historical restricted gene flow is a plausible explanation for the geographical association of clades. Coalescent-based simulations of genealogical relationships between populations of R. solani AG-3 from potato and tobacco were used to estimate the amount and directionality of historical migration patterns in time, and the ages of mutations of populations. Low rates of historical movement of genes were observed between the potato and tobacco populations of R. solani AG-3.Conclusion: the two sisters populations of the basidiomycete fungus R. solani AG-3 from potato and tobacco represent two genetically distinct and historically divergent lineages that have probably evolved within the range of their particular related Solanaceae hosts as sympatric species.

Molecular organization of 5S rDNA in fishes of the genus Brycon

There are few reports on the genomic organization of 5S rDNA in fish species. To characterize the 5S rDNA nucleotide sequence and chromosomal localization in the Neotropical fishes of the genus Brycon, 5S rDNA copies from seven species were generated by PCR. The nucleotide sequences of the coding region (5S rRNA gene) and the nontranscribed spacer (NTS) were determined, revealing that the 5S rRNA genes were highly conserved, while the NTSs were widely variable among the species analyzed. Moreover, two classes of NTS were detected in each species, characterized by base substitutions and insertions-deletions. Using fluorescence in situ hybridization (FISH), two 5S rDNA chromosome loci that could be related to the two 5S rDNA NTS classes were observed in at least one of the species studied. 5S rDNA sequencing and chromosomal localization permitted the characterization of Brycon spp. and suggest a higher similarity among some of them. The data obtained indicate that the 5S rDNA can be an useful genetic marker for species identification and evolutionary studies.

The generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags

Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define approximate to23,500 genes, of which only approximate to1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that <1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers.

Expression and variability of molecular chaperones in the sugarcane expressome

Molecular chaperones perform folding assistance in newly synthesized polypeptides preventing aggregation processes, recovering proteins from aggregates, among other important cellular functions. Thus their study presents great biotechnological importance. The present work discusses the mining for chaperone-related sequences within the sugarcane EST genome project database, which resulted in approximately 300 different sequences. Since molecular chaperones are highly conserved in most organisms studied so far, the number of sequences related to these proteins in sugarcane was very similar to the number found in the Arabidopsis thaliana genome. The Hsp70 family was the main molecular chaperone system present in the sugarcane expressome. However, many other relevant molecular chaperones systems were also present. A digital RNA blot analysis showed that 5'ESTs from all molecular chaperones were found in every sugarcane library, despite their heterogeneous expression profiles. The results presented here suggest the importance of molecular chaperones to polypeptide metabolism in sugarcane cells, based on their abundance and variability. Finally, these data have being used to guide more in deep analysis, permitting the choice of specific targets to study. (c) 2006 Elsevier GmbH. All rights reserved.

Molecular characterization of Acidithiobacillus ferrooxidans and A-thiooxidans strains isolated from mine wastes in Brazil

Nineteen strains of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, including 12 strains isolated from coal, copper, gold and uranium mines in Brazil, strains isolated from similar sources in other countries and the type strains of the two species were characterized together with the type strain of A. caldus by using a combination of molecular systematic methods, namely ribotyping, BOX- and ERIC-PCR and DNA-DNA hybridization assays. Data derived from the molecular fingerprinting analyses showed that the tested strains encompassed a high degree of genetic variability. Two of the Brazilian A. ferrooxidans organisms (strains SSP and PCE) isolated from acid coal mine waste and uranium mine effluent, respectively, and A. thiooxidans strain DAMS, isolated from uranium mine effluent, were the most genetically divergent organisms. The DNA-DNA hybridization data did not support the allocation of Acidithiobacillus strain SSP to the A. ferrooxidans genomic species, as it shared only just over 40% DNA relatedness with the type strain of the species. Acidithiobacillus strain SSP was not clearly related to A. ferrooxidans in the 16S rDNA tree.

Molecular and Serologic Detection of Ehrlichia spp. in Endangered Brazilian Wild Captive Felids

Ehrlichiosis, an emergent tickborne disease that affects both humans and animals, may represent a threat to the survival and preservation of wild felids in Brazil There are few studies of ehrlichiosis in wild felids in Brazil, but Ehrlichia spp are present in domestic cats Antibodies to Ehrlichia canis have been reported in a puma (Puma concolor) In this study we assessed the presence of these hemoparasites in the blood of Brazilian wild captive felids of the 72 animals tested, 5 (7%) were seropositive for the E cams antigen, and L1 (15%) were positive for E emirs DNA sequences We also performed sequence alignment to establish the identity of the parasite species infecting these animals using 16S rRNA and omp-1 genes Sequences based on 16S rRNA were similar to those found in dogs and cats from Thailand, Brazil, China, and Taiwan and with E canis obtained from a single individual (human) in Venezuela Ehrlichia sp sequence from sampled felines based on omp-1. gene was similar to the p28 and p30 multigene family of E canis To our knowledge, this is the first study of molecular detection of Ehrlichia sp in Brazilian wild feline species

Molecular and serological detection of Ehrlichia canis and Babesia vogeli in dogs in Colombia

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

MOLECULAR PHYLOGENY of THE GENUS KUMANOA (BATRACHOSPERMALES, RHODOPHYTA)

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

Molecular tools and analytical approaches for the characterization of farm animal genetic diversity

Genetic studies of livestock populations focus on questions of domestication, within- and among-breed diversity, breed history and adaptive variation. In this review, we describe the use of different molecular markers and methods for data analysis used to address these questions. There is a clear trend towards the use of single nucleotide polymorphisms and whole-genome sequence information, the application of Bayesian or Approximate Bayesian analysis and the use of adaptive next to neutral diversity to support decisions on conservation.