Descripción: Some strains of Bacillus sphaericus are entomopathogenic to mosquito larvae, which transmit diseases, such as filariasis and malaria, affecting millions of people worldwide. This species is unable to use hexoses and pentoses as unique carbon sources, which was proposed to be due to the lack of glycolytic enzymes, such as 6-phosphofructokinase (PFK). In this study, PFK activity was detected and the pfk gene was cloned and sequenced. Furthermore, this gene was shown to be present in strains belonging to all the homology groups of this heterogeneous species, in which PFK activity was also detected. A careful sequence analysis revealed the conservation of different catalytic and regulatory residues, as well as the enzyme's phylogenetic affiliation with the family of allosteric ATP-PFK enzymes.

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: Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are members of the MAPK family and participate in the transduction of stimuli in cellular responses. Their long-term actions are accomplished by promoting the expression of specific genes whereas faster responses are achieved by direct phosphorylation of downstream effectors located throughout the cell. In this study we determined that hERK1 translocates to the mitochondria of HeLa cells upon a proliferative stimulus. In the mitochondrial environment, hERK1 physically associates with (i) at least 5 mitochondrial proteins with functions related to transport (i.e. VDAC1), signalling, and metabolism; (ii) histones H2A and H4; and (iii) other cytosolic proteins. This work indicates for the first time the presence of diverse ERK-complexes in mitochondria and thus provides a new perspective for assessing the functions of ERK1 in the regulation of cellular signalling and trafficking in HeLa cells. © 2009 Galli et al.

Descripción: Strains derived from HfrH carrying the arcA2 null mutation exhibit a higher respiratory rate, enhanced glucose consumption, and a more-reduced intracellular redox state than arcA deletion mutants of a different lineage. The phenotype of the arcA2 mutants was due to the presence of a creC constitutive mutation introduced by P1 transduction. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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: A genetic locus from Acetobacter xylinum involved in acetan polysaccharide synthesis has been characterized. The chromosomal region was identified by screening a genomic library of A. xylinum in a Xanthomonas campestris mutant defective in xanthan polysaccharide synthesis. The A. xylinum cosmid clone can functionally complement a xanthan-negative mutant. The polymer produced by the recombinant strain was found to be indistinguishable from xanthan. Insertion mutagenesis and subcloning of the cosmid clone combined with complementation studies allowed the identification of a 2.3-kb fragment of A. xylinum chromosomal DNA. The nucleotide sequence of this fragment was analyzed and found to contain an open reading frame (aceA) of 1,182 bp encoding a protein of 43.2 kDa. Results from biochemical and genetic analyses strongly suggest that the aceA gene encodes the GDP- mannose:cellobiosyl-diphosphopolyprenol α-mannosyltransferase enzyme, which is responsible for the transfer of an α-mannosyl residue from GDP-Man to cellobiosyl-diphosphopolyprenol. A search for similarities with other known mannosyltransferases revealed that all bacterial α-mannosyltransferases have a short COOH-termina1 amino acid sequence in common.

Descripción: We describe useful vectors to select double-crossover events directly in site-directed marker exchange mutagenesis in gram-negative bacteria. These vectors contain the gusA marker gene, providing colorimetric screens to identify bacteria harboring those sequences. The applicability of these vectors was shown by mapping the 3' end of the Xanthomonas campestris gum operon, involved in biosynthesis of xanthan.

Descripción: The high-altitude Andean lakes (HAAL) in the Argentinean Puna-high Andes region represent an almost unexplored ecosystem exposed to extreme conditions (high UV irradiation, hypersalinity, drastic temperature changes, desiccation, and high pH). Here we present the first genome sequence, a Sphingomonas sp., isolated from this extreme environment. © 2011, American Society for Microbiology.

Descripción: The proopiomelanocortin gene (POMC) is expressed in a group of neurons present in the arcuate nucleus of the hypothalamus. Neuron-specific POMC expression in mammals is conveyed by two distal enhancers, named nPE1 and nPE2. Previous transgenic mouse studies showed that nPE1 and nPE2 independently drive reporter gene expression to POMC neurons. Here, we investigated the evolutionary mechanisms that shaped not one but two neuron- specific POMC enhancers and tested whether nPE1 and nPE2 drive identical or complementary spatiotemporal expression patterns. Sequence comparison among representative genomes of most vertebrate classes and mammalian orders showed that nPE1 is a placental novelty. Using in silico paleogenomics we found that nPE1 originated from the exaptation of a mammalian- apparent LTR retrotransposon sometime between the metatherian/ eutherian split (147 Mya) and the placental mammal radiation (≈90 Mya). Thus, the evolutionary origin of nPE1 differs, in kind and time, from that previously demonstrated for nPE2, which was exapted from a CORE-short interspersed nucleotide element (SINE) retroposon before the origin of prototherians, 166 Mya. Transgenic mice expressing the fluorescent markers tomato and EGFP driven by nPE1 or nPE2, respectively, demonstrated coexpression of both reporter genes along the entire arcuate nucleus. The onset of reporter gene expression guided by nPE1 and nPE2 was also identical and coincidental with the onset of Pomc expression in the presumptive mouse diencephalon. Thus, the independent exaptation of two unrelated retroposons into functional analogs regulating neuronal POMC expression constitutes an authentic example of convergent molecular evolution of cell-specific enhancers.

Descripción: The stress response involves complex physiological mechanisms that maximize behavioral efficacy during attack or defense and is highly conserved in all vertebrates. Key mediators of the stress response are pituitary hormones encoded by the proopiomelanocortin gene (POMC). Despite conservation of physiological function and expression pattern of POMC in all vertebrates, phylogenetic footprinting analyses at the POMC locus across vertebrates failed to detect conserved noncoding sequences with potential regulatory function. To investigate whether ortholog POMC promoters from extremely distant vertebrates are functionally conserved, we used 5′-flanking sequences of the teleost fish Tetraodon nigroviridis POMCα gene to produce transgenic mice. Tetraodon POMCα promoter targeted reporter gene expression exclusively to mouse pituitary cells that normally express Pomc. Importantly, transgenic expression in mouse corticotrophs was increased after adrenalectomy. To understand how conservation of precise gene expression mechanisms coexists with great sequence divergence, we investigated whether very short elements are still conserved in all vertebrate POMC promoters. Multiple local sequence alignments that consider phylogenetic relationships of ortholog regions identified a unique 10-bp motif GTGCTAA(T/G)CC that is usually present in two copies in POMC 5′-flanking sequences of all vertebrates. Underlined nucleotides represent totally conserved sequences. Deletion of these paired motifs from Tetraodon POMCα promoter markedly reduced its transcriptional activity in a mouse corticotropic cell line and in pituitary POMC cells of transgenic mice. In mammals, the conserved motifs correspond to reported binding sites for pituitary-specific nuclear proteins that participate in POMC transcriptional regulation. Together, these results demonstrate that mechanisms that participate in pituitary-specific and hormonally regulated expression of POMC have been preserved since mammals and teleosts diverged from a common ancestor 450 million years ago despite great promoter sequence divergence. Copyright © 2007 by The Endocrine Society.

Descripción: The role of GAs in promoting seed germination is well known and experiments with seeds from different species have suggested the requirement of de novo synthesis of GAs upon imbibition for germination. There are also strong indications that the enhancement of GA synthesis is part of the mechanism through which environmental signals (i.e. light) induce germination. Since along the GA biosynthetic pathway, oxidation at C-20 carried out by GA 20-oxidases is thought to be a site of regulation, a cDNA clone encoding a GA 20-oxidase was isolated from embryos of sorghum (SbGA 20ox). Expression analysis of this gene in embryos within imbibed caryopses with low dormancy showed detectable amounts of the specific mRNA early upon incubation, increasing thereafter. In contrast, it remained barely detectable in embryos from dormant caryopses. Changes in endogenous GA4 levels were in agreement with those of SbGA 20ox mRNA, suggesting that GA production might be regulated differentially at the level of transcription of this gene. The expression of SbGA 20ox was enhanced in incubated embryos isolated from either type of caryopses, illustrating a physiological control exerted by the surrounding seed tissues on gene expression. The results also show that ABA leads to a suppression of transcription of this gene.

Descripción: In the same way that cry genes, coding for larvicidal delta endotoxins, constitute a large and diverse gene family, the cyt genes for hemolytic toxins seem to compose another set of highly related genes in Bacillus thuringiensis. Although the occurrence of Cyt hemolytic factors in B. thuringiensis has been typically associated with mosquitocidal strains, we have recently shown that cyt genes are also present in strains with different pathotypes; this is the case for the morrisoni subspecies, which includes strains biologically active against dipteran, lepidopteran, and coleopteran larvae. In addition, while one Cyt type of protein has been described in all of the mosquitocidal strains studied so far, the present study confirms that at least two Cyt toxins coexist in the more toxic antidipteran strains, such as B. thuringiensis subsp. israelensis and subsp. morrisoni PG14, and that this could also be the case for many others. In fact, PCR screening and Western blot analysis of 50 B. thuringiensis strains revealed that cyt2-related genes are present in all strains with known antidipteran activity, as well as in some others with different or unknown host ranges. Partial DNA sequences for several of these genes were determined, and protein sequence alignments revealed a high degree of conservation of the structural domains. These findings point to an important biological role for Cyt toxins in the final in vivo toxic activity of many B. thuringiensis strains.

Descripción: The proopiomelanocortin gene (POMC) is expressed in the pituitary gland and the ventral hypothalamus of all jawed vertebrates, producing several bioactive peptides that function as peripheral hormones or central neuropeptides, respectively. We have recently determined that mouse and human POMC expression in the hypothalamus is conferred by the action of two 5′ distal and unrelated enhancers, nPE1 and nPE2. To investigate the evolutionary origin of the neuronal enhancer nPE2, we searched available vertebrate genome databases and determined that nPE2 is a highly conserved element in placentals, marsupials, and monotremes, whereas it is absent in nonmammalian vertebrates. Following an in silico paleogenomic strategy based on genome-wide searches for paralog sequences, we discovered that opossum and wallaby nPE2 sequences are highly similar to members of the superfamily of CORE-short interspersed nucleotide element (SINE) retroposons, in particular to MAR1 retroposons that are widely present in marsupial genomes. Thus, the neuronal enhancer nPE2 originated from the exaptation of a CORE-SINE retroposon in the lineage leading to mammals and remained under purifying selection in all mammalian orders for the last 170 million years. Expression studies performed in transgenic mice showed that two nonadjacent nPE2 subregions are essential to drive reporter gene expression into POMC hypothalamic neurons, providing the first functional example of an exapted enhancer derived from an ancient CORE-SINE retroposon. In addition, we found that this CORE-SINE family of retroposons is likely to still be active in American and Australian marsupial genomes and that several highly conserved exonic, intronic and intergenic sequences in the human genome originated from the exaptation of CORESINE retroposons. Together, our results provide clear evidence of the functional novelties that transposed elements contributed to their host genomes throughout evolution. © 2007 Santangelo et al.

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: Pseudomonas sp. 14-3 accumulates polyhydroxybutyrate (PHB) from octanoate, but not from glucose. To elucidate this unusual phenotype, genes responsible for the synthesis of PHB were cloned and analyzed. A PHB polymerase gene (phaC) was found downstream from genes coding for a β-ketothiolase (phaA), an acetoacetyl-coenzyme A reductase (phaB) and a putative transcriptional regulator (phaR). All genes were similar to pha genes from several related species, but differences were observed in the distal region of phaA. Complementation with heterologous β-ketothiolase genes from Azotobacter sp. FA8 or Pseudomonas putida GPp104 restored the capability of Pseudomonas sp. 14-3 to synthesize PHB from glucose, demonstrating that its β-ketothiolase was nonfunctional. Analysis of the genome sequences of other Pseudomonas species has revealed the existence of putative β-ketothiolase genes. The functionality of one of these thiolase genes, belonging to P. putida GPp104, was experimentally demonstrated. Pseudomonas sp. 14-3 is the first natural phaA mutant described, that despite this mutation accumulates high amounts of PHB when growing on fatty acids. © 2006 Federation of European Microbiological Societies.

Descripción: Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp. FA8 were cloned and analyzed. A PHB polymerase gene (phbC) was found downstream from genes coding for β-ketothiolase (phbA) and acetoacetyl-coenzyme A reductase (phbB). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The phbC gene from this strain was introduced into Raktonia eutropha PHB-4 (phbC-negative mutant), and the recombinant accumulated PHB when either glucose or oclanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chuin-length hydroxyalkanoates into PHB.

Descripción: Background: Human β-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the β-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information.Results: Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl), showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels. Conclusions: cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation. © 2010 Fustiñana et al; licensee BioMed Central Ltd.

Descripción: Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. It is composed of polymerized pentasaccharide repeating malts which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (L. Ielpi, R. O. Couso, and M. A. Dankert, J. Bacteriol. 175:2490- 2500, 1993). A cluster of 12 genes in a region designated xpsI or gum has been suggested to encode proteins involved in the synthesis and polymerization of the lipid intermediate. However, no experimental evidence supporting this suggestion has been published. In this work, from the biochemical analysis of a defined set of X. campestris gum mutants, we report experimental data for assigning functions to the products of the gum genes. We also show that the first step in the assembly of the lipid-linked intermediate is severely affected by the combination of certain gum and non- gum mutations. In addition, we provide evidence that the C-terminal domain of the gumD gene product is sufficient for its glucosyl-1-phosphate transferase activity. Finally, we found that alterations in the later stages of xanthan biosynthesis reduce the aggressiveness of X. campestris against the plant.

Descripción: We report the cloning and characterization of rat α10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the α10 nAChR subunit gene is most similar to the rat α9 nAChR, and both α9 and α10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with α10 cRNA alone or in pairwise combinations with either α2-α6 or β2-β4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of α9 and α10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric α9 channels, the α9α10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct current-voltage relationship, and a biphasic response to changes in extracellular Ca2+ ions. The pharmacological profiles of homomeric α9 and heteromeric α9α10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous cholinergic eceptors found in vertebrate hair cells. Our data suggest that efferent modulation of hair cell function occurs, at least in part, through heteromeric nAChRs assembled from both α9 and α10 subunits.