The copper resistance operon copAB from Xanthomonas axonopodis pathovar citri: gene inactivation results in copper sensitivity

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker and the completion of the Xac genome sequence has opened up the possibility of investigating basic cellular mechanisms at the genomic level. Copper compounds have been extensively used in agriculture to control plant diseases. The copA and copB genes, identified by annotation of the Xac genome, encode homologues of proteins involved in copper resistance. A gene expression assay by Northern blotting revealed that copA and copB are expressed as a unique transcript specifically induced by copper. Synthesis of the gene products was also induced by copper, reaching a maximum level at 4 h after addition of copper to the culture medium. CopA was a cytosolic protein and CopB was detected in the cytoplasmic membrane. The gene encoding CopA was disrupted by the insertion of a transposon, leading to mutant strains that were unable to grow in culture medium containing copper, even at the lowest CUSO4 concentration tested (0.25 mM), whereas the wild-type strain was able to grow in the presence of 1 mM copper. Cell suspensions of the wild-type and mutant strains in different copper concentrations were inoculated in lemon leaves to analyse their ability to induce citrus canker symptoms. Cells of mutant strains showed higher sensitivity than the wild-type strain in the presence of copper, i.e. they were not able to induce citrus canker symptoms at high copper concentrations and exhibited a more retarded growth in planta.

cAMP signaling pathway controls glycogen metabolism in Neurospora crassa by regulating the glycogen synthase gene expression and phosphorylation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Clarifications on breakpoints in HSAX and BTAX by comparative mapping of F9, HPRT, and XIST in cattle

The coagulation factor IX gene (179), the hypoxanthine phosphoribosyl transferase 1 gene (HPRT1), and the X-inactive specific transcript gene (XIST) were physically assigned in cattle to analyze chromosomal breakpoints on BTAX recently identified by radiation hybrid (RH) mapping experiments. Whereas the FISH assignment of XIST indicates a similar location on the q-arm of the human and cattle X chromosomes, the locus of HPRT1 supported the assumption of a chromosome rearrangement between the distal half of the q-arm of HSAX and the p-arm of BTAX identified by RH mapping. F9 previously located on the Cl-arm of BTAX was assigned to the p-arm of BTAX using RH mapping and FISH. The suggested new position of F9 close to HPRT I supports the homology between HSAXq and BTAXp. The F9 locus corresponds with the gene order found in the homologous human chromosome segment. XIST was assigned on BTAXq23, HPRT1 and F9 were mapped to BTAXp22, and the verification of the location of F9 in a 5000 rad cattle-hamster whole genome radiation hybrid panel linked the gene to markers URB10 and HPRT1. Copyright (C) 2003 S. Karger AG, Basel.

The putative telomerase reverse transcriptase component of Leishmania amazonensis: gene cloning and characterization

The Leishmania amazonensis telomerase gene was cloned by a polymerase chain reaction-based strategy using primers designed from a Leishmania major sequence that shared similarities with conserved telomerase motifs. The genes from three other species were cloned for comparative purposes. A ClustalW multiple-sequence alignment demonstrated that the Leishmania telomerases show greater homology with each other than with the proteins of other kinetoplastids and eukaryotes. Characterization experiments indicated that the putative Leishmania telomerase gene was probably in single copy and located in the largest chromosomes. A single messenger ribonucleic acid transcript was found in promastigotes. Phylogenetic analysis suggested that Leishmania telomerase might represent a liaison between the oldest and the newest branches of telomerases.

The contribution of transposable elements to Bos taurus gene structure

In an effort to identify the contribution of TEs to bovine genome evolution, the abundance, distribution and insertional orientation of TEs were examined in all bovine nuclear genes identified in sequence build 2.1 (released October 11, 2005). Exons, introns and promoter segments (3 kb upstream the transcription initiation sites) were screened with the RepeatMasker program. Most of the genes analyzed contained TE insertions, with an average of 18 insertions/gene. The majority of TE insertions identified were classified as retrotransposons and the remainder classified as DNA transposons. TEs were inserted into exons and promoter segments infrequently, while insertion into intron sequences was strikingly more abundant. The contribution of TEs to exon sequence is of great interest because TE insertions can directly influence the phenotype by altering protein sequences. We report six cases where the entire exon sequences of bovine genes are apparently derived from TEs and one of them, the insertion of Charlie into a bovine transcript similar to the zinc finger 452 gene is analyzed in detail. The great similarity of the TE-cassette sequence to the ZNF452 protein and phylogenetic relationship strongly suggests the occurrence of Charlie 10 DNA exaptation in the mammalian zinc finger 452 gene. (c) 2006 Published by Elsevier B.V.

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.

NtWBC1, an ABC transporter gene specifically expressed in tobacco reproductive organs

To identify genes specifically or predominantly expressed in the stigmas/styles and to establish their possible function in the reproductive process of plants, a tobacco stigma/style cDNA library was constructed and differentially screened, resulting in the isolation of several cDNA clones. The molecular characterization of one of these clones is described here. After sequencing the cDNA and the isolated genomic clone, it was determined that the corresponding gene encodes a protein containing an ATP-binding cassette, characteristic of ABC transporters. This gene, designated as NtWBC1 (Nicotiana tabacum ABC transporter of the White-Brown Complex subfamily), encodes a protein that contains the typical structure of the 'half-transporters' of the White subfamily. To establish the spatial expression pattern of the NtWBC1 gene, northern blot and real-time RT-PCR analyses with total RNA from roots, stems, leaves, sepals, petals, stamens, stigmas/styles, ovaries, and seeds were performed. The result revealed a transcript of 2.5 kb present at high levels in stigmas and styles and a smaller transcript (2.3 kb) present at a lower level in stamens. NtWBC1 expression is developmentally regulated in stigmas/styles, with mRNA accumulation increasing toward anthesis. In situ hybridization experiments demonstrated that NtWBC1 is expressed in the stigmatic secretory zone and in anthers, at the stomium region and at the vascular bundle. NtWBC1 is the first ABC transporter gene with specific expression in plant reproductive organs to be identified and its expression pattern suggests important role(s) in the reproductive process.

The Paracoccidioides brasiliensis gp70 antigen is encoded by a putative member of the flavoproteins monooxygenase family

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Localization and hormonal regulation of endometrial matrix metalloproteinase-26 in the rhesus macaque

The current understanding of hormonal regulation of matrix metalloproteinase-26 (MMP-26) in the primate endometrium is incomplete. The goal of this work was to clarify estrogen and progesterone regulation of MMP-26 in the endometrium of ovariectomized, hormone-treated rhesus macaques.Ovariectomized rhesus macaques (n 66) were treated with estradiol (E-2), E-2 plus progesterone, E-2 followed by progesterone alone or no hormone. Endometrium was collected from the hormone-treated animals during the early, mid- and late proliferative and secretory phases of the artificial menstrual cycle. MMP-26 expression was quantified by real-time PCR, and MMP-26 transcript and protein were localized by in situ hybridization and immunohistochemistry and correlated with estrogen receptor 1 and progesterone receptor (PGR).MMP-26 was localized to glandular epithelium and was undetectable in the endometrial stroma and vasculature. MMP-26 transcript levels were minimal in the hormone-deprived macaques and treatment with E-2 alone did not affect MMP-26 levels. Treatment with progesterone both in the presence and absence of E-2 stimulated MMP-26 expression in the early and mid-secretory phases (P 0.001). MMP-26 expression preceded decidualization of endometrial stroma. MMP-26 levels then declined to baseline in the late secretory phase (P 0.01) despite continued E-2 plus progesterone treatment. Loss of detectable MMP-26 expression in the late secretory phase was correlated with late secretory phase loss of glandular epithelial PGR.Endometrial MMP-26 expression is dependent on the presence of progesterone in the early secretory phase and then gradually becomes refractory to progesterone stimulation in the late secretory phase. In the macaque, MMP-26 is a marker of the pre-decidual, secretory endometrium. During the second half of the late secretory phase, and during decidualization, MMP-26 loses its response to progesterone concurrent with the loss of epithelial PGR. The decline in MMP-26 levels between the mid- and late secretory phases may play a role in the receptive window for embryo implantation.

Selective Ablation of the Androgen Receptor in Mouse Sertoli Cells Affects Sertoli Cell Maturation, Barrier Formation and Cytoskeletal Development

The observation that mice with a selective ablation of the androgen receptor (AR) in Sertoli cells (SC) (SCARKO mice) display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli) and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin). Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2). It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development.

Conditional reasoning: A key to assessing computer-based knowledge-building communication processes

This article describes a methodological approach to conditional reasoning in online asynchronous learning environments such as Virtual-U VGroups, developed by SFU, BC, Canada, consistent with the notion of meaning implication: If part of a meaning C is embedded in B and a part of a meaning B is embedded in A, then A implies C in terms of meaning [Piaget 91]. A new transcript analysis technique was developed to assess the flows of conditional meaning implications and to identify the occurrence of hypotheses and connections among them in two human science graduate mixed-mode online courses offered in the summer/spring session of 1997 by SFU. Flows of conditional meaning implications were confronted with Virtual-U VGroups threads and results of the two courses were compared. Findings suggest that Virtual-U VGroups is a knowledge-building environment although the tree-like Virtual-U VGroups threads should be transformed into neuronal-like threads. Findings also suggest that formulating hypotheses together triggers a collaboratively problem-solving process that scaffolds knowledge-building in asynchronous learning environments: A pedagogical technique and an built-in tool for formulating hypotheses together are proposed. © Springer Pub. Co.

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.

ZmPUMP encodes a maize mitochondrial uncoupling protein that is induced by oxidative stress

A cDNA clone (designated ZmPUMP) encoding an uncoupling protein (UCP) from maize (Zea mays) was identified by searching for homologous sequences among the expressed sequence tags of the GenBank database. The ZmPUMP cDNA contains a single open reading frame of 933 nucleotides encoding 310 amino acids. Several features identified the predicted ZmPUMP protein as a member of the mitochondrial UCP subfamily of mitochondrial carriers. Expression studies demonstrated that ZmPUMP is ubiquitously expressed in maize tissues and its transcript level is not altered in early stages of embryo germination. In contrast to known UCP genes, ZmPUMP is not responsive to cold stress. Instead its expression is increased in response to H 2O 2- or menadione-induced oxidative stress. © 2003 Elsevier Science Ireland Ltd. All rights reserved.

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.

In silico evaluation of the Eucalyptus transcriptome

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