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PubMed1990

The BM12 mutation and autoantibodies to dsDNA in NZB.H-2bm12 mice.

Molecular and genetic tools have been used to shed light on the genes that contribute to susceptibility to murine lupus and the mechanisms that lead to immunopathology. The MHC genes and their products have been consistently shown to contribute toward the development of disease. To understand the contribution of MHC-class II genes, our laboratory had derived two inbred strains of mice, NZB.H-2bm12 and NZB.H-2b. These new colonies of mice were studied and compared in the 10th generation backcross; inbreeding was serially followed by H-2 typing, responses to beef/porcine insulin, and the presence of the B6 Ig allotype, IgG2ab. Of great interest is the finding that NZB.H-2bm12, in contrast to NZB.H-2b or NZB (H-2d), mice develop high titer autoantibodies to dsDNA. This result is unique because NZB (H-2d) mice, unliked NZB x NZW (NZB/W F1) or NZB x SWR (SNF1) hybrids do not develop autoantibodies to dsDNA, even after immunization. NZB mice, in contrast, are characterized only by autoantibodies to ssDNA. Our observation is also striking because the gene conversion that resulted in the I-A beta bm12 mutation occurred at amino acid residues 68, 71, and 72 of I-E beta b. Recently the contribution of NZW to accelerated autoimmunity in the NZB x NZW F1 hybrid has also been linked to H-2 and a single amino acid change at amino acid 72 of I-E beta. Thus, amino acid residue 72 may be a hot spot for disorders of immune regulation when superimposed on the appropriate genetic background. NZB mice expressing the I-Abm12 mutation will allow specific dissection of the requirements for autoantibody production to dsDNA uncomplicated by heterozygosity.

Amino Acid Sequence↗

PubMed2003

An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi.

The hyperthermophilic euryarchaeon Pyrococcus abyssi and the related species Pyrococcus furiosus and Pyrococcus horikoshii, whose genomes have been completely sequenced, are presently used as model organisms in different laboratories to study archaeal DNA replication and gene expression and to develop genetic tools for hyperthermophiles. We have performed an extensive re-annotation of the genome of P. abyssi to obtain an integrated view of its phylogeny, molecular biology and physiology. Many new functions are predicted for both informational and operational proteins. Moreover, several candidate genes have been identified that might encode missing links in key metabolic pathways, some of which have unique biochemical features. The great majority of Pyrococcus proteins are typical archaeal proteins and their phylogenetic pattern agrees with its position near the root of the archaeal tree. However, proteins probably from bacterial origin, including some from mesophilic bacteria, are also present in the P. abyssi genome.

Adaptation, Physiological↗

PubMed1997

Introduction of unmarked mutations in the Helicobacter pylori vacA gene with a sucrose sensitivity marker.

Research on Helicobacter pylori has been hindered by the lack of useful genetic tools. Using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows introduction of unmarked mutations in H. pylori. A kan-sacB cassette, consisting of the sacB gene expressed from the H. pylori flagellin promoter and the kanamycin resistance module, was introduced by homologous recombination into a target H. pylori gene. The resultant strains were sucrose sensitive and kanamycin resistant. Following transformation with a mutated allele, growth in sucrose-containing medium allowed the selection of strains that had lost the kan-sacB module and had integrated the unmarked allele. We have used this cassette to perform a site-directed modification of two histidine residues encoded by the vacA gene in a two-step procedure. This system should prove useful in the site-directed mutagenesis of H. pylori genes.

Alleles↗

PubMed2001

Use of operon fusions in Mannheimia haemolytica to identify environmental and cis-acting regulators of leukotoxin transcription.

The leukotoxin of Mannheimia haemolytica is an important virulence factor that contributes to much of the pathology observed in the lungs of animals with bovine shipping fever pneumonia. We believe that identification of factors that regulate leukotoxin expression may provide insight into M. haemolytica pathogenicity. The DNA sequence upstream of the leukotoxin operon is divergently shared by P(lapT), which transcribes an arginine permease gene. The intergenic region contains several elements that are potential sites for transcriptional modulation of the promoters. We have developed plasmid-borne chloramphenicol acetyltransferase (cat) operon fusions, as well as lktC::cat chromosomal fusions, to study transcription initiation in M. haemolytica. Using these genetic tools, we have identified cis-acting sequences and environmental conditions that modulate transcription of the leukotoxin and lapT promoters. By deletion analysis, promoters were shown to rely on sequences upstream of their -10 and -35 regions for full activity. Direct repeats of the sequence TGT-N(11)-ACA and a static bend region caused by phased adenine tracts were necessary for full activation of P(lkt). A computer-generated model of the promoter's structure shows how DNA bending brings the repeat sequences within close proximity to the P(lkt) RNA polymerase, and we hypothesize that these repeats are a binding site for an activator of leukotoxin transcription. The lktC::cat operon fusion was also used to demonstrate that, like that of other RTX toxins, leukotoxin transcription is environmentally regulated. Roles for iron deprivation and temperature change were identified.

Bacterial Toxins↗

PubMed2006

Population dynamics of an endogenous meiotic drive system in Aedes aegypti in Trinidad.

An endogenous meiotic drive system was previously reported to be segregating in the yellow fever mosquito Aedes aegypti L. (Diptera: Culicidae) population in Trinidad. The meiotic driver (M(D)) is tightly linked to the male determining locus and selectively targets sensitive responders linked to the female determining allele, causing fragmentation of female gametes. This results in highly male-biased progeny. The M(D) system was initially studied as a genetic tool for population control with limited success, but recently interest has focused on its potential for population replacement. This study examines the distribution and dynamics of the M(D) system in Trinidad natural populations. We obtained ovitrap samples from seven geographically distinct regions and determined the allele frequencies of the driver (M(D)) and sensitive (m(s)) versus insensitive (m(i)) responders, respectively. Frequencies of the M(D) allele ranged from 0.1 to 0.5 and were low at the two major port cities, Port of Spain and San Fernando, suggesting the effects of frequent immigration by non-driving genotypes. Frequencies of the m(i) allele ranged from 0.4 to 0.7, suggesting the effects of strong selection by the driver. In addition, our results show that the driver and sensitivity of responders in the Trinidad populations are highly polymorphic. Continued studies of the dynamics of the M(D) system in natural populations are critical to considerations of its use in population replacement.

Aedes↗

PubMed2006

Identification of virulence-associated genes of Pseudomonas viridiflava activated during infection by use of a novel IVET promoter probing plasmid.

Analysis of virulence mechanisms of plant pathogens is often limited by the lack of genetic tools that can be used to identify genes that are preferentially expressed during their interactions with plants. In the present study, we used the newly constructed IVET (in vivo expression technique) plasmid pIviGK and the corresponding antibiotic resistance-based selection method to identify genes that encode pathogenicity factors of the soft rot-causing bacterium Pseudomonas viridiflava. These included pel, the gene encoding pectate lyase, which is responsible for the development of soft rot symptoms. We have also isolated and characterized the gene mviNpv encoding a putative novel membrane associated virulence factor of P. viridiflava. A mutation in mviNpv was shown to influence motility as well as virulence of P. viridiflava. The mviNpv gene is expressed to a moderate level in LB media and its expression increases under inducing conditions as was shown by measuring in planta expression dynamics of the fused gfp reporter gene.

Capsicum↗

PubMed2004

Approach for functional analysis of glycan using RNA interference.

The elucidation of the biological role of glycan is one of the most important issues to be resolved following the genome project. RNA interference is becoming an efficient reverse genetic tool for studying gene function in model organisms, including C.elegans and Drosophila melanogaster. Our molecular evolutionary study has shown that a prototype of glycosyltransferases, which synthesize a variety of glycan structures in the Golgi apparatus, was conserved between mammals and Drosophila. For analyses of the basic physiological functions of glycans, we established the Drosophila inducible RNAi knockdown system and applied it to one glycosyltransferase and one transporter, proteoglycan UDP-galactose: beta-xylose beta1,4galactosyltransferase I and the PAPS-transporter, respectively. If on the silencing of each gene induced ubiquitously under the control of a cytoplasmic actin promoter, the RNAi knockdown fly died, then the protein was indispensable for life. The expression of the target gene was disrupted specifically and the degree of interference was well correlated with the phenotype. The inducible RNAi knockdown fly obtained using the GAL4-UAS system will pave the way for the functional analysis of glycans.

Actins↗

PubMed2007

Adaptation of a luciferase gene reporter and lac expression system to Borrelia burgdorferi.

The development of new genetic systems for studying the complex regulatory events that occur within Borrelia burgdorferi is an important goal of contemporary Lyme disease research. Although recent advancements have been made in the genetic manipulation of B. burgdorferi, there still remains a paucity of basic molecular systems for assessing differential gene expression in this pathogen. Herein, we describe the adaptation of two powerful genetic tools for use in B. burgdorferi. The first is a Photinus pyralis firefly luciferase gene reporter that was codon optimized to enhance translation in B. burgdorferi. Using this modified reporter, we demonstrated an increase in luciferase expression when B. burgdorferi transformed with a shuttle vector encoding the outer surface protein C (OspC) promoter fused to the luciferase reporter was cultivated in the presence of fresh rabbit blood. The second is a lac operator/repressor system that was optimized to achieve the tightest degree of regulation. Using the aforementioned luciferase reporter, we assessed the kinetics and maximal level of isopropyl-beta-D-thiogalactopyranoside (IPTG)-dependent gene expression. This lac-inducible expression system also was used to express the gene carried on lp25 required for borrelial persistence in ticks (bptA). These advancements should be generally applicable for assessing further the regulation of other genes potentially involved in virulence expression by B. burgdorferi.

Animals↗

PubMed2005

Chemotaxis behavior mediated by single larval olfactory neurons in Drosophila.

BACKGROUND: Odorant receptors (ORs) are thought to act in a combinatorial fashion, in which odor identity is encoded by the activation of a subset of ORs and the olfactory sensory neurons (OSNs) that express them. The extent to which a single OR contributes to chemotaxis behavior is not known. We investigated this question in Drosophila larvae, which represent a powerful genetic system to analyze the contribution of individual OSNs to odor coding. RESULTS: We identify 25 larval OR genes expressed in 21 OSNs and generate genetic tools that allow us to engineer larvae missing a single OSN or having only a single or a pair of functional OSNs. Ablation of single OSNs disrupts chemotaxis behavior to a small subset of the odors tested. Larvae with only a single functional OSN are able to chemotax robustly, demonstrating that chemotaxis is possible in the absence of the remaining elements of the combinatorial code. We provide behavioral evidence that an OSN not sufficient to support chemotaxis behavior alone can act in a combinatorial fashion to enhance chemotaxis along with a second OSN. CONCLUSIONS: We conclude that there is extensive functional redundancy in the olfactory system, such that a given OSN is necessary and sufficient for the perception of only a subset of odors. This study is the first behavioral demonstration that formation of olfactory percepts involves the combinatorial integration of information transmitted by multiple ORs.

Animals↗

PubMed2002

RNA interference: applicability in tick research.

The rapid development of new genetic tools has boosted the gene discovery machinery. RNA interference (RNAi), a gene silencing process, has been recently used in several eukaryotic organisms to elucidate the function(s) of unknown genes and biochemical pathways. We used the dsRNA technique in Amlyomma americanum female ticks to test the applicability of the RNAi approach in ticks. Incubation of tick salivary glands (TSGs) in vitro and in vivo injection into whole female ticks with histamine binding protein (HBP) dsRNA led to a reduction in the HBP transcripts in the dsRNA treated groups. The dsRNA-injected ticks had a profound difference in their feeding pattern compared to control ticks that might reflect an increase in local histamine concentrations at the feeding sites. To our knowledge, this is the first RNAi study in ticks. In conclusion, RNAi can be applied in ticks and might be used to test the function of key proteins crucial for avoiding host defense at the tick-host interface.

Animals↗

PubMed1999

Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata.

The opportunistic pathogen Candida glabrata causes significant disease in humans. To develop genetic tools to investigate the pathogenicity of this organism, we have constructed ura3 and his3 auxotrophic strains by deleting the relevant coding regions in a C. glabrata clinical isolate. Linearized plasmids carrying a Saccharomyces cerevisiae URA3 gene efficiently transformed the ura3 auxotroph to prototrophy. Homologous recombination events were observed when the linearized plasmid carried short terminal regions homologous with the chromosome. In contrast, in the absence of any chromosomal homology, the plasmid integrated by illegitimate recombination into random sites in the genome. Sequence analysis of the target sites revealed that for the majority of illegitimate transformants there was no microhomology with the integration site. Approximately 0.25% of the insertions resulted in amino acid auxotrophy, suggesting that insertion was random at a gross level. Sequence analysis suggested that illegitimate recombination is nonrandom at the single-gene level and that the integrating plasmid has a preference for inserting into noncoding regions of the genome. Analysis of the relative numbers of homologous and illegitimate recombination events suggests that C. glabrata possesses efficient systems for both homologous and nonhomologous recombination.

Base Sequence↗

PubMed2006

Baculovirus-mediated gene silencing in insect cells using intracellularly produced long double-stranded RNA.

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetics tool to silence gene expression in multiple organisms, including plants, nematodes and insects. In this study, DNA vectors capable of promoting the synthesis of long hairpin dsRNAs in vivo from a DNA template to suppress gene expression in insect cells have been successfully constructed. The inhibition of the expression of a gene encoding enhanced green fluorescent protein (eGFP) in insect cells was demonstrated by using plasmid or baculovirus vectors. Both plasmid and baculovirus vectors were able to inhibit eGFP expression in a dose dependent manner. Complete inhibition was obtained when co-transfection ratios of target plasmid to inhibition plasmid were 1:1 and 1:0.1. Eighty percent suppression was still maintained even when the ratio of eGFP plasmid to 'hairpin' plasmid was as high as 1:0.01. When the hairpin dsRNAs were encoded in a baculovirus, the suppression was about 50% when the ratio of 'target' baculovirus to 'inhibition' baculovirus reached 1:10. Therefore, the designed plasmid and baculovirus vectors are useful to induce RNAi in insect cell systems.

Animals↗

PubMed1994

Gene expression in mycobacteria: transcriptional fusions based on xylE and analysis of the promoter region of the response regulator mtrA from Mycobacterium tuberculosis.

Understanding promoter regulation and signal-transduction systems in pathogenic mycobacteria is critical for uncovering the processes that govern interactions of these bacteria with the human host. In order to develop additional genetic tools for analysis of mycobacterial promoters, the xyIE gene from Pseudomonas was tested as a transcriptional fusion reporter in fast- and slow-growing mycobacteria. Initially, its utility was demonstrated by expression behind the hsp60 promoter in Mycobacterium smegmatis and Mycobacterium bovis BCG. The presence of an active promoter in front of the promoterless xyIE cassette on a plasmid was scored by development of a bright yellow colour upon spraying of mycobacterial colonies on plates with a solution of catechol. The gene product of xyIE, catechol 2,3 dioxygenase, was measurable in sonic extracts and whole cells, permitting quantitative determination of promoter activity in both fast- and slow-growing mycobacteria. The xyIE-based mycobacterial transcriptional fusion plasmid pRCX3 was constructed and used to assess promoter activity within the sequences located upstream of the newly characterized Mycobacterium tuberculosis H37Rv response regulator mtrA, a member of the superfamily of bacterial signal-transduction systems.

Bacterial Proteins↗

PubMed2006

Molecular characterization of teflon, a gene required for meiotic autosome segregation in male Drosophila melanogaster.

Drosophila melanogaster males lack recombination and have evolved a mechanism of meiotic chromosome segregation that is independent of both the chiasmatic and achiasmatic segregation systems of females. The teflon (tef) gene is specifically required in males for proper segregation of autosomes and provides a genetic tool for understanding recombination-independent mechanisms of pairing and segregation as well as differences in sex chromosome vs. autosome segregation. Here we report on the cloning of the tef gene and the molecular characterization of tef mutations. Rescue experiments using a GAL4-driven pUAS transgene demonstrate that tef corresponds to predicted Berkeley Drosophila Genome Project (BDGP) gene CG8961 and that tef expression is required in the male germ line prior to spermatocyte stage S4. Consistent with this early prophase requirement, expression of tef was found to be independent of regulators of meiotic M phase initiation or progression. The predicted Tef protein contains three C2H2 zinc-finger motifs, one at the amino terminus and two in tandem at the carboxyl terminus. In addition to the zinc-finger motifs, a 44- to 45-bp repeat is conserved in three related Drosophila species. On the basis of these findings, we propose a role for Tef as a bridging molecule that holds autosome bivalents together via heterochromatic connections.

Amino Acid Sequence↗

PubMed2006

Enhancement of virus-induced gene silencing in tomato by low temperature and low humidity.

Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studying gene function in plants. We showed that silencing of a phytoene desaturase (PDS) gene is maintained throughout TRV-PDS-inoculated tomato plants as well as in their flowers and fruit and is enhanced by low temperature (15 degrees C) and low humidity (30%). RT-PCR analysis of the PDS gene revealed a dramatic reduction in the level of PDS mRNA in leaves, flowers and fruits. Silencing of PDS results in the accumulation of phytoene, the desaturase substrate. In addition, the content of chlorophyll a, chlorophyll b and total chlorophyll in the leaves of PDS-silenced plants was reduced by more than 90%. We also silenced the LeEIN2 gene by infecting seedlings, and this suppressed fruit ripenning. We conclude that this VIGS approach should facilitate large-scale functional analysis of genes involved in the development and ripening of tomato.

Carotenoids↗

PubMed2005

DISTORTED3/SCAR2 is a putative arabidopsis WAVE complex subunit that activates the Arp2/3 complex and is required for epidermal morphogenesis.

In a plant cell, a subset of actin filaments function as a scaffold that positions the endomembrane system and acts as a substrate on which organelle motility occurs. Other actin filament arrays appear to be more dynamic and reorganize in response to growth signals and external cues. The distorted group of trichome morphology mutants provides powerful genetic tools to study the control of actin filament nucleation in the context of morphogenesis. In this article, we report that DISTORTED3 (DIS3) encodes a plant-specific SCAR/WAVE homolog. Null alleles of DIS3, like those of other Arabidopsis thaliana WAVE and Actin-Related Protein (ARP) 2/3 subunit genes, cause trichome distortion, defects in cell-cell adhesion, and reduced hypocotyl growth in etiolated seedlings. DIS3 efficiently activates the actin filament nucleation and branching activity of vertebrate Arp2/3 and functions within a WAVE-ARP2/3 pathway in vivo. DIS3 may assemble into a WAVE complex via a physical interaction with a highly diverged Arabidopsis Abi-1-like bridging protein. These results demonstrate the utility of the Arabidopsis trichome system to understand how the WAVE and ARP2/3 complexes translate signaling inputs into a coordinated morphogenetic response.

Actin-Related Protein 2↗

PubMedDate not supplied

From DNA sequence to application: possibilities and complications.

The development of sophisticated genetic tools during the past 15 years have facilitated a tremendous increase of fundamental and application-oriented knowledge of lactic acid bacteria (LAB) and their bacteriophages. This knowledge relates both to the assignments of open reading frames (ORF's) and the function of non-coding DNA sequences. Comparison of the complete nucleotide sequences of several LAB bacteriophages has revealed that their chromosomes have a fixed, modular structure, each module having a set of genes involved in a specific phase of the bacteriophage life cycle. LAB bacteriophage genes and DNA sequences have been used for the construction of temperature-inducible gene expression systems, gene-integration systems, and bacteriophage defence systems. The function of several LAB open reading frames and transcriptional units have been identified and characterized in detail. Many of these could find practical applications, such as induced lysis of LAB to enhance cheese ripening and re-routing of carbon fluxes for the production of a specific amino acid enantiomer. More knowledge has also become available concerning the function and structure of non-coding DNA positioned at or in the vicinity of promoters. In several cases the mRNA produced from this DNA contains a transcriptional terminator-antiterminator pair, in which the antiterminator can be stabilized either by uncharged tRNA or by interaction with a regulatory protein, thus preventing formation of the terminator so that mRNA elongation can proceed. Evidence has accumulated showing that also in LAB carbon catabolite repression in LAB is mediated by specific DNA elements in the vicinity of promoters governing the transcription of catabolic operons. Although some biological barriers have yet to be solved, the vast body of scientific information presently available allows the construction of tailor-made genetically modified LAB. Today, it appears that societal constraints rather than biological hurdles impede the use of genetically modified LAB.

Bacteriophages↗

PubMed1997

Management of patients and subjects at risk for multiple endocrine neoplasia type 1: MEN 1. GENEM 1. Groupe d'Etude des Néoplasies Endocriniennes Multiples de type 1.

Multiple endocrine neoplasia type 1 (MEN 1) is characterized by the combined occurrence, to variable degree, of hyperparathyroidism (HPT) (85.7% of cases according to the French Registry of GENEM 1), tumors of the endocrine pancreas (49.6%), pituitary adenomas (38.4%) and, less frequently, adrenal tumors (9.6%) and neuroendocrine tumors (5.8%). Currently, diagnosis of MEN 1 is done in the fourth decade of life, but familial screening (using genetic tools whose diagnostic accuracy approaches 100%) has lowered the age of diagnosis. Screening for MEN 1 in a patient harboring an apparently sporadic tumor will depend on the endocrine gland involved. Extensive screening for MEN 1 in the presence of HPT will be conducted only when the familial history is suggestive, when parathyroid glands are hyperplastic or when multiple parathyroid adenomas have been found at surgery. All patients with an endocrine pancreas tumor need to be investigated for the presence of other endocrine lesions of MEN 1. Extensive screening for MEN 1 is only recommended when a patient with a pituitary tumor or an adrenal tumor has a familial history suggestive of MEN 1. Otherwise regular measurement of blood calcium and PTH levels seem sufficient. Extensive screening for endocrine lesions when MEN 1 is suspected involves hormone measurements and imaging procedures. For the diagnosis of HPT, calcemia and PTH 1-84 must be measured. In the absence of clinical symptoms, basal measurement of serum gastrin, glucose, insulin, glucagon, VIP, somatostatin and pancreatic polypeptide levels are combined with abdominal ultrasonography. When symptoms suggest the Zollinger-Ellison syndrome, the secretin stimulation test is recommended. The diagnosis of a pituitary tumor is made by pituitary imaging and selected hormone assays (mainly PRL). To detect an adrenal tumor, CT scan is recommended, combined with serum potassium, urinary free cortisol and androgen measurement. When the diagnosis of MEN 1 is made, clinical and hormonal follow-up (once a year) and imaging surveillance (every 3-5 years) may be sufficient to detect new other endocrinopathies (unless suggestive clinical symptoms arise). Surgical management of each endocrine lesion must be done by skilled surgeons according to therapeutic protocols which have been discussed in detail. Genetic screening is an integral part of familial screening which may be conducted in collateral and in the offspring of MEN 1 patients. Obviously ethical principles (informed consent, etc.) must be respected. As it is now possible to detect presymptomatic gene carriers with a high degree of accuracy, follow-up is needed to make appropriate management decisions. The marked anxiety provoked by screening in an overtly asymptomatic healthy subject must not be underestimated. Conversely, a negative genetic diagnosis helps to reassure the subject and avoid repetitive and costly follow-up.

France↗