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PubMed2006

Apolipoprotein E-specific innate immune response in astrocytes from targeted replacement mice.

BACKGROUND: Inheritance of the three different alleles of the human apolipoprotein (apo) E gene (APOE) are associated with varying risk or clinical outcome from a variety of neurologic diseases. ApoE isoform-specific modulation of several pathogenic processes, in addition to amyloid beta metabolism in Alzheimer's disease, have been proposed: one of these is innate immune response by glia. Previously we have shown that primary microglia cultures from targeted replacement (TR) APOE mice have apoE isoform-dependent innate immune activation and paracrine damage to neurons that is greatest with TR by the epsilon4 allele (TR APOE4) and that derives from p38 mitogen-activated protein kinase (p38MAPK) activity. METHODS: Primary cultures of TR APOE2, TR APOE3 and TR APOE4 astrocytes were stimulated with lipopolysaccharide (LPS). ApoE secretion, cytokine production, and nuclear factor-kappa B (NF-kappaB) subunit activity were measured and compared. RESULTS: Here we showed that activation of primary astrocytes from TR APOE mice with LPS led to TR APOE-dependent differences in cytokine secretion that were greatest in TR APOE2 and that were associated with differences in NF-kappaB subunit activity. CONCLUSION: Our results suggest that LPS activation of innate immune response in TR APOE glia results in opposing outcomes from microglia and astrocytes as a result of TR APOE-dependent activation of p38MAPK or NF-kappaB signaling in these two cell types.

Journal Article↗
PubMed2000

Regions outside of the leucine-rich repeats of flax rust resistance proteins play a role in specificity determination.

Multiple alleles controlling different gene-for-gene flax rust resistance specificities occur at the L locus of flax. At least three distinct regions can be recognized in the predicted protein products: the Toll/interleukin-1 receptor homology (TIR) region, a nucleotide binding site (NBS) region, and a leucine-rich repeat (LRR) region. Replacement of the TIR-encoding region of the L6 allele with the corresponding regions of L2 or LH by recombination changed the specificity of the allele from L6 to L7. Replacement of the TIR and most of the NBS-encoding region of L10 with the equivalent region of L2 or L9 generated recombinant alleles having a novel specificity. However, replacement of the L10 TIR-encoding region with the TIR-encoding region of L2 gave rise to an allele with no detectable specificity. These data indicate that non-LRR regions can determine specificity differences between allelic gene products and that functional specificity involves interactions between coadapted polymorphic regions in the protein products of the alleles. Evidence for the action of diversifying selection on the TIR region is observed.

Adaptation, Physiological↗
PubMed1997

Microsatellite-based cancer detection using capillary array electrophoresis and energy-transfer fluorescent primers.

The development of sensitive, rapid, and accurate methods and apparatus for high-throughput short tandem repeat (STR) analysis will be critical for the use of microsatellite alteration in cancer screening. Here we show that STR-based bladder cancer diagnosis can be performed using capillary array electrophoresis and two-color labeling with energy-transfer (ET) fluorescent primers. Rapid (< or = 35 min) separations are achieved on capillary arrays using replaceable separation matrices and the allelic ratios are quantitatively determined with a precision of +/- 10%. With this precision, a variation of 20% was considered diagnostically significant. These methods provide a significant improvement in the speed, ease, and precision of STR analyses compared to slab gel electrophoresis.

DNA, Neoplasm↗
PubMed1999

The F plasmid centromere, sopC, is required for full repression of the sopAB operon.

The SopB protein of the F plasmid has a dual role in the partition of F plasmid copies to daughter cells prior to division. It binds to the sopC centromere site to form the partition complex needed for stabilizing the plasmid, and it interacts with SopA to repress transcription of the sopAB operon, thus preventing the destabilization that results from excess SopB. We have isolated sop mutants by screening for unstable inheritance of mutagenized mini-F DNA. Four of the mutants resulted from different missense mutations in sopB. All four were deficient, to varying degrees, in autoregulation of Sop protein synthesis. The mutant proteins showed diminished capacity for reducing the linking number of mini-F and for destabilizing a plasmid carrying sopC, indicating that reduced ability to form a normal complex with sopC might underlie the autoregulation defect. Repression of the transcription of a sop promoter- lacZ fusion by SopA and SopB was strongly enhanced in the presence of sopC, in cis or in trans, and the enhancement was reduced or nullified when wild-type sopB was replaced by the mutant sopB alleles. A single 43 bp unit of sopC was almost as effective as sopC itself in enhancing repression. The results show that sopC is necessary for full repression of the sop promoter. They thus indicate a previously unsuspected role for this centromere site, and suggest that autoregulation and partition might normally be coordinated processes.

Bacterial Proteins↗
PubMed2001

Transformation: a tool for studying fungal pathogens of plants.

Plant diseases caused by plant pathogenic fungi continuously threaten the sustainability of global crop production. An effective way to study the disease-causing mechanisms of these organisms is to disrupt their genes, in both a targeted and random manner, so as to isolate mutants exhibiting altered virulence. Although a number of techniques have been employed for such an analysis, those based on transformation are by far the most commonly used. In filamentous fungi, the introduction of DNA by transformation typically results in either the heterologous (illegitimate) integration or the homologous integration of the transforming DNA into the target genome. Homologous integration permits a targeted gene disruption by replacing the wild-type allele on the genome with a mutant allele on transforming DNA. This process has been widely used to determine the role of newly isolated fungal genes in pathogenicity. The heterologous integration of transforming DNA causes a random process of gene disruption (insertional mutagenesis) and has led to the isolation of many fungal mutants defective in pathogenicity. A big advantage of insertional mutagenesis over the more traditional chemical or radiation mutagenesis procedures is that the mutated gene is tagged by transforming DNA and can subsequently be cloned using the transforming DNA. The application of various transformation-based techniques for fungal gene manipulation and how they have increased our understanding and appreciation of some of the most serious plant pathogenic fungi are discussed.

Agrobacterium tumefaciens↗
PubMed1991

Cloning and characterization of the Saccharopolyspora erythraea fdxA gene encoding ferredoxin.

The Saccharopolyspora erythraea gene (fdxA) corresponding to a previously purified ferredoxin [Shafiee and Hutchinson, J. Bacteriol., 170 (1988) 1548-1553] was cloned using an oligodeoxyribonucleotide probe based on the N-terminal sequence of the ferredoxin. The nucleotide sequence of a 1.3-kb segment encompassing fdxA indicates that the corresponding protein, SeFdI, is 105 amino acids long, and very similar to other 7Fe ferredoxins. A partial open reading frame closely linked to fdxA was also detected. Disruption of fdxA was attempted by replacing the wild-type allele with an in vitro mutated copy. The failure to construct an fdxA mutant strain suggests that fdxA lies in an essential region of the S. erythraea chromosome.

Amino Acid Sequence↗
PubMed1991

A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica.

A new suicide vector (pKNG101) that facilitates the positive selection of double recombination events in Gram-bacteria has been developed. It contains a conditional origin of replication (oriR6K), the strAB genes encoding the streptomycin phosphotransferase (SmR), an origin of transfer (mobRK2), the sacB gene mediating sucrose sensitivity, and multiple cloning sites. It was used to mutate the blaA gene of Yersinia enterocolitica, by marker-exchange mutagenesis. To do this, we have first cloned into the suicide vector pKNG101, a 2.5-kb fragment of Y. enterocolitica chromosomal DNA encoding the 20-kDa beta-lactamase A. Gene blaA was then mutated in vitro by insertion of luxAB, which resulted in pKNG105. The disrupted blaA gene was then reintroduced into Y. enterocolitica chromosome by homologous recombinations in two steps. First, E. coli SM10 lambda pir (pKNG105) was mated with strains of Y. enterocolitica. This led to the integration of pKNG105 into the chromosome, by a single homologous recombination event. The transconjugants, selected for SmR, were sensitive to sucrose due to the synthesis of levans (toxic compounds), catalysed by levansucrase, the product of sacB. For the second step, a single colony from the first step was grown in rich medium deprived of antibiotic, allowing the occurrence of a second crossing-over that replaced the wild-type allele blaA with the mutant one, and then excised the plasmid-borne sacB from the chromosome. Such blaA mutants were selected on their ability to grow on TSA medium containing 5% sucrose.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Replication↗
PubMed2005

Membrane tumor necrosis factor confers partial protection to Listeria infection.

Tumor necrosis factor (TNF) plays a critical role in the host response to the intracellular pathogen Listeria monocytogenes (LM). TNF exists in soluble and membrane-bound forms and exhibits both unique and overlapping activities. We examined the role of membrane TNF in the absence of secreted TNF for host resistance in knockin mice in which the endogenous TNF was replaced by a regulated, noncleavable allele (mem-TNF). Macrophages expressing mem-TNF produced nitric oxide and displayed normal bactericidal activity. Although mice completely deficient in TNF (TNF(-/-)) succumbed to LM infection within 4 days, mem-TNF mice controlled LM infection at a low dose (10(4) CFU) but succumbed at a higher dose of infection (10(5) CFU). In contrast to complete TNF deficiency, mem-TNF mice developed confined microabscesses that expressed inducible nitric oxide synthase. The transfer of lymphocytes from immunized mem-TNF, but not TNF(-/-), mice protected TNF(-/-) mice from fatal infection. Taken together the data suggest that in the absence of soluble TNF, the presence of membrane-expressed TNF on phagocytes and lymphocytes partially restores host defense to LM infection.

Animals↗
PubMed2003

Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli.

We have developed a method called "gene gorging" to make precise mutations in the Escherichia coli genome at frequencies high enough (1-15%) to allow direct identification of mutants by PCR or other screen rather than by selection. Gene gorging begins by establishing a donor plasmid carrying the desired mutation in the target cell. This plasmid is linearized by in vivo expression of the meganuclease I-SceI, providing a DNA substrate for lambda Red mediated recombination. This results in efficient replacement of the wild type allele on the chromosome with the modified sequence. We demonstrate gene gorging by introducing amber stop codons into the genes xylA, melA, galK, fucI, citA, ybdO, and lacZ. To compliment this approach we developed an arabinose inducible amber suppressor tRNA. Controlled expression mediated by the suppressor was demonstrated for the lacZ and xylA amber mutants.

Anti-Bacterial Agents↗
PubMed1997

Characterization of the rpoC gene of Streptomyces coelicolor A3(2) and its use to develop a simple and rapid method for the purification of RNA polymerase.

The Streptomyces coelicolor rpoC gene, that encodes the beta' subunit of RNA polymerase, was isolated using the Escherichia coli rpoC gene as a hybridization probe. Comparison of the predicted amino acid sequence of the S. coelicolor beta' subunit to those characterized from other bacteria revealed three distinct subfamilies of beta' subunits, one of which consists of the S. coelicolor subunit and those from Mycobacterium leprae and Mycoplasma genitalium. Using site-directed mutagenesis, the carboxy terminus of the S. coelicolor beta' subunit was modified to contain six histidine residues. The histidine-tagged gene, rpoCHIS, was used to replace the wild-type allele in the chromosome of S. coelicolor and S. lividans. These strains were unaffected in growth and sporulation, demonstrating that the histidine-tagged RNA polymerase was competent to carry out all essential in-vivo functions. During a 1-day procedure, highly purified RNA polymerase was obtained by nickel-NTA agarose affinity chromatography followed by heparin-sepharose chromatography. Using in-vitro run-off transcription assays, the affinity purified RNA polymerase was shown to initiate transcription correctly from the S. lividans galP1 and galP2 promoters, and the Bacillus subtilus veg and ctc promoters. An extension of this procedure yielded highly-purified core RNA polymerase. To facilitate introduction of the rpoCHIS allele into other genetic backgrounds, a mutation in the adjacent gene, rpoB (rifA), conferring rifampin-resistance, was isolated in S. coelicolor to provide a genetic marker to follow transfer of the rpoCHIS allele. The use of this affinity chromatography procedure, in combination with the ability to introduce the rpoCHIS allele into different Streptomyces strains by transformation, will greatly facilitate the in-vitro analysis of transcription in members of this genus.

Amino Acid Sequence↗
PubMed2004

Renal apolipoprotein A-I amyloidosis associated with a novel mutant Leu64Pro.

Apolipoprotein A-I amyloidosis (Apo A-I) is an inherited systemic disease that results from the pathologic deposition in tissues throughout the body of fibrils composed of Apo A-I-related molecules. This disorder has been linked to mutations occurring within the coding region of the Apo A-I gene and, to date, 11 such substitutions have been documented. In 4 of these cases, the kidney was the target organ of the disease process. The authors report their studies on a patient with renal amyloidosis and a heretofore undescribed alteration in the amyloidogenic precursor protein. Analyses of genomic DNA evidenced a transition in the second base of codon 64 (T-->C) in one Apo A-I allele that resulted in the replacement of leucine by proline at position 64 (Leu64Pro). Additionally, fibrils extracted from the kidney and characterized chemically were found to be composed almost exclusively of an approximately 96-residue N-terminal Apo A-I fragment that contained the Leu64Pro substitution. These studies have provided further evidence for Apo A-I amyloidogenicity and the propensity of certain mutants to deposit in renal parenchyma.

Amino Acid Substitution↗
PubMedDate not supplied

Development of mice with osteoblast-specific connexin43 gene deletion.

Genetic deficiency of Cx43 in vivo causes skeletal developmental defects, osteoblast dysfunction and perinatal lethality. To determine the role of Cx43 in the adult skeleton, we developed two models of osteoblast-specific Cx43 gene deletion using Cre mediated replacement of a "floxed" Cx43 allele with a LacZ reporter gene. Cre recombinase expression in osteoblasts was driven by either the osteocalcin OG2 promoter or the 2.3 kb fragment of the Colalpha1(I) promoter. Homozygous Cx43(fl/fl) mice, in which the Cx43 coding region is flanked by two loxP sites, were crossed with Cre expressing mice in a heterozygous Cx43-null background [Cx43(+/-); Colalpha1(I)-Cre or Cx43(+/-); OG2-Cre]. Cx43 gene ablation was demonstrated in tissues by selective X-gal staining of cells lining the endosteal surface, and in cultured osteoblastic cells from calvaria using different approaches. Although no LacZ expression was observed in proliferating calvaria cells, before osteoblast differentiation begins, post-proliferative cells isolated from conditional knockout mice [Cx43(fl/-); Colalpha1(I)-Cre or Cx43(fl/-); OG2-Cre] developed strong LacZ expression as they differentiated, in parallel to a progressive disappearance of Cx43 mRNA and protein abundance relative to controls. Selective Cre mediated Cx43 gene inactivation in bone forming cells will be useful to determine the role of Cx43 in adult skeletal homeostasis and overcome the perinatal lethality of the conventional null model.

Animals↗
PubMed1995

The PapG-adhesin at the tip of P-fimbriae provides Escherichia coli with a competitive edge in experimental bladder infections of cynomolgus monkeys.

Human urinary tract infection is an infectious disease that depends on a series of host-microbial interactions. The bacteria first colonize the colon and then the periurethral/vaginal areas; they ascend to and infect first the bladder and then the kidneys. Expression of Escherichia coli P-fimbriae constitutes the strongest correlation to renal pathogenicity, but is also related to first-time cystitis in children. The role of P-fimbriae in the preceding steps in the infectious process is unknown. To examine this, we constructed, from a P-fimbriated E. coli strain with a class II G-adhesin preferentially binding to globoside, one isogenic mutant lacking the G-adhesin and another isogenic mutant in which we replaced the papG class II allele with a class III adhesin preferentially binding to the Forssman antigen. We report here the comparison of the adhesin knockout mutant (DS17-8) and the class-switch mutant (DS17-1) with the wild-type (DS17) for in vivo colonization of the gut, vagina, and bladder of cynomolgus monkeys. It was recently shown that the class II tip G-adhesin is a prerequisite for acute pyelonephritis to occur in the monkey model in the absence of other kidney-specific adhesins or obstruction of the urinary flow. Here we show that it is not required for bladder infection but gives a competitive advantage in mixed infections. In the vagina and colon, the G-adhesin gives no competitive advantage.

Adhesins, Escherichia coli↗
PubMed1997

Cellular and molecular neurosurgery: pathways from concept to reality--part I: target disorders and concept approaches to gene therapy of the central nervous system.

Recent advances in cellular and molecular biology and better understanding of genetic and biochemical bases of different central nervous system (CNS) disorders have made gene therapy of the CNS a realistic goal. Concept approaches for gene therapy of CNS disorders are reviewed and include the following: 1) gene replacement with a single normal allele to correct the inherited global neurodegenerative disorders, such as enzyme deficiencies; 2) brain repair to restore the function of a particular subset of cells that were lost because of a neurodegenerative process; 3) gene therapy of brain tumors; and 4) gene therapy of stroke. Techniques of viral vector-mediated CNS transfer of a therapeutic gene, transplantation of genetically modified cells, fetal embryonic implantation and/or implantation of genetically engineered neural progenitor cells, and production of a specific enzyme, neurotransmitter, and/or growth factor are discussed with respect to the therapeutic potential for global and localized CNS neurodegenerative disorders and stroke. Transfection of the CNS tumor cells with the drug susceptibility ("suicide") gene and/or "toxic" gene and antisense strategies and a concept of adoptive immunotherapy of brain tumors are also discussed. Other approaches, such as transfer of drug-resistant genes and monoclonal antibody gene transfer, are briefly discussed. In addition to summarizing current principles of gene therapy for several groups of CNS disorders, the issues that remain to be resolved in clinical reality, such as delivery of the genetic material and regulation of the cellular expression of the transgene, and the negatives associated with the concepts of gene therapy, such as transient gene expression, toxicity of viral proteins, drawbacks of antisense therapy, and the problem of immune response to the transfected protein, have been also identified.

Animals↗
PubMed1999

Analysis of the nucleus-encoded and chloroplast-targeted rieske protein by classic and site-directed mutagenesis of Chlamydomonas.

Three mutants of the alga Chlamydomonas reinhardtii affected in the nuclear PETC gene encoding the Rieske iron-sulfur protein 2Fe-2S subunit of the chloroplast cytochrome b(6)f complex have been characterized. One has a stable deletion that eliminates the protein; two others carry substitutions Y87D and W163R that result in low accumulation of the protein. Attenuated expression of the stromal protease ClpP increases accumulation and assembly into b(6)f complexes of the Y87D and W163R mutant Rieske proteins in quantities sufficient for analysis. Electron-transfer kinetics of these complexes were 10- to 20-fold slower than those for the wild type. The deletion mutant was used as a recipient for site-directed mutant petC alleles. Six glycine residues were replaced by alanine residues (6G6A) in the flexible hinge that is critical for domain movement; substitutions were created near the 2Fe-2S cluster (S128 and W163); and seven C-terminal residues were deleted (G171och). Although the 6G6A and G171och mutations affect highly conserved segments in the chloroplast Rieske protein, photosynthesis in the mutants was similar to that of the wild type. These results establish the basis for mutational analysis of the nuclear-encoded and chloroplast-targeted Rieske protein of photosynthesis.

Amino Acid Sequence↗
PubMed1996

Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors.

The expression of sacB, the Bacillus subtilis gene encoding levansucrase, is lethal to mycobacteria in the presence of 10% sucrose. In this study, we describe the use of sacB as a marker for positive selection of gene-replacement events into Mycobacterium smegmatis. A sucrose counter-selectable suicide plasmid was used to deliver an inactivated copy of the pyrF gene (pyrF::K(m)) into the M. smegmatis genome. Only uracil auxotroph clones, resulting from replacement of the endogenous pyrF allele, survived in a one-step selection on plates containing kanamycin and 10% sucrose. This demonstrated that selection on sucrose against the maintenance of the vector bearing the sacB gene is 100% efficient, enabling the positive selection of allelic-exchange mutants. Two-step selection is also feasible; it was used to construct unmarked pyrF mutants in which the gene was inactivated by a frameshift mutation. This method of generating unmarked, directed mutations is rapid and simple, making it a powerful tool for the genetic characterization of mycobacteria.

Alleles↗
PubMed2002

Cellular localisation of the clamp protein during DNA replication.

The beta subunit of Escherichia coli DNA polymerase III holoenzyme was fused to the green fluorescent protein GFP. The gene fusion under the control of the heterologous lac promoter was used to replace the wild-type allele in the chromosome. The formation of GFP-beta fluorescent foci in GFP-beta expressing cells required DNA replication and their number per cell was dependent on cell growth. Examination of GFP-beta foci in a synchronous round of replication suggested that DNA replication was accompanied by the recruitment of GFP-beta foci near the midcell, followed by the rapid migration of the foci in opposite directions to the 1/4 and 3/4 positions during DNA replication.

Chromosomes, Bacterial↗
PubMed1998

Characterization of mutations contributing to sulfathiazole resistance in Escherichia coli.

A sulfathiazole-resistant dihydropteroate synthase (DHPS) present in two different laboratory strains of Escherichia coli repeatedly selected for sulfathiazole resistance was mapped to folP by P1 transduction. The folP mutation in each of the strains was shown to be identical by nucleotide sequence analysis. A single C-->T transition resulted in a Pro-->Ser substitution at amino acid position 64. Replacement of the mutant folP alleles with wild-type folP significantly reduced the level of resistance to sulfathiazole but did not abolish it, indicating the presence of an additional mutation(s) that contributes to sulfathiazole resistance in the two strains. Transfer of the mutant folP allele to a wild-type background resulted in a strain with only a low level of resistance to sulfathiazole, suggesting that the presence of the resistant DHPS was not in itself sufficient to account for the overall sulfathiazole resistance in these strains of E. coli. Additional characterization of an amplified secondary resistance determinant, sur, present in one of the strains, identified it as the previously identified bicyclomycin resistance determinant bcr, a member of a family of membrane-bound multidrug resistance antiporters. An additional mutation contributing to sulfathiazole resistance, sux, has also been identified and has been shown to affect the histidine response to adenine sensitivity displayed by these purU strains.

Amino Acid Sequence↗