Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Allele replacement”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8Linked to original sources
PubMed1993

TnMax--a versatile mini-transposon for the analysis of cloned genes and shuttle mutagenesis.

A series of Tn1721-based mini-transposons (TnMax) has been developed which are suitable for the targeting of cloned genes, shuttle mutagenesis, identification of protein export signals and the rescue of chromosomal markers. TnMax mini-Tn consist of two 38-bp inverted repeats (IR) flanking a resolution site (res), a suicide replication origin (orifd), and a gene conferring resistance to either chloramphenicol (TnMax1) or erythromycin (TnMax2). Other versions of TnMax (TnMax3 and TnMax4) carry a promoterless alkaline phosphatase-encoding gene (phoA') useful for the identification of protein export signals. The various mini-Tn cartridges are fused on the same plasmid with a common transposition control unit (TCU) comprising the tnpA (transposase) and tnpR (resolvase) genes under control of the inducible Ptrc promoter. This configuration causes a high frequency of transposition in Escherichia coli (approx. 10(-2) events/copy of target plasmid) and a minimum size of the mini-Tn. Like Tn1721, TnMax variants prefer random insertion into plasmids rather than into the E. coli chromosome, thus representing superb tools for the insertion mutagenesis of cloned genes. The TnMax-borne orifd simplifies the identification of targeted plasmids and facilitates shuttle mutagenesis, i.e., suicide delivery of a mutated gene with subsequent allelic replacement of a corresponding resident gene, in a variety of microorganisms. Rescue of such targeted chromosomal genes is easily accomplished by the excision of TnMax plus flanking segments using appropriate restriction endonucleases, ligation, and transformation of an E. coli host permissive for orifd-directed replication.

Base Sequence↗
PubMed1993

Evidence for a fourteen-gene, phnC to phnP locus for phosphonate metabolism in Escherichia coli.

The Escherichia coli phn (psiD) locus consists of a large gene cluster encoding proteins necessary for the use of phosphonates (Pn) as a sole phosphorus source. On the basis of nucleotide (nt) sequence analysis, the phn locus contains a 12.6-kb operon of seventeen genes named, in alphabetical order, phnA to phnQ [Chen et al., J. Biol. Chem. 265 (1990) 4461-4471]. New Pn+ plasmids were made which are suitable for mutational analysis of this gene cluster. These plasmids contain the R6K origin for DNA replication, can be conjugatively transferred, contain the tetAR genes, and therefore provide a way for allele replacement. The construction of these plasmids showed that phnA and phnB have no role in Pn metabolism. Also, these plasmids were employed to introduce nonpolar phnD::lacZ and phnD::uidA fusions into the chromosome, which allowed us to show that phnD probably has a role in transport. In addition, it was shown that phnP is the most distal gene required for Pn use. This was done by testing the effect of phn::uidA insertions in or near the 3' end of phnP on Pn use. Altogether, these results show that all genes required for Pn use are in the 10.9-kb, fourteen-gene, phnCDEFGHIJKLMNOP locus.

Amino Acid Sequence↗
PubMed2006

DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps.

The innate immune response plays a crucial role in satisfactory host resolution of bacterial infection. In response to chemotactic signals, neutrophils are early responding cells that migrate in large numbers to sites of infection. The recent discovery of secreted neutrophil extracellular traps (NETs) composed of DNA and histones opened a novel dimension in our understanding of the microbial killing capacity of these specialized leukocytes. M1 serotype strains of the pathogen Group A Streptococcus (GAS) are associated with invasive infections including necrotizing fasciitis (NF) and express a potent DNase (Sda1). Here we apply a molecular genetic approach of allelic replacement mutagenesis, single gene complementation, and heterologous expression to demonstrate that DNase Sda1 is both necessary and sufficient to promote GAS neutrophil resistance and virulence in a murine model of NF. Live fluorescent microscopic cell imaging and histopathological analysis are used to establish for the first time a direct linkage between NET degradation and bacterial pathogenicity. Inhibition of GAS DNase activity with G-actin enhanced neutrophil clearance of the pathogen in vitro and reduced virulence in vivo. The results demonstrate a significant role for NETs in neutrophil-mediated innate immunity, and at the same time identify a novel therapeutic target against invasive GAS infection.

Actins↗
PubMed2005

Enterococcal surface protein Esp does not facilitate intestinal colonization or translocation of Enterococcus faecalis in clindamycin-treated mice.

Enterococcal surface protein (Esp) is a cell wall-associated protein of Enterococcus faecalis that has been identified as a potential virulence factor. We used a mouse model to examine whether Esp facilitates intestinal colonization or translocation of E. faecalis to mesenteric lymph nodes. After clindamycin treatment, similar levels of high-density colonization were established after orogastric inoculation of an E. faecalis isolate containing the esp gene within a large pathogenicity island and an isogenic mutant created by allelic replacement of the esp gene with a chloramphenicol resistance cassette (P=0.7); translocation to mesenteric lymph nodes was detected in 3 of 12 (25%) mice in both groups. Isogenic mutants of FA2-2 (a plasmid-free derivative of E. faecalis strain JH2) with or without the esp gene failed to establish colonization of clindamycin-treated mice. These results suggest that Esp does not facilitate intestinal colonization or translocation of E. faecalis.

Animals↗
PubMed2005

A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum.

Toward more efficient L-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background. Following the creation of a new L-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway. Comparative genomic analysis for the pathway between a classically derived L-lysine producer and its parental wild-type identified several mutations. Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for L-lysine production. Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased L-lysine production. Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, D-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme. Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during L-lysine production. These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for L-lysine biosynthesis, thereby leading to improved product formation.

Corynebacterium↗
PubMed2005

Efficient gene inactivation in Bacillus anthracis.

A procedure for high-efficiency gene inactivation in Bacillus anthracis has been developed. It is based on a highly temperature-sensitive plasmid vector carrying kanamycin resistance cassette surrounded by DNA fragments flanking the desired insertion site. The approach was tested by constructing glutamate racemase E1 (racE1), glutamate racemase E2 (racE2) and comEC knock-out mutants of B. anthracis strain DeltaANR. Allelic replacements were observed at high frequencies, ranging from approximately 0.5% for racE2 up to 50% for racE1 and comEC. The system can be used for genetic validation of potential drug targets.

Amino Acid Isomerases↗
PubMed2004

Fitness of Toxoplasma gondii is not related to DHFR single-nucleotide polymorphism during congenital toxoplasmosis.

Factors that regulate the pathogenesis of Toxoplasma gondii in humans are poorly understood. When acquired during pregnancy, toxoplasmosis can be disastrous, leading to fetal loss or conversely to subclinical disease. In congenitally infected infants, evolution is highly unpredictable. Genotype based virulence patterns have been described in mice, but in humans this classification does not correlate with the gravity of the disease. Mutations on DHFR-TS loci have recently been reported to confer T. gondii fitness cost. In this study, we investigated the relationship between the virulence of the parasite, as measured by clinical outcome in the fetus or newborn, fitness, as measured by parasitic load in amniotic fluid, and allelic polymorphism in DHFR. Six cases of severe congenital toxoplasmosis and 23 cases of mild congenital infections were included in the study. Quantitative PCR was performed to evaluate total T. gondii DNA load in amniotic fluid and detection of mutations was carried out with a LightCycler using hybridisation probes. Parasitic load was significantly higher in severe infections than in mild diseases. Among isolates from severe or non-severe cases of congenital toxoplasmosis, no polymorphism could be detected at loci 36, 83 or 245 of the DHFR gene. The virulent RH strain presented the same melting temperature as the non-virulent PRU strain for codons 36, 83 and 245. Only mutated clones, M2M3 and M2M4 with allelic replacement at these positions, displayed different profiles allowing a clear distinction between wild and mutant types. We concluded that the DHFR gene mutations we investigated do not regulate T. gondii fitness in humans.

Animals↗
PubMed2005

Mutation in mce operons attenuates Mycobacterium tuberculosis virulence.

On the Mycobacterium tuberculosis genome there are four mce operons, all of which are similar in sequence and organization, and code for putatively exported proteins. To investigate whether Mce proteins are essential for virulence, we generated knock-out mutants in mce1, mce2 and mce3 operons of M. tuberculosis and evaluated their ability to multiply in a mammalian host. The allelic replacement was confirmed in each mutant strain by Southern blotting. RT-PCR experiments demonstrated the lack of in vitro expression of mutated genes in Deltamce1 and Deltamce2 mutants. On the other hand, no expression of mce3 was detected in either the wild-type or mutant strains. Similar doubling time and growth characteristics in in vitro culture were observed for mutants and parental strains. The intratracheal route was used to infect BALB/c mice with the Deltamce3, Deltamce2 and Deltamce1 mutants. Ten weeks after infection, all mice infected with the Deltamce mutants survived, while those infected with the wild-type strain died. This long survival correlated with very low counts of colony-forming units (CFU) in the lungs. Deltamce1-infected mice developed very few and small granulomas, while animals infected with Deltamce3 or Deltamce2 mutants showed delayed granuloma formation. Mice infected with Deltamce1 did not develop pneumonia, while animals infected with Deltamce3 and Deltamce2 mutants showed small pneumonic patches. In spleens, bacterial counts of mutant strains were less reduced than in lungs, compared with those of wild-type. In contrast, no such attenuation was observed when the intraperitoneal route was used for infection. Moreover, Deltamce1 mutants appear to be more virulent in lungs after intraperitoneal inoculation. In conclusion, mce operons seem to affect the virulence of M. tuberculosis in mice, depending on the route of infection. Hypotheses are discussed to explain this last issue. Thus, mutants in these genes seem to be good candidates for vaccine testing.

Animals↗
PubMed2005

Conserved virulence of C to B capsule switched Neisseria meningitidis clinical isolates belonging to ET-37/ST-11 clonal complex.

Capsule switching in Neisseria meningitidis is thought to occur by horizontal DNA exchange between meningococcal strains. Antigenic variants may be generated by allelic replacement of the siaD gene; the variants may then be selected by specific immunity against the capsular antigen. There were several vaccination campaigns against serogroup C in France in 2002, following an increase in the prevalence of invasive isolates of serogroup C of the phenotype C:2a:P1.5 and C:2a:P1.5,2 belonging to the ET-37/ST-11 clonal complex. We evaluated the emergence of capsule variants by the detection of B:2a:P1.5 and B:2a:P1.5,2 meningococcal isolates of the ET-37/ST-11 clonal complex. These isolates were significantly more frequent after the year 2002. Pulsed field gel electrophoresis profiles of the serogroup B (ET-37/ST-11) isolates differed from that of serogroup C (ET-37/ST-11) isolates by the bands that harbor the siaD genes responsible for the serogroup specificity. However, serogroup B and C, ET37/ST-11 isolates both express similar virulence as assessed from colonization and invasiveness in a mouse model. Our results indicate that capsule switching events within the same clonal complex can arise frequently with no alteration in virulence. This justifies an enhanced system of surveillance by molecular typing of such isolates, particularly after serogroup-specific vaccination.

Adolescent↗
PubMed2004

Identification and characterisation of hyaluronate lyase from Streptococcus suis.

Hyaluronate lyase, which catalyses the degradation of hyaluronic acid (HA), has been described from several pathogenic streptococcal species. We describe, for the first time, identification and purification of hyaluronate lyase from the zoonotic pig pathogen Streptococcus suis. We have cloned the hyaluronate lyase gene from S. suis and used it to generate an allelic replacement knock-out mutant of S. suis serotype 7 that can no longer biosynthesise the enzyme. Interestingly, a limited strain survey indicates that hyaluronate lyase activity is not present in all disease isolates of S. suis. Polyclonal anti-hyaluronate lyase anti-serum raised against our recombinant hyaluronate lyase has been used in Western blots, showing that hyaluronate lyase activity is always associated with the presence of protein of the expected size, whereas lack of hyaluronate lyase activity is due to truncation or absence of the enzyme. We show that hyaluronate lyase activity is required for S. suis to use HA polymer as a carbon source and that supplying exogenous recombinant hyaluronate lyase to all S. suis strains tested allowed fermentation of the resultant HA breakdown products.

Blotting, Western↗
PubMed2006

An operon in Streptococcus pneumoniae containing a putative alkylhydroperoxidase D homologue contributes to virulence and the response to oxidative stress.

Analysis of the pneumococcal genome sequences from strains R6 and TIGR4 identified a putative alkylhydroperoxidase homologue RT-PCR showed this gene to be expressed in an operon with the downstream open reading frame. No probable function for this second gene is suggested although it appears to be an integral membrane protein. An allelic replacement mutant lacking this two-gene operon in strain D39 was attenuated in competitive infections with the wild type parent. This operon is, therefore, a novel pneumococcal virulence determinant. In line with a role in the response to oxidative stress, this mutant showed enhanced resistance to killing by hydrogen peroxide, a phenotype shared by alkylhydroperoxidase mutants in other bacterial species. The analysis of non-polar single mutants shows that both genes contribute to these phenotypes. Finally, an important role in pneumococcal biology is suggested by the presence of this operon in all 20 clinical isolates examined and the highly conserved sequence of the two genes.

Amino Acid Sequence↗
PubMed2006

Single, double and triple mutants of Salmonella enterica serovar Typhimurium degP (htrA), degQ (hhoA) and degS (hhoB) have diverse phenotypes on exposure to elevated temperature and their growth in vivo is attenuated to different extents.

DegP (HtrA) is a well-studied protease involved in survival of bacteria under stress conditions in vitro and in vivo. There are two paralogues of DegP in the Salmonella enterica serovar Typhimurium genome, DegQ and DegS. In order to understand more about the biological significance of this gene family, a series of deg-deletion mutants was generated in S. Typhimurium strain SL3261 by allelic replacement. At elevated temperature in vitro, the viability of degP and degS mutants was reduced when compared with the parent strain whereas the viability of a degQ mutant was not significantly affected. The viability of a double degP-degS mutant at elevated temperature was severely decreased when compared with the respective single mutants or, interestingly, with a triple degP-degQ-degS mutant. All the deg deletions were transduced into the mouse-virulent strain SL1344 and the resultant mutants were injected intravenously into BALB/c mice to test virulence. degP and degS single mutants and all combinations of double and triple mutants were attenuated to different degrees, whereas the single degQ mutant was as virulent as the wild-type strain. Thus, within this gene family, degP and degS appear important for survival at elevated temperature and are necessary for full virulence, whereas a single degQ deletion appears to have no clear role in survival and growth at elevated temperature or in mice.

Animals↗
PubMed2026

Long-term saline-alkaline selection rewires the growth-survival trade-off in Priestia megaterium.

Saline-alkaline soils impose persistent osmotic, ionic, pH, and nutrient stress on soil microorganisms, but the evolutionary routes by which beneficial bacteria adapt to such conditions remain poorly resolved. We performed adaptive laboratory evolution to examine the adaption of the plant growth-promoting rhizobacterium Priestia megaterium HA22 to long-term oligotrophic saline-alkaline selection. After 175 serial transfers, the evolved lineage proliferated stably at 40 g L-1 Na2SO4 at pH 10.0, whereas the wild-type strain failed to proliferate. Genome resequencing and allelic replacement revealed a 5-bp insertion in spo0A, the master sporulation regulator, as a major adaptive mutation. This mutation abolished sporulation; shortened the lag phase; and enhanced vegetative growth, nutrient uptake, and expression of tricarboxylic acid cycle and nitrogen metabolism gene under saline-alkaline stress. According to untargeted metabolomics, adaptation was accompanied by increased amino acid metabolism and aminoacyl-tRNA biosynthesis, with proline, isoleucine and pantothenic acid functionally promoting growth. A point mutation in ugpB enhanced glycerol-3-phosphate uptake, increased peptidoglycan and wall teichoic acid levels, and partially rescued the survival cost of the spo0A mutation. In greenhouse assays under combined saline-alkaline stress, the evolved strain increased soybean shoot dry weight and root dry weight by 56.08% and 27.02%, respectively. These results indicate that prolonged, predictable saline-alkaline selection can favor active growth rather than dormancy when compensatory cell envelope reinforcement buffers survival costs.

Adaptive laboratory evolution↗
PubMed2002

The methods to generate transgenic animals and to control transgene expression.

Transgenic animals have been used for years to study gene function and to create models for the study of human diseases. This approach has become still more justified after the complete sequencing of several genomes. Transgenic animals are ready to become industrial bioreactors for the preparation of pharmaceuticals in milk and probably in the future in egg white. Improvement of animal production by transgenesis is still in infancy. Despite its intensive use, animal transgenesis is still suffering from technical limitations. The generation of transgenics has recently become easier or possible for different species thanks to the use of transposons or retrovirus, to incubation of sperm which DNA followed by fertilization by intracellular sperm injection or not and to the use of the cloning technique using somatic cells in which genes have been added or inactivated. The Cre-LoxP system is more and more used to withdraw a given sequence from the genome or to target the integration of a foreign DNA. The tetracycline system has been improved and can more and more frequently be used to obtain faithful expression of transgenes. Several tools: RNA forming a triple helix with DNA, antisense RNA including double strand RNA inducing RNA interference and ribozymes, and also expression of proteins having a negative transdominant effect, are tentatively being improved to inhibit specifically the expression of host or viral genes.All these techniques are expected to offer experimenters new and more precise models to study gene function even in large animals. Improvement of breeding by transgenesis has become more plausible including through the precise allele replacement in farm animals.

Animals↗
PubMed1996

Minimal functional system required for expression of erythromycin resistance by msrA in Staphylococcus aureus RN4220.

Previous studies have suggested that inducible erythromycin (Er) resistance in staphylococci mediated by the plasmid-borne ABC-transporter msrA is dependent on additional unidentified chromosomally encoded transmembrane (TM) domains. The requirement for two S. aureus candidate sequences, stpC and smpC, highly similar to sequences adjacent to msrA on the original S. epidermidis plasmid was investigated. Deletion of the sequences by allelic replacement was accomplished by electroporation of S. aureus RN4220 with a nonreplicating suicide vector. S. aureus strains carrying a delta(stpC-smpC) mutation showed an identical ErR phenotype to those arising from single crossover events and unmutated RN4220 containing msrA. This proves that neither stpC nor smpC is required for ErR. To further define the minimal functional unit required for MSR, the control region within the leader sequence of msrA was deleted. This resulted in constitutive resistance to Er and type B streptogramins (Sg), proving that SgR does not require the presence of Er. Deletion constructs containing the N- or C-terminal ABC regions of MsrA did not confer ErR in RN4220 singly or in combination.

Anti-Bacterial Agents↗
PubMed2003

Role of suilysin in pathogenesis of Streptococcus suis capsular serotype 2.

Three suilysin (SLY) knockout mutant strains of Streptococcus suis serotype 2 were generated by allelic replacement from one North American and two European wild type strains. The mutants were characterized by Southern blot, Western blot and phenotyping. In vitro bactericidal testing showed that both wild type and SLY mutants were resistant to bactericidal factors in whole pig blood. To demonstrate the role of SLY during S. suis infection, four animal trials were carried out using young pigs. Either high dose (4 x 10(6)CFU/ml/pig) or low dose (0.5 x 10(6)CFU/ml/pig) live cell aerosol was applied to the pharynx. In one trial, a low challenge dose of North American strain SX332 and its isogenic sly(-) mutant strain (SX932) resulted in acute disease in 3/5 of pigs exposed to the wild type strain, while 5/5 of pigs exposed to the mutant strain survived the trial. In the repeat trial, 1/8 of pigs in wild type group and 6/8 of pigs in mutant group developed disease. The high dose trial with 332/932 pair showed that 4/8 pigs challenged with wild type and 5/8 of pigs challenged with mutant strain developed disease respectively. The third low dose trial, using European strain 31533 and its isogenic sly(-) mutant strain SX911, showed that 1/8 of pigs challenged with the wild type strain and 4/8 of pigs challenged with the corresponding mutant strain developed disease. All the diseased pigs showed fever, clinical signs and developed septicemia. S. suis was isolated from tissue samples such as brain, submandibular lymph node, lung, spleen, liver, heart or joint. Serum antibody titer against cell surface proteins changed little while the antibody titer against SLY increased only in the wild type group after challenge. sly gene was cloned and expressed in E. coli. The recombinant SLY (rSLY) protein showed 800 hemolysin units per microg protein. In vitro study showed that rSLY triggered TNFalpha production by human monocytes and IL-6 production by pig pulmonary alveolar macrophages and monocytes. Thus, the results of this study suggest that SLY does not seem to be a critical virulence factor for S. suis serotype 2 respiratory infection, but by stimulating cytokine release it may play a role in innate immunity.

Aerosols↗
PubMed2003

Characterization of a bovine lactoferrin binding protein of Streptococcus uberis.

The interaction between Streptococcus uberis and bovine lactoferrin (bLf) has been characterized. The binding of 125I-bLf to S. uberis was time-dependent and displaceable by unlabeled bLf. The Scatchard plot was linear and approximately 7,800 binding sites were expressed by each bacterial cell, with an affinity of 1.0 x 10(-7) M. Both heat and protease treatment of bacterial cells reduced bLf-binding significantly, indicating the presence of a cell surface localized protein receptor for the glycoprotein. One protein was identified from the cell wall of S. uberis as the functionally active bLf-binding protein and it existed in both monomeric and dimeric forms. The recombinant protein expressed in E. coli cells was able to bind bLf and had molecular weights similar to that of native S. uberis. Deletion analysis located the bLf-binding domain to a 200 amino acid region at the amino terminus of Lbp. Analysis of the primary and secondary structure suggested that Lbp is an M-like protein. An isogenic mutant of S. uberis lacking the internal sequence of the lbp gene was constructed by allele replacement. Adherence experiments with wild type S. uberis and the lbp mutant revealed that Lbp is not responsible for attachment of S. uberis to host epithelial cells.

Amino Acid Sequence↗
PubMed2005

The synthetic genetic interaction spectrum of essential genes.

The nature of synthetic genetic interactions involving essential genes (those required for viability) has not been previously examined in a broad and unbiased manner. We crossed yeast strains carrying promoter-replacement alleles for more than half of all essential yeast genes to a panel of 30 different mutants with defects in diverse cellular processes. The resulting genetic network is biased toward interactions between functionally related genes, enabling identification of a previously uncharacterized essential gene (PGA1) required for specific functions of the endoplasmic reticulum. But there are also many interactions between genes with dissimilar functions, suggesting that individual essential genes are required for buffering many cellular processes. The most notable feature of the essential synthetic genetic network is that it has an interaction density five times that of nonessential synthetic genetic networks, indicating that most yeast genetic interactions involve at least one essential gene.

Gene Expression Regulation, Fungal↗