scholarly journals Efficient Targeted Mutagenesis inBorrelia burgdorferi

2000 ◽  
Vol 182 (9) ◽  
pp. 2445-2452 ◽  
Author(s):  
James L. Bono ◽  
Abdallah F. Elias ◽  
John J. Kupko ◽  
Brian Stevenson ◽  
Kit Tilly ◽  
...  

ABSTRACT Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is gyrBr , a mutated form of the chromosomal gyrB gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumermycin A1. The utility of the coumermycin-resistantgyrBr gene for targeted gene disruption is limited by a high frequency of recombination with the endogenousgyrB gene. A kanamycin resistance gene (kan) was introduced into B. burgdorferi, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen. B. burgdorferi transformants with the kan gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with thekan gene expressed from either the B. burgdorferi flaB or flgB promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in B. burgdorferi and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.

Metabolites ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 49 ◽  
Author(s):  
Ana Molina-Márquez ◽  
Marta Vila ◽  
Javier Vigara ◽  
Ana Borrero ◽  
Rosa León

Genetic manipulation shows great promise to further boost the productivity of microalgae-based compounds. However, selection of microalgal transformants depends mainly on the use of antibiotics, which have raised concerns about their potential impacts on human health and the environment. We propose the use of a synthetic phytoene desaturase-encoding gene (CRTIop) as a selectable marker and the bleaching herbicide norflurazon as a selective agent for the genetic transformation of microalgae. Bacterial phytoene desaturase (CRTI), which, unlike plant and algae phytoene desaturase (PDS), is not sensitive to norflurazon, catalyzes the conversion of the colorless carotenoid phytoene into lycopene. Although the expression of CRTI has been described to increase the carotenoid content in plant cells, its use as a selectable marker has never been testedin algae or in plants. In this study, a version of the CRTI gene adapted to the codon usage of Chlamydomonas has been synthesized, and its suitability to be used as selectable marker has been shown. The microalgae were transformed by the glass bead agitation method and selected in the presence of norflurazon. Average transformation efficiencies of 550 colonies µg−1 DNA were obtained. All the transformants tested had incorporated the CRTIop gene in their genomes and were able to synthesize colored carotenoids.


1980 ◽  
Vol 180 (2) ◽  
pp. 487-488 ◽  
Author(s):  
Alexander M. Slutsky ◽  
Pyotre M. Rabinovich ◽  
Leonid Z. Yakubov ◽  
Irene V. Sineokaya ◽  
Anatoly I. Stepanov ◽  
...  

2003 ◽  
Vol 69 (5) ◽  
pp. 3020-3023 ◽  
Author(s):  
Guojie Li ◽  
S. Kathariou

ABSTRACT Listeria monocytogenes is a gram-positive, facultative intracellular bacterium implicated in severe food-borne illness (listeriosis) in humans. The construction of well-defined gene replacements in the genome of L. monocytogenes has been instrumental to several genetic studies of the virulence and other attributes of the organism. Construction of such mutations by currently available procedures, however, tends to be labor intensive, and gene replacement mutants are sometimes difficult to recover due to lack of direct selection for the construct. In this study we describe the construction and use of plasmid vector pGF-EM, which can be conjugatively transferred from Escherichia coli S17-1 to L. monocytogenes and which provides the genetic means for direct selection of gene replacements.


Genetics ◽  
1993 ◽  
Vol 134 (3) ◽  
pp. 717-728 ◽  
Author(s):  
W A Fonzi ◽  
M Y Irwin

Abstract Genetic manipulation of Candida albicans is constrained by its diploid genome and asexual life cycle. Recessive mutations are not expressed when heterozygous and undesired mutations introduced in the course of random mutagenesis cannot be removed by genetic back-crossing. To circumvent these problems, we developed a genotypic screen that permitted identification of a heterozygous recessive mutation at the URA3 locus. The mutation was introduced by targeted mutagenesis, homologous integration of transforming DNA, to avoid introduction of extraneous mutations. The ura3 mutation was rendered homozygous by a second round of transformation resulting in a Ura- strain otherwise isogenic with the parental clinical isolate. Subsequent mutation of the Ura- strain was achieved by targeted mutagenesis using the URA3 gene as a selectable marker. URA3 selection was used repeatedly for the sequential introduction of mutations by flanking the URA3 gene with direct repeats of the Salmonella typhimurium hisG gene. Spontaneous intrachromosomal recombination between the flanking repeats excised the URA3 gene restoring a Ura- phenotype. These Ura- segregants were selected on 5-fluoroorotic acid-containing medium and used in the next round of mutagenesis. To permit the physical mapping of disrupted genes, the 18-bp recognition sequence of the endonuclease I-SceI was incorporated into the hisG repeats. Site-specific cleavage of the chromosome with I-SceI revealed the position of the integrated sequences.


Confectionery sunflower - a special area of use of sunflower, which requires the creation of marketable seeds quality features. One of the possible ways to create large-fruited sunflower is to create production hybrids and lines. Objective: to evaluate the created new large-fruited sunflower lines by a complex of morphological characters and determine the best lines for use as large-seeds hybrids as parent components or source material. In 2016-2019 years on the basis of the Institute of Oilseed Crops NAAS a study was conducted to assess the economic characteristics of large-fruited sunflower lines. We studied a collection of 27 lines of large-seeds sources. The lines were created by direct selection or crossing and sampling: Reyny of Argentinean origin, Zaporizhzhya confectionery variety, confectionery hybrid with striped pericarp color of Israeli origin, white seed of Turkish origin, synthetic population - donor of complex resistance. To study from the collection, lines were drawn that went through at least 7 generations with selection for seed size. Experience has shown that the shortest growing season for lines 174d and KP11 was 99 days, and the longest for lines I2K670 was 109 days. In the studied collection, the greatest mass of 1000 seeds has the KP11-146.47g line, which is the mother component and does not have branching. The second by weight of 1000 seeds (109 g) stood out line 168v, which also had branches and pollen fertility restoration genes and will be used as the paternal form. The third largest is also one basket line ZKN51-100. The collection included lines originating from the same combination, but with a different morphotype for the presence and absence of branching. So, based on the combination of KP11 x Zaporizhzhya Confectionery, three lines were obtained. A mass of 1000 seeds was observed in 98-86 g, with the branching line having the largest mass of 1000 seeds. The lines created with one combination VK678 x ZKN32: with a branch 168a had a mass of 1000 seeds 95g, and a line 168b - without a branch 109 g. Of the two lines obtained from the descendants of the combination KP11 x the striped hybrid both had branches, but the seeds were much smaller (weight of 1000 seeds 59 and 79 g). The collection also studied samples created on the basis of varieties and populations 160c, 174, 175b, the mass of 1000 seeds of which turned out to be more acceptable for large-fruited use from 83 to 99 g. Summing up the results of studying the collection of newly created lines, we can highlight the lines 162d, 168v, 175b, KP11 that are potentially promising for use in hybrids. The selections showed that large-fruited lines can be obtained from large-fruited varieties, self-pollination of large-fruited hybrids and crossing lines with hybrids and varieties. Self-pollination and selection of large-fruited lines in several generations does not provide the necessary variability for positive changes in selections. The result of the selection by weight of 1000 seeds in the offspring from crosses and from populations creates opportunities for new large-seeds sunflower.


Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1379-1391
Author(s):  
Monique A Johnson ◽  
Hans R Waterham ◽  
Galyna P Ksheminska ◽  
Liubov R Fayura ◽  
Joan Lin Cereghino ◽  
...  

Abstract We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.


2006 ◽  
Vol 397 (3) ◽  
pp. 509-518 ◽  
Author(s):  
Andreas Fischbach ◽  
Stephan Adelt ◽  
Alexander Müller ◽  
Günter Vogel

myo-Inositol and its downstream metabolites participate in diverse physiological processes. Nevertheless, considering their variety, it is likely that additional roles are yet to be uncovered. Biosynthesis of myo-inositol takes place via an evolutionarily conserved metabolic pathway and is strictly dependent on inositol-3-phosphate synthase (EC 5.5.1.4). Genetic manipulation of this enzyme will disrupt the cellular inositol supply. Two methods, based on gene deletion and antisense strategy, were used to generate mutants of the cellular slime mould Dictyostelium discoideum. These mutants are inositol-auxotrophic and show phenotypic changes under inositol starvation. One remarkable attribute is their inability to live by phagocytosis of bacteria, which is the exclusive nutrient source in their natural environment. Cultivated on fluid medium, the mutants lose their viability when deprived of inositol for longer than 24 h. Here, we report a study of the alterations in the first 24 h in cellular inositol, inositol phosphate and phosphoinositide concentrations, whereby a rapidly accumulating phosphorylated compound was detected. After its identification as 2,3-BPG (2,3-bisphosphoglycerate), evidence could be found that the internal disturbances of inositol homoeostasis trigger the accumulation. In a first attempt to characterize this as a physiologically relevant response, the efficient in vitro inhibition of a D. discoideum inositol-polyphosphate 5-phosphatase (EC 3.1.3.56) by 2,3-BPG is presented.


1994 ◽  
Vol 14 (6) ◽  
pp. 4011-4019
Author(s):  
J A Nelson ◽  
P B Savereide ◽  
P A Lefebvre

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.


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