scholarly journals Hypoxanthine Incorporation Is Nonmutagenic in Escherichia coli

2006 ◽  
Vol 188 (18) ◽  
pp. 6553-6560 ◽  
Author(s):  
Brian Budke ◽  
Andrei Kuzminov
Keyword(s):  
Escherichia Coli ◽  
Nitrous Acid ◽  
Double Mutant ◽  
The Other ◽  
Wild Type ◽  
Purine Base ◽  
Spontaneous Mutagenesis ◽  
Endonuclease V ◽  
Mutant Strains ◽  
Induced Mutagenesis

ABSTRACT Endonuclease V, encoded by the nfi gene, initiates removal of the base analogs hypoxanthine and xanthine from DNA, acting to prevent mutagenesis from purine base deamination within the DNA. On the other hand, the RdgB nucleotide hydrolase in Escherichia coli is proposed to prevent hypoxanthine and xanthine incorporation into DNA by intercepting the noncanonical DNA precursors dITP and dXTP. Because many base analogs are mutagenic when incorporated into DNA, it is intuitive to think of RdgB as acting to prevent similar mutagenesis from deaminated purines in the DNA precursor pools. To test this idea, we used a set of Claire Cupples' strains to detect changes in spontaneous mutagenesis spectra, as well as in nitrous acid-induced mutagenesis spectra, in wild-type cells and in rdgB single, nfi single, and rdgB nfi double mutants. We found neither a significant increase in spontaneous mutagenesis in rdgB and nfi single mutants or the double mutant nor any changes in nitrous acid-induced mutagenesis for rdgB mutant strains. We conclude that incorporation of deaminated purines into DNA is nonmutagenic.

scholarly journals Novel Escherichia coli umuD′ Mutants: Structure-Function Insights into SOS Mutagenesis

1998 ◽  
Vol 180 (17) ◽  
pp. 4658-4666 ◽  
Author(s):  
Mary McLenigan ◽  
Thomas S. Peat ◽  
Ekaterina G. Frank ◽  
John P. McDonald ◽  
Martín Gonzalez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Potential Effect ◽  
Wild Type ◽  
Cleavage Reaction ◽  
Nonsense Mutations ◽  
Spontaneous Mutagenesis ◽  
Sos Mutagenesis ◽  
Low Copy Number ◽  
Induced Mutagenesis

ABSTRACT Although it has been 10 years since the discovery that theEscherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD′, the function of UmuD′ has yet to be determined. In an attempt to elucidate the role of UmuD′ in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD′ plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD′ mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD′, three were nonsense mutations that resulted in a truncated UmuD′ protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomalumuD1, umuD44, and umuD77 alleles into umuD′. All 17 umuD′ mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD′.

scholarly journals An msbB Homologue Carried in Plasmid pO157 Encodes an Acyltransferase Involved in Lipid A Biosynthesis in Escherichia coli O157:H7

2004 ◽  
Vol 72 (2) ◽  
pp. 1174-1180 ◽  
Author(s):  
Sang-Hyun Kim ◽  
Wenyi Jia ◽  
Russell E. Bishop ◽  
Carlton Gyles
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Double Mutant ◽  
Lipid A ◽  
Escherichia Coli O157 ◽  
Wild Type ◽  
Single Mutant ◽  
Double Mutant Strain ◽  
Mutant Strains ◽  
Lipid A Biosynthesis

ABSTRACT Escherichia coli O157:H7 carries a chromosomal msbB1 and a plasmid-encoded msbB2 gene. We characterized msbB2 function as a homologue of msbB1 by examination of wild-type organisms and mutant strains that lacked functional msbB1, msbB2, and both msbB1 and msbB2. The msbB double-mutant strain generated pentaacyl lipid A, while the single-mutant strains synthesized hexaacyl lipid A. Complementation with overexpressed msbB2 converted pentaacyl into hexaacyl lipid A in the double-mutant strain. The transcription of both msbB genes occurred simultaneously. Lack of MsbB2 activity slightly increased the microheterogeneity of the lipid A species. These results suggest that the msbB2 gene plays a role not only in the routine generation of fully hexaacylated lipid A but also in suppressing the microheterogeneity of lipid A species, the endotoxic determinant of the organism.

scholarly journals act Operon Control of Developmental Gene Expression in Myxococcus xanthus

2002 ◽  
Vol 184 (4) ◽  
pp. 1172-1179 ◽  
Author(s):  
Thomas M. A. Gronewold ◽  
Dale Kaiser
Keyword(s):  
Fruiting Body ◽  
Myxococcus Xanthus ◽  
The Other ◽  
Loss Of Function ◽  
Wild Type ◽  
Developmental Gene ◽  
Developmental Gene Expression ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Signal Production

ABSTRACT Cell-bound C-signal guides the building of a fruiting body and triggers the differentiation of myxospores. Earlier work has shown that transcription of the csgA gene, which encodes the C-signal, is directed by four genes of the act operon. To see how expression of the genes encoding components of the aggregation and sporulation processes depends on C-signaling, mutants with loss-of-function mutations in each of the act genes were investigated. These mutations were found to have no effect on genes that are normally expressed up to 3 h into development and are C-signal independent. Neither the time of first expression nor the rate of expression increase was changed in actA, actB, actC, or actD mutant strains. Also, there was no effect on A-signal production, which normally starts before 3 h. By contrast, the null act mutants have striking defects in C-signal production. These mutations changed the expression of four gene reporters that are related to aggregation and sporulation and are expressed at 6 h or later in development. The actA and actB null mutations substantially decreased the expression of all these reporters. The other act null mutations caused either premature expression to wild-type levels (actC) or delayed expression (actD), which ultimately rose to wild-type levels. The pattern of effects on these reporters shows how the C-signal differentially regulates the steps that together build a fruiting body and differentiate spores within it.

scholarly journals IC138 Defines a Subdomain at the Base of the I1 Dynein That Regulates Microtubule Sliding and Flagellar Motility

2009 ◽  
Vol 20 (13) ◽  
pp. 3055-3063 ◽  
Author(s):  
Raqual Bower ◽  
Kristyn VanderWaal ◽  
Eileen O'Toole ◽  
Laura Fox ◽  
Catherine Perrone ◽  
...  
Keyword(s):  
Double Mutant ◽  
Essential Role ◽  
Null Allele ◽  
Flagellar Motility ◽  
Wild Type ◽  
Gene Encoding ◽  
Radial Spoke ◽  
Mutant Strains ◽  
Dynein Arms ◽  
Image Averaging

To understand the mechanisms that regulate the assembly and activity of flagellar dyneins, we focused on the I1 inner arm dynein (dynein f) and a null allele, bop5-2, defective in the gene encoding the IC138 phosphoprotein subunit. I1 dynein assembles in bop5-2 axonemes but lacks at least four subunits: IC138, IC97, LC7b, and flagellar-associated protein (FAP) 120—defining a new I1 subcomplex. Electron microscopy and image averaging revealed a defect at the base of the I1 dynein, in between radial spoke 1 and the outer dynein arms. Microtubule sliding velocities also are reduced. Transformation with wild-type IC138 restores assembly of the IC138 subcomplex and rescues microtubule sliding. These observations suggest that the IC138 subcomplex is required to coordinate I1 motor activity. To further test this hypothesis, we analyzed microtubule sliding in radial spoke and double mutant strains. The results reveal an essential role for the IC138 subcomplex in the regulation of I1 activity by the radial spoke/phosphorylation pathway.

Sensitivity of normal and mutant strains of Escherichia coli to actinomycin-D

1972 ◽  
Vol 18 (6) ◽  
pp. 909-915 ◽  
Author(s):  
A. P. Singh ◽  
K.-J. Cheng ◽  
J. W. Costerton ◽  
E. S. Idziak ◽  
J. M. Ingram
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Wild Type Strain ◽  
Actinomycin D ◽  
Cytoplasmic Membrane ◽  
Cell Envelope ◽  
Coli Strain ◽  
Wild Type ◽  
Mutant Strains ◽  
In The Wild

The site of the cell barrier to actinomycin-D uptake was studied using a wild-type Escherichia coli strain P and its cell envelope-defective filamentous mutants, strains 6γ and 12γ, both of which 'leak' β-galactosidase and alkaline phosphatase into the medium during growth indicating both membrane and cell-wall defects. Actinomycin-D entered the cells of these two mutant strains as evidenced by the inhibition of both 14C-uracil incorporation and synthesis of the induced β-galactosidase system. Under similar conditions, no inhibition occurred in the wild-type strain and its sucrose-lysozyme prepared spheroplasts. Actinomycin-D did, however, inhibit the above-mentioned systems in the wild-type sucrose-lysozyme spheroplasts prepared in the presence of 2 mM EDTA. The experimental data indicate that although the cell wall may act as a primary barrier or sieve to actinomycin-D, the cytoplasmic membrane should be considered the final and determinative barrier to this antibiotic.

Abscisic acid resistant mutants in the fern Ceratopteris: characterization and genetic analysis

1985 ◽  
Vol 63 (9) ◽  
pp. 1582-1585 ◽  
Author(s):  
Leslie G. Hickok
Keyword(s):  
Abscisic Acid ◽  
Genetic Analysis ◽  
Sexual Differentiation ◽  
Double Mutant ◽  
Acid Resistance ◽  
The Other ◽  
Additive Effects ◽  
Wild Type ◽  
Resistant Mutants ◽  
Moderate Resistance

Abscisic acid normally inhibits growth and male sexual differentiation (antheridia formation) in gametophytes of the fern Ceratopteris. Abscisic acid resistant mutants show increased growth and sexual differentiation in comparison with the wild type when cultured in the presence of abscisic acid. Two different mutants that confer resistance to the effects of abscisic acid have been fully characterized. One shows moderate resistance and the other strong resistance. The mutations involve separate but linked loci. Recombination between the loci yields double mutant (cis) recombinants that exhibit additive effects and show exceptional levels of abscisic acid resistance.

scholarly journals An Epistasis Analysis of recA and recN in Escherichia coli K-12

Genetics ◽  
2020 ◽  
Vol 216 (2) ◽  
pp. 381-393
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Double Mutant ◽  
Strand Break ◽  
Wild Type ◽  
Epistasis Analysis ◽  
Chromatid Cohesion ◽  
K 12 ◽  
Structural Maintenance Of Chromosomes ◽  
Sos Protein

RecA is essential for double-strand-break repair (DSBR) and the SOS response in Escherichia coli K-12. RecN is an SOS protein and a member of the Structural Maintenance of Chromosomes family of proteins thought to play a role in sister chromatid cohesion/interactions during DSBR. Previous studies have shown that a plasmid-encoded recA4190 (Q300R) mutant had a phenotype similar to ∆recN (mitomycin C sensitive and UV resistant). It was hypothesized that RecN and RecA physically interact, and that recA4190 specifically eliminated this interaction. To test this model, an epistasis analysis between recA4190 and ∆recN was performed in wild-type and recBC sbcBC cells. To do this, recA4190 was first transferred to the chromosome. As single mutants, recA4190 and ∆recN were Rec+ as measured by transductional recombination, but were 3-fold and 10-fold decreased in their ability to do I-SceI-induced DSBR, respectively. In both cases, the double mutant had an additive phenotype relative to either single mutant. In the recBC sbcBC background, recA4190 and ∆recN cells were very UVS (sensitive), Rec−, had high basal levels of SOS expression and an altered distribution of RecA-GFP structures. In all cases, the double mutant had additive phenotypes. These data suggest that recA4190 (Q300R) and ∆recN remove functions in genetically distinct pathways important for DNA repair, and that RecA Q300 was not important for an interaction between RecN and RecA in vivo. recA4190 (Q300R) revealed modest phenotypes in a wild-type background and dramatic phenotypes in a recBC sbcBC strain, reflecting greater stringency of RecA’s role in that background.

Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster 

2003 ◽  
Vol 99 (4) ◽  
pp. 867-875 ◽  
Author(s):  
Sumiko Gamo ◽  
Junya Tomida ◽  
Katsuyuki Dodo ◽  
Dai Keyakidani ◽  
Hitoshi Matakatsu ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Double Mutant ◽  
Developmental Stages ◽  
Northern Blotting ◽  
General Anesthetics ◽  
Wild Type ◽  
Mutant Strains ◽  
Low Expression

Background Various species, e.g., Caenorhabditis elegans, Drosophila melanogaster, and mice, have been used to explore the mechanisms of action of general anesthetics in vivo. The authors isolated a Drosophila mutant, ethas311, that was hypersensitive to diethylether and characterized the calreticulin (crc) gene as a candidate of altered anesthetic sensitivity. Methods Molecular analysis of crc included cloning and sequencing of the cDNA, Northern blotting, and in situ hybridization to accomplish the function of the gene and its mutation. For anesthetic phenotype assay, the 50% anesthetizing concentrations were determined for ethas311, revertants, and double-mutant strains (wild-type crc transgene plus ethas311). Results Expression of the crc 1.4-kb transcript was lower in the mutant ethas311 than in the wild type at all developmental stages. The highest expression at 19 h after pupation was observed in the brain of the wild type but was still low in the mutant at that stage. The mutant showed resistance to isoflurane as well as hypersensitivity to diethylether, whereas it showed the wild phenotype to halothane. Both mutant phenotypes were restored to the wild type in the revertants and double-mutant strains. Conclusion ethas311 is a mutation of low expression of the Drosophila calreticulin gene. The authors demonstrated that hypersensitivity to diethylether and resistance to isoflurane are associated with low expression of the gene. In Drosophila, calreticulin seems to mediate these anesthetic sensitivities, and it is a possible target for diethylether and isoflurane, although the predicted anesthetic targets based on many studies in vitro and in vivo are the membrane proteins, such as ion channels and receptors.

scholarly journals Production of 3-Nitrosoindole Derivatives by Escherichia coli during Anaerobic Growth

2009 ◽  
Vol 191 (17) ◽  
pp. 5369-5376 ◽  
Author(s):  
Young-Man Kwon ◽  
Bernard Weiss
Keyword(s):  
Escherichia Coli ◽  
Nitrous Acid ◽  
Anaerobic Growth ◽  
Functional Genes ◽  
Initial Reaction ◽  
Endonuclease V ◽  
Tryptophanase Activity ◽  
K 12 ◽  
Metabolic Reduction ◽  
Dna Repair Mutants

ABSTRACT When Escherichia coli K-12 is grown anaerobically in medium containing tryptophan and sodium nitrate, it produces red compounds. The reaction requires functional genes for trytophanase (tnaA), a tryptophan permease (tnaB), and a nitrate reductase (narG), as well as a natural drop in the pH of the culture. Mass spectrometry revealed that the purified chromophores had mass/charge ratios that closely match those for indole red, indoxyl red, and an indole trimer. These compounds are known products of chemical reactions between indole and nitrous acid. They are derived from an initial reaction of 3-nitrosoindole with indole. Apparently, nitrite that is produced from the metabolic reduction of nitrate is converted in the acid medium to nitrous acid, which leads to the nitrosation of the indole that is generated by tryptophanase. An nfi (endonuclease V) mutant and a recA mutant were selectively killed during the period of chromophore production, and a uvrA strain displayed reduced growth. These effects depended on the addition of nitrate to the medium and on tryptophanase activity in the cells. Unexpectedly, the killing of a tnaA + nfi mutant was not accompanied by marked increases in mutation frequencies for several traits tested. The vulnerability of three DNA repair mutants indicates that a nitrosoindole or a derivative of a nitrosoindole produces lethal DNA damage.

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