Developing a Genetic System in Deinococcus radiodurans for Analyzing Mutations

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 661-668
Author(s):  
Mandy Kim ◽  
Erika Wolff ◽  
Tiffany Huang ◽  
Lilit Garibyan ◽  
Ashlee M Earl ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Deinococcus Radiodurans ◽  
Genetic System ◽  
Base Change ◽  
Base Substitution ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
Radiation Induced ◽  
Induced Mutagenesis

Abstract We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the β-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

scholarly journals HOMOLOGOUS RECOMBINATION IN ESCHERICHIA COLI: DEPENDENCE ON SUBSTRATE LENGTH AND HOMOLOGY

Genetics ◽  
1986 ◽  
Vol 112 (3) ◽  
pp. 441-457 ◽  
Author(s):  
Ping Shen ◽  
Henry V Huang
Keyword(s):  
Dna Sequences ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
In Vivo Recombination ◽  
Recombinant Frequency ◽  
Genetic Length ◽  
Efficient Processing ◽  
Low Levels

ABSTRACT We studied the in vivo recombination between homologous DNA sequences cloned in phage lambda and a pBR322-derived plasmid by assaying for the formation of phage-plasmid cointegrates by a single (or an odd number of) reciprocal exchange. (1) Recombination proceeds by the RecBC pathway in wild-type cells and by low levels of a RecF-dependent pathway in recBC  - cells. The RecE pathway appears not to generate phage-plasmid cointegrates. (2) Recombination is linearly dependent on the length of the homologous sequences. In both RecBC and RecF-dependent pathways there is a minimal length, called the minimal efficient processing segment (MEPS), below which recombination becomes inefficient. The length of MEPS is between 23-27 base pairs (bp) and between 44-90 bp for the RecBC- and RecF-dependent pathways, respectively. A model, based on overlapping MEPS, of the correlation of genetic length with physical length is presented. The bases for the different MEPS length of the two pathways are discussed in relationship to the enzymes specific to each pathway. (3) The RecBC and the RecF-dependent pathways are each very sensitive to substrate homology. In wild-type E. coli, reduction of homology from 100% to 90% decreases recombinant frequency over 40-fold. The homology dependence of the RecBC and RecF-dependent pathways are similar. This suggests that a component common to both, probably recA, is responsible for the recognition of homology.

scholarly journals Coexistence of SOS-Dependent and SOS-Independent Regulation of DNA Repair Genes in Radiation-Resistant Deinococcus Bacteria

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 924
Author(s):  
Laurence Blanchard ◽  
Arjan de Groot
Keyword(s):  
Dna Repair ◽  
Deinococcus Radiodurans ◽  
Dna Repair Genes ◽  
Lesion Bypass ◽  
Radiation Resistant ◽  
Radiation Induced ◽  
Induced Mutagenesis ◽  
High Doses ◽  
Translesion Polymerases ◽  
Repair Genes

Deinococcus bacteria are extremely resistant to radiation and able to repair a shattered genome in an essentially error-free manner after exposure to high doses of radiation or prolonged desiccation. An efficient, SOS-independent response mechanism to induce various DNA repair genes such as recA is essential for radiation resistance. This pathway, called radiation/desiccation response, is controlled by metallopeptidase IrrE and repressor DdrO that are highly conserved in Deinococcus. Among various Deinococcus species, Deinococcus radiodurans has been studied most extensively. Its genome encodes classical DNA repair proteins for error-free repair but no error-prone translesion DNA polymerases, which may suggest that absence of mutagenic lesion bypass is crucial for error-free repair of massive DNA damage. However, many other radiation-resistant Deinococcus species do possess translesion polymerases, and radiation-induced mutagenesis has been demonstrated. At least dozens of Deinococcus species contain a mutagenesis cassette, and some even two cassettes, encoding error-prone translesion polymerase DnaE2 and two other proteins, ImuY and ImuB-C, that are probable accessory factors required for DnaE2 activity. Expression of this mutagenesis cassette is under control of the SOS regulators RecA and LexA. In this paper, we review both the RecA/LexA-controlled mutagenesis and the IrrE/DdrO-controlled radiation/desiccation response in Deinococcus.

scholarly journals THE DNA OF CAENORHABDITIS ELEGANS

Genetics ◽  
1974 ◽  
Vol 77 (1) ◽  
pp. 95-104
Author(s):  
J E Sulston ◽  
S Brenner
Keyword(s):  
Caenorhabditis Elegans ◽  
Chemical Analysis ◽  
Dna Sequences ◽  
Base Composition ◽  
Nematode Caenorhabditis Elegans ◽  
5S Rna ◽  
Base Pairs ◽  
Small Component ◽  
E Coli ◽  
The Mean

ABSTRACT Chemical analysis and a study of renaturation kinetics show that the nematode, Caenorhabditis elegans, has a haploid DNA content of 8 x IO7 base pairs (20 times the genome of E. coli). Eighty-three percent of the DNA sequences are unique. The mean base composition is 36% GC; a small component, containing the rRNA cistrons, has a base composition of 51% GC. The haploid genome contains about 300 genes for 4s RNA, 110 for 5s RNA, and 55 for (18 + 28)S RNA.

scholarly journals Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals.

1996 ◽  
Vol 40 (10) ◽  
pp. 2380-2386 ◽  
Author(s):  
M J Everett ◽  
Y F Jin ◽  
V Ricci ◽  
L J Piddock
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Quinolone Resistance ◽  
Fluoroquinolone Resistance ◽  
Polymorphism Analysis ◽  
Single Mutation ◽  
Wild Type ◽  
Single Strand Conformational Polymorphism ◽  
E Coli ◽  
High Level

Twenty-eight human isolates of Escherichia coli from Argentina and Spain and eight veterinary isolates received from the Ministry of Agriculture Fisheries and Foods in the United Kingdom required 2 to > 128 micrograms of ciprofloxacin per ml for inhibition. Fragments of gyrA and parC encompassing the quinolone resistance-determining region were amplified by PCR, and the DNA sequences of the fragments were determined. All isolates contained a mutation in gyrA of a serine at position 83 (Ser83) to an Leu, and 26 isolates also contained a mutation of Asp87 to one of four amino acids: Asn (n = 14), Tyr (n = 6), Gly (n = 5), or His (n = 1). Twenty-four isolates contained a single mutation in parC, either a Ser80 to Ile (n = 17) or Arg (n = 2) or a Glu84 to Lys (n = 3). The role of a mutation in gyrB was investigated by introducing wild-type gyrB (pBP548) into all isolates; for three transformants MICs of ciprofloxacin were reduced; however, sequencing of PCR-derived fragments containing the gyrB quinolone resistance-determining region revealed no changes. The analogous region of parE was analyzed in 34 of 36 isolates by single-strand conformational polymorphism analysis and sequencing; however, no amino acid substitutions were discovered. The outer membrane protein and lipopolysaccharide profiles of all isolates were compared with those of reference strains, and the concentration of ciprofloxacin accumulated (with or without 100 microM carbony cyanide m-chlorophenylhydrazone [CCCP] was determined. Twenty-two isolates accumulated significantly lower concentrations of ciprofloxacin than the wild-type E. coli isolate; nine isolates accumulated less then half the concentration. The addition of CCCP increased the concentration of ciprofloxacin accumulated, and in all but one isolate the percent increase was greater than that in the control strains. The data indicate that high-level fluoroquinolone resistance in E. coli involves the acquisition of mutations at multiple loci.

Organization of the noncontiguous promoter components of adenovirus VAI RNA gene is strikingly similar to that of eucaryotic tRNA genes

1983 ◽  
Vol 3 (11) ◽  
pp. 1996-2005
Author(s):  
R A Bhat ◽  
B Metz ◽  
B Thimmappaya
Keyword(s):  
Dna Sequences ◽  
Transcriptional Control ◽  
Transcriptional Activity ◽  
Evolutionary Relationship ◽  
Trna Genes ◽  
Wild Type ◽  
Base Pairs ◽  
Internal Promoter ◽  
Cell Extracts

The intragenic transcriptional control region (internal promoter) of the adenovirus type 2 VAI RNA gene was mutated by deletion, insertion, and substitution of DNA sequences at the plasmid level. The mutant plasmids were assayed for in vitro transcriptional activity by using HeLa cell extracts. The mutant clones with substitution or insertion of DNA sequences or both between nucleotides +18 and +53 of the VAI RNA gene were all transcriptionally active, although to various extents. Substitution of unrelated DNA sequences up to +26 or between +54 and +61 abolished the transcriptional activity completely. Based on these results, the intragenic promoter sequences of the VAI RNA gene can be subdivided into two components: element A, +10 to +18; and element B, +54 to +69. The distance between the A and B components could be enlarged from its normal 35 base pairs to 75 base pairs without destroying the transcriptional activity. However, a deletion of 4 or 6 base pairs in the DNA segment separating the A and B components (segment C) reduced the transcriptional activity of the genes to less than 2% of that of the wild type. When the VAI RNA gene with its element A or B was substituted for the corresponding element A or B of the Xenopus laevis tRNAMet gene, the hybrid genes transcribed close to the level of the wild-type VAI RNA gene and about 10- to 20-fold more efficiently than the tRNAMet gene. Thus, the organization of DNA sequences in the internal promoter of the VAI RNA gene appears to be very similar to that of eucaryotic tRNA genes. This similarity suggests an evolutionary relationship of the VAI RNA gene to tRNA genes.

scholarly journals Dihydropteroate Synthase of Mycobacterium leprae and Dapsone Resistance

2000 ◽  
Vol 44 (6) ◽  
pp. 1530-1537 ◽  
Author(s):  
Diana L. Williams ◽  
Laynette Spring ◽  
Eugene Harris ◽  
Paul Roche ◽  
Thomas P. Gillis
Keyword(s):  
Dna Sequences ◽  
Inhibitory Concentration ◽  
Mycobacterium Leprae ◽  
Clinical Isolates ◽  
Missense Mutations ◽  
Wild Type ◽  
Knockout Mutant ◽  
Dihydropteroate Synthase ◽  
E Coli ◽  
Bacterial Lysates

ABSTRACT Two Mycobacterium leprae genes, folP1 andfolP2, encoding putative dihydropteroate synthases (DHPS), were studied for enzymatic activity and for the presence of mutations associated with dapsone resistance. Each gene was cloned and expressed in a folP knockout mutant of Escherichia coli(C600ΔfolP::Kmr). Expression ofM. leprae folP1 in C600ΔfolP::Kmr conferred growth on a folate-deficient medium, and bacterial lysates exhibited DHPS activity. This recombinant displayed a 256-fold-greater sensitivity to dapsone (measured by the MIC) than wild-type E. coli C600, and 50-fold less dapsone was required to block (expressed as the 50% inhibitory concentration [IC50]) the DHPS activity of this recombinant. When the folP1 genes of several dapsone-resistant M. leprae clinical isolates were sequenced, two missense mutations were identified. One mutation occurred at codon 53, substituting an isoleucine for a threonine residue (T53I) in the DHPS-1, and a second mutation occurred in codon 55, substituting an arginine for a proline residue (P55R). Transformation of the C600ΔfolP::Kmr knockout with plasmids carrying either the T53I or the P55R mutant allele did not substantially alter the DHPS activity compared to levels produced by recombinants containing wild-type M. leprae folP1. However, both mutations increased dapsone resistance, with P55R having the greatest affect on dapsone resistance by increasing the MIC 64-fold and the IC50 68-fold. These results prove that thefolP1 of M. leprae encodes a functional DHPS and that mutations within this gene are associated with the development of dapsone resistance in clinical isolates of M. leprae. Transformants created with M. leprae folP2 did not confer growth on the C600ΔfolP::Kmrknockout strain, and DNA sequences of folP2 from dapsone-susceptible and -resistant M. leprae strains were identical, indicating that this gene does not encode a functional DHPS and is not involved in dapsone resistance in M. leprae.

scholarly journals Overlapping oriC and centromere-like functions in secondary genome replicons determine their maintenance independent of chromosome I in Deinococcus radiodurans

2020 ◽  
Author(s):  
Ganesh K Maurya ◽  
Hari S. Misra
Keyword(s):  
Radiation Resistance ◽  
Copy Number ◽  
Deinococcus Radiodurans ◽  
Specific Interactions ◽  
Wild Type ◽  
Γ Radiation ◽  
E Coli ◽  
Copy Numbers ◽  
Type Pattern ◽  
Chromosome I

AbstractThe Deinococcus radiodurans multipartite genome system (MGS) consists of chromosome I (ChrI) and secondary genome elements; Chr II and megaplasmid (MP). The sequences upstream to parAB operons in Chr II (cisII) and MP (cisMP) helped an E. coli plasmid maintenance in D. radiodurans and showed sequence specific interactions with DnaA and ParBs. The cells devoid of cisII (ΔcisII) or cisMP (ΔcisMP) showed reduced γ radiation resistance and copy number of Chr II and MP. Fluorescent Reporter-Operator System (FROS) developed for ChrI, ChrII and MP in ΔcisII or ΔcisMP mutants showed no change in wild type pattern of Chr I localization. However, the relative copy numbers of Chr II and MP had reduced while anucleate cells had increased in mutants. These results suggested that cisII and cisMP elements contain both ori and centromere-like functions, and like other MGS bacteria, the Chr I and secondary genome are maintained independently in D. radiodurans.

scholarly journals Genetic evidence for promoter competition in Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (11) ◽  
pp. 4608-4615 ◽  
Author(s):  
J E Hirschman ◽  
K J Durbin ◽  
F Winston
Keyword(s):  
Transcription Initiation ◽  
Initiation Site ◽  
Base Change ◽  
Wild Type ◽  
Base Pairs ◽  
Cis Acting ◽  
Initiation Of Transcription ◽  
In Trans ◽  
Tata Sequence ◽  
Transcription Signals

The his4-912 delta mutation is an insertion of the long terminal repeat (delta) of the yeast retrotransposon Ty into the HIS4 promoter region, such that the delta is 97 base pairs upstream of the HIS4 transcription initiation site. Strains carrying the his4-912 delta allele are His- at 23 degrees C; this phenotype can be reversed either by growth at 37 degrees C or by mutations in trans-acting SPT genes. Under conditions in which his4-912 delta confers a His- phenotype. HIS4 transcription initiates at the delta initiation site, rather than at the HIS4 initiation site, producing a longer, nonfunctional transcript. Under conditions in which the strain is His+, transcription initiates at the wild-type HIS4 initiation site. To understand how transcription is balanced between the delta and HIS4 promoters, we have selected for cis-acting suppressors of his4-912 delta. Two classes defined by six independent mutations restore synthesis of a functional HIS4 transcript. The first class is an A-to-G base change 1 base upstream of the proposed delta TATA sequence. These mutants do not synthesize the delta-initiated transcript; instead, they synthesize only the wild-type HIS4 transcript. The second class of mutations alters base pairs surrounding the functional HIS4 TATA sequence. The two strongest His+ mutants of this class synthesize the wild-type HIS4 transcript at levels consistent with their His+ phenotype. Surprisingly, these two mutants also have a reduced level of the delta-initiated transcript relative to the his4-912 delta parent. Analysis of these mutants indicates that the level of transcription from one promoter can affect the level of transcription from the other promoter and suggests that delta and HIS4 transcription signals compete for initiation of transcription from each site.

Cloning of promoter-like sequences from Lactobacillus paracasei subsp. paracasei CG11 and their expression in Escherichia coli, Lactococcus lactis, and Lactobacillus reuteri

1994 ◽  
Vol 40 (12) ◽  
pp. 1043-1050 ◽  
Author(s):  
Gordana Djordjevic ◽  
Bojana Bojovic ◽  
Ana Banina ◽  
Ljubisa Topisirovic
Keyword(s):  
Escherichia Coli ◽  
Lactococcus Lactis ◽  
Dna Sequences ◽  
Lactobacillus Reuteri ◽  
Lactobacillus Paracasei ◽  
Translational Efficiency ◽  
Broad Host Range ◽  
Chromosomal Dna ◽  
Base Pairs ◽  
E Coli

Fragments of chromosomal DNA from Lactobacillus paracasei subsp. paracasei CG11 (formerly Lactobacillus casei CG11) capable of functioning as promoters were isolated using the broad host range, promoter-probe vector pGKV210. Five such fragments designated P61, P79, P80, P116, and P144 were completely sequenced and analyzed. Fragment P61 had the highest transcriptional efficiency in Escherichia coli and Lactobacillus reuteri whereas P80 was the most active in Lactococcus lactis. In general, the orders of the transcriptional strengths were almost identical in E. coli and Lactobacillus reuteri but different from that in Lactococcus lactis. Mapping of the 5′ end of cat mRNA showed that different regions of fragments P79 and P144 were used as promoters in Lactococcus lactis than in E. coli and Lactobacillus reuteri. Analysis of these DNA sequences revealed that the putative −35 and −10 hexanucleotides resembled those of E. coli, Bacillus subtilis, and lactococci. The spacing between these two hexanucleotides and between the putative −10 hexanucleotide and the transcriptional start point (A residues predominated) ranged from 17 to 18 base pairs and from 5 to 7 base pairs, respectively. Each of the cloned Lactobacillus paracasei CG11 promoter-like fragments contained an AT-rich sequence upstream of the putative −35 region (from 60 to 73%).Key words: Lactobacillus, promoter-like sequences, transcriptional efficiency, translational efficiency.

scholarly journals Transcription-Dependent Increase in Multiple Classes of Base Substitution Mutations in Escherichia coli

2002 ◽  
Vol 184 (24) ◽  
pp. 6866-6872 ◽  
Author(s):  
Joanna Klapacz ◽  
Ashok S. Bhagwat
Keyword(s):  
Escherichia Coli ◽  
Genetic System ◽  
Base Substitution ◽  
E Coli ◽  
Base Substitutions ◽  
Selection For ◽  
Substitution Mutations ◽  
High Level ◽  
C To T Mutations ◽  
Genetic Assay

ABSTRACT We showed previously that transcription in Escherichia coli promotes C · G-to-T · A transitions due to increased deamination of cytosines to uracils in the nontranscribed but not the transcribed strand (A. Beletskii and A. S. Bhagwat, Proc. Natl. Acad. Sci. USA 93:13919-13924, 1996). To study mutations other than that of C to T, we developed a new genetic assay that selects only base substitution mutations and additionally excludes C · G to T · A transitions. This novel genetic reversion system is based on mutations in a termination codon and involves positive selection for resistance to bleomycin or kanamycin. Using this genetic system, we show here that transcription from a strong promoter increases the level of non-C-to-T as well as C-to-T mutations. We find that high-level transcription increases the level of non-C-to-T mutations in DNA repair-proficient cells in three different sequence contexts in two genes and that the rate of mutation is higher by a factor of 2 to 4 under these conditions. These increases are not caused by a growth advantage for the revertants and are restricted to genes that are induced for transcription. In particular, high levels of transcription do not create a general mutator phenotype in E. coli. Sequence analysis of the revertants revealed that the frequency of several different base substitutions increased upon transcription of the bleomycin resistance gene and that G · C-to-T · A transversions dominated the spectrum in cells transcribing the gene. These results suggest that high levels of transcription promote many different spontaneous base substitutions in E. coli.

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