scholarly journals Deletion Formation Between the Two Salmonella typhimurium Flagellin Genes Encoded on the Mini F Plasmid: Escherichia coli ssb Alleles Enhance Deletion Rates and Change Hot-Spot Preference for Deletion Endpoints

Genetics ◽  
1997 ◽  
Vol 145 (3) ◽  
pp. 563-572 ◽  
Author(s):  
Takafumi Mukaihara ◽  
Masatoshi Enomoto
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Hot Spots ◽  
Hot Spot ◽  
Direct Repeats ◽  
Replication Slippage ◽  
Detection Systems ◽  
Deletion Formation ◽  
Homologous Sequences ◽  
Deletion Detection

Deletion formation between the 5′-mostly homologous sequences and between the 3′-homeologous sequences of the two Salmonella typhimurium flagellin genes was examined using plasmid-based deletion-detection systems in various Escherichia coli genetic backgrounds. Deletions in plasmid pLC103 occur between the 5′ sequences, but not between the 3′ sequences, in both RecA-independent and RecA-dependent ways. Because the former is predominant, deletion formation in a recA background depends on the length of homologous sequences between the two genes. Deletion rates were enhanced 30- to 50-fold by the mismatch repair defects, mutS, mutL and uvrD, and 250-fold by the ssb-3 allele, but the effect of the mismatch defects was canceled by the ΔrecA allele. Rates of the deletion between the 3′ sequences in plasmid pLC107 were enhanced 17- to 130-fold by ssb alleles, but not by other alleles. For deletions in pLC107, 96% of the endpoints in the recA+ background and 88% in ΔrecA were in the two hot spots of the 60- and 33-nucleotide (nt) homologous sequences, whereas in the ssb-3 background >50% of the endpoints were in four- to 14-nt direct repeats dispersed in the entire 3′ sequences. The deletion formation between the homeologous sequences is RecA-independent but depends on the length of consecutive homologies. The mutant ssb allele lowers this dependency and results in the increase in deletion rates. Roles of mutant SSB are discussed with relation to misalignment in replication slippage.

Hairpins involving both inverted and direct repeats are associated with homoplasious indels in non-coding chloroplast DNA of Taraxacum (Lactuceae: Asteraceae)

Genome ◽  
2000 ◽  
Vol 43 (4) ◽  
pp. 634-641 ◽  
Author(s):  
T HM Mes ◽  
P Kuperus ◽  
J Kirschner ◽  
J Stepanek ◽  
P Oosterveld ◽  
...  
Keyword(s):  
Chloroplast Dna ◽  
Chloroplast Genome ◽  
Hot Spot ◽  
Phylogenetic Signal ◽  
Direct Repeats ◽  
Nucleotide Substitutions ◽  
Replication Slippage ◽  
Coding Regions ◽  
Chloroplast Genomes ◽  
Structural Mutations

Sequence variation in 2.2 kb of non-coding regions of the chloroplast genome of eight dandelions (Taraxacum: Lactuceae) from Asia and Europe is interpreted in the light of the phylogenetic signal of base substitutions vs. indels (insertions-deletions). The four non-coding regions displayed a total of approximately 30 structural mutations of which 9 are potentially phylogenetically informative. Insertions, deletions, and an inversion were found that involved consecutive stretches of up to 172 bases. When compared to phylogenetic relationships of the chloroplast genomes based on nucleotide substitutions only, many homoplasious indels (33%) were detected that differed considerably in length and did not comprise simple sequence repeats typically associated with replication slippage. Though many indels in the intergenic spacers were associated with direct repeats, frequently, the variable stretches participated in inverted repeat stabilized hairpins. In each intergenic spacer or intron examined, nucleotide stretches ranging from 30 to 60 bp were able to fold into stabilized secondary structures. When these indels were homoplasious, they always ranked among the most stabilized hairpins in the non-coding regions. The association of higher order structures that involve both classes of repeats and parallel structural mutations in hot spot regions of the chloroplast genome can be used to differentiate among mutations that differ in phylogenetic reliability.Key words: Taraxacum, indel, non-coding chloroplast DNA, hairpin, evolution.

scholarly journals Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli.

2020 ◽  
Author(s):  
Tingting Ding ◽  
Chaoyong Huang ◽  
Zeyu Liang ◽  
Xiaoyan Ma ◽  
Ning Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombination Event ◽  
Plasmid Stability ◽  
Direct Repeats ◽  
Replication Slippage ◽  
High Frequencies ◽  
Plasmid Construction ◽  
Guide Rnas ◽  
Cloning Strategy ◽  
Chromosome Fragments

Abstract Background: Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (DRs) which are susceptible to self-homologous recombination. As a result, the paired-gRNA plasmids cannot remain stable, which greatly prevents scalable application of the CRISPR/Cas9 system. Results: To address this limitation, different DRs-involved paired-gRNA plasmids were designed and the events of recombination were characterized. Deletion between DRs occurred with high frequencies during plasmid construction and subsequent plasmid propagation. This recombination event was RecA-independent, which agreed with the replication slippage model. To increase plasmid stability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs), which completely eliminated DRs-induced recombination. Using RPGPs, rapid deletion of chromosome fragments up to 100 kb with an efficiency of 83.33% was achieved in Escherichia coli. Conclusions: The RPGPs cloning strategy serves as a general solution to avoid plasmid RecA-independent recombination. It can be adapted to applications that rely on paired gRNAs or repeated genetic parts.

scholarly journals Mu and IS1 Transpositions Exhibit Strong Orientation Bias at the Escherichia coli bgl Locus

2001 ◽  
Vol 183 (11) ◽  
pp. 3328-3335 ◽  
Author(s):  
Dipankar Manna ◽  
Xiuhua Wang ◽  
N. Patrick Higgins
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Hot Spots ◽  
Protein Complexes ◽  
Hot Spot ◽  
High Affinity Site ◽  
Mu Transposition ◽  
Orientation Bias

ABSTRACT The region upstream of the Escherichia coli bgloperon is an insertion hot spot for several transposons. Elements as distantly related as Tn1, Tn5, and phage Mu home in on this location. To see what characteristics result in a high-affinity site for transposition, we compared in vivo and in vitro Mu transposition patterns near the bgl promoter. In vivo, Mu insertions were focused in two narrow zones of DNA nearbgl, and both zones exhibited a striking orientation bias. Five hot spots upstream of the bgl cyclic AMP binding protein (CAP) binding site had Mu insertions exclusively with the phage oriented left to right relative to the direction of bgl transcription. One hot spot within the CAP binding domain had the opposite (right-to-left) orientation of phage insertion. The DNA segment lying between these two Mu hot-spot clusters is extremely A/T rich (80%) and is an efficient target for insertion sequences during stationary phase. IS1 insertions that activate the bgl operon resulted in a decrease in Mu insertions near the CAP binding site. Mu transposition in vitro differed significantly from the in vivo transposition pattern, having a new hot-spot cluster at the border of the A/T-rich segment. Transposon hot-spot behavior and orientation bias may relate to an asymmetry of transposon DNA-protein complexes and to interactions with proteins that produce transcriptionally silenced chromatin.

Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli

2019 ◽  
Author(s):  
Tingting Ding ◽  
Chaoyong Huang ◽  
Zeyu Liang ◽  
Xiaoyan Ma ◽  
Ning Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Editing ◽  
High Efficiency ◽  
Direct Repeats ◽  
Replication Slippage ◽  
High Frequencies ◽  
Plasmid Instability ◽  
Plasmid Construction ◽  
Cloning Strategy ◽  
Chromosome Fragments

SummaryA growing number of CRISPR-Cas9 associated applications require co-expression of two distinct gRNAs. However, coexpressing paired gRNAs under the driving of independent but identical promoters in the same direction triggers plasmid instability, due to the presence of direct repeats (DRs). In this study, deletion between DRs occurred with high frequencies during plasmid construction and duplication processes, when three DRs-involved paired-gRNA plasmids cloning strategies were tested. This recombination phenomenon was RecA-independent, in agreement with the replication slippage model. To completely eliminate the DRs-induced plasmid instability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs). Using RPGPs, we achieved a rapid deletion of chromosome fragments up to 100 kb with high efficiency of 83.33% in Escherichia coli. This study provides general solutions to construct stable plasmids containing short DRs, which can improve the performances of CRISPR systems that relied on paired gRNAs, and also facilitate other applications involving repeated genetic parts.

Local DNA Sequence Control of Deletion Formation in Escherichia coli Plasmid pBR322

Genetics ◽  
1987 ◽  
Vol 115 (1) ◽  
pp. 41-49
Author(s):  
Ujjala DasGupta ◽  
Kathleen Weston-Hafer ◽  
Douglas E Berg
Keyword(s):  
Escherichia Coli ◽  
Dna Sequence ◽  
Specific Interactions ◽  
Plasmid Pbr322 ◽  
Direct Repeats ◽  
Ampicillin Resistance ◽  
Deletion Formation ◽  
Coli Plasmid ◽  
Insertion Mutations ◽  
Dna Processing

ABSTRACT The specificity of deletion formation was studied using tests involving reversion of palindromic insertion mutations. Insertions of a Tn5-related transposon at 13 sites in the ampicillin-resistance (amp) gene of plasmid pBR322 were shortened to a nested set of perfect palindromes, 22, 32 and 90 bp long. We monitored frequencies of reversion to Ampr, which is the result of deletion of the palindrome plus one copy of the flanking 9 bp direct repeats (which had been formed by transposition). Revertant frequencies were found to depend on the location and the sequence of the palindromic insert. Changing a 45-kb interrupted palindrome to a 22-bp perfect palindrome stimulated deletion formation by factors of from fourfold to 545-fold among the 13 sites, while elongation of the perfect palindrome from 22 to 90 bp stimulated deletion formation by factors of from eight- to 18,000-fold. We conclude that deletion formation is strongly affected by subtle features of DNA sequence or conformation, both inside and outside the deleted segment, and that these effects may reflect specific interactions of DNA processing proteins with template DNAs.

scholarly journals Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli

2020 ◽  
Author(s):  
Tingting Ding ◽  
Chaoyong Huang ◽  
Zeyu Liang ◽  
Xiaoyan Ma ◽  
Ning Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombination Event ◽  
Plasmid Stability ◽  
Direct Repeats ◽  
Replication Slippage ◽  
High Frequencies ◽  
Plasmid Construction ◽  
Guide Rnas ◽  
Cloning Strategy ◽  
Chromosome Fragments

Abstract Background: Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (DRs) which are susceptible to self-homologous recombination. As a result, the paired-gRNA plasmids cannot remain stable, which greatly prevents scalable application of the CRISPR/Cas9 system. Results: To address this limitation, different DRs-involved paired-gRNA plasmids were designed and the events of recombination were characterized. Deletion between DRs occurred with high frequencies during plasmid construction and subsequent plasmid propagation. This recombination event was RecA-independent, which agreed with the replication slippage model. To increase plasmid stability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs), which completely eliminated DRs-induced recombination. Using RPGPs, rapid deletion of chromosome fragments up to 100 kb with an efficiency of 83.33% was achieved in Escherichia coli.Conclusions: The RPGPs cloning strategy serves as a general solution to avoid plasmid RecA-independent recombination. It can be adapted to applications that rely on paired gRNAs or repeated genetic parts.

scholarly journals Palindromy and the location of deletion endpoints in Escherichia coli.

Genetics ◽  
1989 ◽  
Vol 121 (4) ◽  
pp. 651-658 ◽  
Author(s):  
K Weston-Hafer ◽  
D E Berg
Keyword(s):  
Escherichia Coli ◽  
Dna Sequencing ◽  
Dna Synthesis ◽  
Direct Repeat ◽  
Inverted Repeats ◽  
Direct Repeats ◽  
Ampicillin Resistance ◽  
Colony Hybridization ◽  
Deletion Formation ◽  
Insertion Mutations

Abstract The contributions of direct and inverted repeats to deletion formation were studied by characterizing Ampr revertants of plasmids with a series of insertion mutations at a specific site in the pBR322 ampicillin resistance (amp) gene. The inserts at this site are palindromic, variable in length, and bracketed by 9- or 10-bp direct repeats of amp sequence. There is an additional direct repeat composed of 4 bp within the insert and 4 bp of adjoining amp sequence. DNA sequencing and colony hybridization of Ampr revertants showed that they contained either the parental amp sequence, implying deletion endpoints in the flanking 9- or 10-bp repeats, or a specific 1-bp substitution, implying endpoints in the 4-bp repeats. Although generally direct repeats seem to be used as deletion endpoints with a frequency proportional to their lengths, we found that with uninterrupted palindromes longer than 32 bp, the majority of deletions ended in the 4 bp, not the 9- or 10-bp repeats. This preferential use of the shorter direct repeats associated with palindromes is interpreted according to a DNA synthesis-error model in which hairpin structures formed by intrastrand pairing foster the slippage of nascent strands during DNA synthesis.

Spatial Hotspot Analysis of Bucharest’s Urban Heat Island (UHI) Using Modis Data

2018 ◽  
Vol 18 (1) ◽  
pp. 14-22 ◽  
Author(s):  
Georgiana Grigoraș ◽  
Bogdan Urițescu
Keyword(s):  
Surface Temperature ◽  
Air Temperature ◽  
Land Surface Temperature ◽  
Hot Spots ◽  
Land Surface ◽  
Hot Spot ◽  
Monitoring Network ◽  
Residential Areas ◽  
Hotspot Analysis ◽  
The City

Abstract The aim of the study is to find the relationship between the land surface temperature and air temperature and to determine the hot spots in the urban area of Bucharest, the capital of Romania. The analysis was based on images from both moderate-resolution imaging spectroradiometer (MODIS), located on both Terra and Aqua platforms, as well as on data recorded by the four automatic weather stations existing in the endowment of The National Air Quality Monitoring Network, from the summer of 2017. Correlation coefficients between land surface temperature and air temperature were higher at night (0.8-0.87) and slightly lower during the day (0.71-0.77). After the validation of satellite data with in-situ temperature measurements, the hot spots in the metropolitan area of Bucharest were identified using Getis-Ord spatial statistics analysis. It has been achieved that the “very hot” areas are grouped in the center of the city and along the main traffic streets and dense residential areas. During the day the "very hot spots” represent 33.2% of the city's surface, and during the night 31.6%. The area where the mentioned spots persist, falls into the "very hot spot" category both day and night, it represents 27.1% of the city’s surface and it is mainly represented by the city center.

The family Orthotrichaceae (Bryophyta) in flora of Russia: results of revision and biogeographical review

2018 ◽  
Vol 52 (2) ◽  
pp. 519-534 ◽  
Author(s):  
V. E. Fedosov
Keyword(s):  
Hot Spots ◽  
Latitudinal Gradient ◽  
Hot Spot ◽  
Spatial Differentiation ◽  
Taxonomic Composition ◽  
Undescribed Species ◽  
Longitudinal Gradient ◽  
Biogeographic Pattern ◽  
The Family ◽  
North Pacific Region

Recent studies on Orthotrichoid mosses in Russia are summarized genus by genus. Orthotrichum furcatum Otnyukova is synonymized with Nyholmiella obtusifolia. Orthotrichum vittii is excluded from the Russian moss flora. Description of O. dagestanicum is amended. Fifty four currently recognized species from 9 genera of the Orthotrichaceae are presently known to occur in Russia; list of species with common synonyms and brief review of distribution in Russia is presented. Numerous problematic specimens with unresolved taxonomy were omitted for future. Revealed taxonomical inconsistencies in the genera Zygodon, Ulota, Lewinskya, Nyholmiella, Orthotrichum are briefly discussed. Main regularities of spatial differentiation of the family Orthotrichaceae in Russia are considered. Recently presented novelties contribute to the certain biogeographic pattern, indicating three different centers of diversity of the family, changing along longitudinal gradient. Unlike European one, continental Asian diversity of Orthotrichaceae is still poorly known, the Siberian specimens which were previously referred to European species in most cases were found to represent other, poorly known or undescribed species. North Pacific Region houses peculiar and poorly understood hot spot of diversity of Orthotrichoid mosses. Thus, these hot spots are obligatory to be sampled in course of revisions of particular groups, since they likely comprise under-recorded cryptic- or semi-cryptic species. Latitudinal gradient also contributes to the spatial differentiation of the revealed taxonomic composition of Orthotrichaceae.

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