scholarly journals New logic facilitated identification of novel phage recombinase function units

2019 ◽  
Author(s):  
Yizhao Chang ◽  
Qian Wang ◽  
Tianyuan Su ◽  
Qingsheng Qi
Keyword(s):  
Genetic Manipulation ◽  
Structural Information ◽  
Mobile Genetic Elements ◽  
Taxonomic Group ◽  
Genetic Tools ◽  
Genetic Elements ◽  
Highly Effective ◽  
Single Strand Annealing ◽  
Function Unit ◽  
Strand Annealing

AbstractPhage recombinase function unit (PRFU) contains one single strand annealing protein (SSAP) and one 5’-3’ exonuclease (EXO). Well-known PRFU such as lambda-Red or Rac RecET has been proved to be useful tool in the recombineering ofEscherichia colibut had limited efficiency in distant species. Therefore, PRFUs from closely related species were often searched and developed as specific genetic tools. However, there are still many bacteria with few PRFU identified. Here, we hypothesised that the occurrence of PRFU might be related to specific strains. Based on this assumption, we managed to identify 59 unique sets of PRFUs in over 23 species of the genusCorynebacterium, a taxonomic group that were reported to be lacking in such system. The identified PRFUs were then classified to show their similarities and relation to species based on both sequential and structural information. Representative PRFUs were verified to be highly effective in mediating recombineering using ssDNA or dsDNA substrates. Analysis of the functional PRFUs indicated that they share similar local genome contexts, which might suggest their common origin. Our findings reveal the relation of PRFUs, species and strains and provide novel candidate genetic manipulation tools in genusCorynebacterium.Author SummaryGenusCorynebacteriumis a taxonomic group with highly diversified species that are of medical, veterinary, or biotechnological relevance. However, being lack of genetic tools limits the study of these species. Our new verified phage recombinase function units (PRFUs) in this genus that are highly effective in mediating recombineering will provide new candidate genetic tools in this genus. Besides, reference genomes were often used for genome study and data mining. While for the mobile genetic elements, their transferable characteristic may affect their occurrence among genomes. Here we provide evidence for this idea by showing that the occurrence of PRFU is strain related but not species related. This may be helpful for the functional genomics study of mobile genetic elements among genomes.

scholarly journals Unlimited genetic switches for cell-type specific manipulation

2018 ◽  
Author(s):  
Jorge Garcia-Marques ◽  
Ching-Po Yang ◽  
Isabel Espinosa-Medina ◽  
Kent Mok ◽  
Minoru Koyama ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Cell Types ◽  
Biological Functions ◽  
Cell Type ◽  
Repair Mechanism ◽  
Genetic Switches ◽  
Single Strand Annealing ◽  
Cell Type Specific ◽  
Strand Annealing ◽  
Genetic Targeting

SummaryGaining independent genetic access to discrete cell types is critical to interrogate their biological functions, as well as to deliver precise gene therapy. Transcriptome analyses have allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types have remained inaccessible to genetic manipulation. Here, we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches that each respond to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell type-specific manipulations and combinatorial genetic targeting. CaSSA is thus a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.

Transgenesis of schistosomes: approaches employing mobile genetic elements

Parasitology ◽  
2007 ◽  
Vol 135 (2) ◽  
pp. 141-153 ◽  
Author(s):  
V. H. MANN ◽  
M. E. MORALES ◽  
K. J. KINES ◽  
P. J. BRINDLEY
Keyword(s):  
Large Scale ◽  
Insertional Mutagenesis ◽  
Genetic Manipulation ◽  
Draft Genome ◽  
Mobile Genetic Elements ◽  
Forward Genetics ◽  
Loss Of Function ◽  
Genetic Elements ◽  
The Past ◽  
Foreign Genes

SUMMARYDraft genome sequences forSchistosoma mansoniandSchistosoma japonicumare now available. However, the identity and importance of most schistosome genes have yet to be determined. Recently, progress has been made towards the genetic manipulation and transgenesis of schistosomes. Both loss-of-function and gain-of-function approaches appear to be feasible in schistosomes based on findings described in the past 5 years. This review focuses on reports of schistosome transgenesis, specifically those dealing with the transformation of schistosomes with exogenous mobile genetic elements and/or their endogenous relatives for the genetic manipulation of schistosomes. Transgenesis mediated by mobile genetic elements offers a potentially tractable route to introduce foreign genes to schistosomes, a means to determine the importance of schistosome genes, including those that could be targeted in novel interventions and the potential to undertake large-scale forward genetics by insertional mutagenesis.

ISSR-PCR markers and mobile genetic elements in horse (Equus caballus) genome

2014 ◽  
pp. 42-57 ◽  
Author(s):  
N.V. Bardukov ◽  
◽  
A.V. Feofilov ◽  
T.T. Glazko ◽  
V.I. Glazko ◽  
...  
Keyword(s):  
Equus Caballus ◽  
Mobile Genetic Elements ◽  
Pcr Markers ◽  
Genetic Elements ◽  
Issr Pcr

Yeastspt6-140Mutation, Affecting Chromatin and Transcription, Preferentially Increases Recombination in Which Rad51p-Mediated Strand Exchange Is Dispensable

Genetics ◽  
2001 ◽  
Vol 158 (2) ◽  
pp. 597-611 ◽  
Author(s):  
Francisco Malagón ◽  
Andrés Aguilera
Keyword(s):  
Strand Exchange ◽  
Micrococcal Nuclease ◽  
Sensitivity Analyses ◽  
Inverted Repeats ◽  
Major Mechanism ◽  
Spontaneous Recombination ◽  
Single Strand Annealing ◽  
Strand Invasion ◽  
Strand Annealing ◽  
Break Induced Replication

AbstractWe have shown that the spt6-140 and spt4-3 mutations, affecting chromatin structure and transcription, stimulate recombination between inverted repeats by a RAD52-dependent mechanism that is very efficient in the absence of RAD51, RAD54, RAD55, and RAD57. Such a mechanism of recombination is RAD1-RAD59-dependent and yields gene conversions highly associated with the inversion of the repeat. The spt6-140 mutation alters transcription and chromatin in our inverted repeats, as determined by Northern and micrococcal nuclease sensitivity analyses, respectively. Hyper-recombination levels are diminished in the absence of transcription. We believe that the chromatin alteration, together with transcription impairment caused by spt6-140, increases the incidence of spontaneous recombination regardless of whether or not it is mediated by Rad51p-dependent strand exchange. Our results suggest that spt6, as well as spt4, primarily stimulates a mechanism of break-induced replication. We discuss the possibility that the chromatin alteration caused by spt6-140 facilitates a Rad52p-mediated one-ended strand invasion event, possibly inefficient in wild-type chromatin. Our results are consistent with the idea that the major mechanism leading to inversions might not be crossing over but break-induced replication followed by single-strand annealing.

scholarly journals The Short Life Span of Saccharomyces cerevisiae sgs1 and srs2 Mutants Is a Composite of Normal Aging Processes and Mitotic Arrest Due to Defective Recombination

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1531-1542 ◽  
Author(s):  
Mitch McVey ◽  
Matt Kaeberlein ◽  
Heidi A Tissenbaum ◽  
Leonard Guarente
Keyword(s):  
Cell Cycle ◽  
Life Span ◽  
Mitotic Cell ◽  
Dna Helicase ◽  
Premature Aging ◽  
Growth Defect ◽  
Short Life Span ◽  
Single Strand Annealing ◽  
Late Generation ◽  
Strand Annealing

Abstract Evidence from many organisms indicates that the conserved RecQ helicases function in the maintenance of genomic stability. Mutation of SGS1 and WRN, which encode RecQ homologues in budding yeast and humans, respectively, results in phenotypes characteristic of premature aging. Mutation of SRS2, another DNA helicase, causes synthetic slow growth in an sgs1 background. In this work, we demonstrate that srs2 mutants have a shortened life span similar to sgs1 mutants. Further dissection of the sgs1 and srs2 survival curves reveals two distinct phenomena. A majority of sgs1 and srs2 cells stops dividing stochastically as large-budded cells. This mitotic cell cycle arrest is age independent and requires the RAD9-dependent DNA damage checkpoint. Late-generation sgs1 and srs2 cells senesce due to apparent premature aging, most likely involving the accumulation of extrachromosomal rDNA circles. Double sgs1 srs2 mutants are viable but have a high stochastic rate of terminal G2/M arrest. This arrest can be suppressed by mutations in RAD51, RAD52, and RAD57, suggesting that the cell cycle defect in sgs1 srs2 mutants results from inappropriate homologous recombination. Finally, mutation of RAD1 or RAD50 exacerbates the growth defect of sgs1 srs2 cells, indicating that sgs1 srs2 mutants may utilize single-strand annealing as an alternative repair pathway.

scholarly journals The Yeast Recombinational Repair Protein Rad59 Interacts With Rad52 and Stimulates Single-Strand Annealing

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 515-525 ◽  
Author(s):  
Allison P Davis ◽  
Lorraine S Symington
Keyword(s):  
Protein A ◽  
Single Strand ◽  
Physical Interaction ◽  
Dna Double Strand Breaks ◽  
Single Stranded Dna ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Recombination Pathway

Abstract The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.

scholarly journals Genomic evolution of antimicrobial resistance in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pimlapas Leekitcharoenphon ◽  
Markus Hans Kristofer Johansson ◽  
Patrick Munk ◽  
Burkhard Malorny ◽  
Magdalena Skarżyńska ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Virulence Genes ◽  
Mobile Genetic Elements ◽  
Whole Genome ◽  
Sequencing Analysis ◽  
Metagenomic Sequencing ◽  
Fecal Samples ◽  
E Coli ◽  
Genetic Elements

AbstractThe emergence of antimicrobial resistance (AMR) is one of the biggest health threats globally. In addition, the use of antimicrobial drugs in humans and livestock is considered an important driver of antimicrobial resistance. The commensal microbiota, and especially the intestinal microbiota, has been shown to have an important role in the emergence of AMR. Mobile genetic elements (MGEs) also play a central role in facilitating the acquisition and spread of AMR genes. We isolated Escherichia coli (n = 627) from fecal samples in respectively 25 poultry, 28 swine, and 15 veal calf herds from 6 European countries to investigate the phylogeny of E. coli at country, animal host and farm levels. Furthermore, we examine the evolution of AMR in E. coli genomes including an association with virulence genes, plasmids and MGEs. We compared the abundance metrics retrieved from metagenomic sequencing and whole genome sequenced of E. coli isolates from the same fecal samples and farms. The E. coli isolates in this study indicated no clonality or clustering based on country of origin and genetic markers; AMR, and MGEs. Nonetheless, mobile genetic elements play a role in the acquisition of AMR and virulence genes. Additionally, an abundance of AMR was agreeable between metagenomic and whole genome sequencing analysis for several AMR classes in poultry fecal samples suggesting that metagenomics could be used as an indicator for surveillance of AMR in E. coli isolates and vice versa.

Sign in / Sign up

Export Citation Format

Share Document