scholarly journals Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4920 ◽  
Author(s):  
Ben A. Evans ◽  
Olivia L. Smith ◽  
Ethan S. Pickerill ◽  
Mary K. York ◽  
Kristen J.P. Buenconsejo ◽  
...  
Keyword(s):  
Protein Function ◽  
Point Mutations ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Model Organisms ◽  
Efficient Manner ◽  
Restriction Digest ◽  
Efficient Detection ◽  
Wide Range ◽  
Restriction Sites

Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the Candida albicans UME6 Zn2+-binding domain, a known regulator of C. albicans filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type C. albicans. Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest.

scholarly journals Transcriptome wide SSR discovery cross-taxa transferability and development of marker database for studying genetic diversity population structure of Lilium species

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Manosh Kumar Biswas ◽  
Mita Bagchi ◽  
Ujjal Kumar Nath ◽  
Dhiman Biswas ◽  
Sathishkumar Natarajan ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Population Structure ◽  
Large Scale ◽  
Polymorphic Information Content ◽  
Pcr Amplification ◽  
Efficient Manner ◽  
Lilium Species ◽  
Population Structure Analysis ◽  
Wide Range ◽  
Functional Molecular Markers

Abstract Lily belongs to family liliaceae, which mainly propagates vegetatively. Therefore, sufficient number of polymorphic, informative, and functional molecular markers are essential for studying a wide range of genetic parameters in Lilium species. We attempted to develop, characterize and design SSR (simple sequence repeat) markers using online genetic resources for analyzing genetic diversity and population structure of Lilium species. We found di-nucleotide repeat motif were more frequent (4684) within 0.14 gb (giga bases) transcriptome than other repeats, of which was two times higher than tetra-repeat motifs. Frequency of di-(AG/CT), tri-(AGG/CTT), tetra-(AAAT), penta-(AGAGG), and hexa-(AGAGGG) repeats was 34.9%, 7.0%, 0.4%, 0.3%, and 0.2%, respectively. A total of 3607 non-redundant SSR primer pairs was designed based on the sequences of CDS, 5′-UTR and 3′-UTR region covering 34%, 14%, 23%, respectively. Among them, a sub set of primers (245 SSR) was validated using polymerase chain reaction (PCR) amplification, of which 167 primers gave expected PCR amplicon and 101 primers showed polymorphism. Each locus contained 2 to 12 alleles on average 0.82 PIC (polymorphic information content) value. A total of 87 lily accessions was subjected to genetic diversity analysis using polymorphic SSRs and found to separate into seven groups with 0.73 to 0.79 heterozygosity. Our data on large scale SSR based genetic diversity and population structure analysis may help to accelerate the breeding programs of lily through utilizing different genomes, understanding genetics and characterizing germplasm with efficient manner.

"Hot spots" mutation analysis of p53 gene in gastrointestinal cancers by amplification of naturally occurring and artificially created restriction sites

1993 ◽  
Vol 39 (10) ◽  
pp. 2186-2191 ◽  
Author(s):  
P H Chen ◽  
S Y Lin ◽  
C K Wang ◽  
Y J Chen ◽  
T C Chen ◽  
...  
Keyword(s):  
Hot Spot ◽  
Point Mutations ◽  
P53 Gene ◽  
Restriction Enzymes ◽  
Cancer Tissue ◽  
Gastrointestinal Cancers ◽  
Simple Method ◽  
Surgical Specimen ◽  
Naturally Occurring ◽  
Restriction Sites

Abstract We developed a rapid, simple method to detect "hot spot" point mutations of the p53 gene. A DNA fragment from cancer tissue of a surgical specimen was selectively amplified with specific oligonucleotide primers and then digested with restriction enzymes that recognized artificial or naturally occurring restriction sites. To detect mutations in codons 273 and 245, we created artificial Bst U1 and BgI I sites by introducing a single nucleotide mismatch into the respective mutagenesis primers. We used the naturally occurring restriction sites of Msp I, Hae III, and Hha I to detect mutations in codons 248, 249, and 175, respectively. In 74 cases of gastrointestinal cancer, 5 of 35 colorectal cancers showed mutations; 1 of 15 cases of gastric cancers showed mutation; and 1 of 24 hepatocellular carcinomas showed mutation. This nonradioactive method is an accurate and clinically useful way to detect hot-spot point mutations of the p53 gene in gastrointestinal cancers.

scholarly journals Refactoring gene sequences for broad assembly standards compatibility

2017 ◽  
Author(s):  
Tyson R. Shepherd
Keyword(s):  
Hot Spots ◽  
Restriction Enzymes ◽  
Open Reading Frames ◽  
Model Organisms ◽  
Protein Coding ◽  
Coding Sequence ◽  
Computational Program ◽  
Restriction Sites ◽  
Recombination Hot Spots ◽  
Reading Frames

AbstractFour cloning standards in synthetic biology are BioBrick, BglBrick, MoClo and GoldenBraid, with each requiring their constitutive parts be compatible with the associated restriction enzymes. To standardize parts for the broadest usage, it would be useful to synthesize genes that are simultaneously compatible with all 4 popular assembly strategies. Here it is shown that using a defined set of rules, implemented in a computational program, any protein coding sequence can be made compatible with all four standards by silent mutations. Using a coding sequence as an input, all BioBrick, BglBrick, MoClo, and GoldenBraid restriction sites and chi recombination hot spots can be destroyed with silent mutations that approximate the codon usage of the organism. As an application, all open reading frames in the model organisms Escherichia Coli and Bacillus Subtilis are computationally refactored, showing the feasibility of implementing this umbrella strategy for synthesizing genes with the broadest compatibility.

scholarly journals On the Origins of Homology Directed Repair in Mammalian Cells

2021 ◽  
Vol 22 (7) ◽  
pp. 3348
Author(s):  
Brett M. Sansbury ◽  
Eric B. Kmiec
Keyword(s):  
Human Genome ◽  
Protein Function ◽  
Mammalian Cells ◽  
Gene Editing ◽  
Single Agent ◽  
Point Mutations ◽  
Human Beings ◽  
Homology Directed Repair ◽  
Dna Oligonucleotides ◽  
Wide Range

Over the course of the last five years, expectations surrounding our capacity to selectively modify the human genome have never been higher. The reduction to practice site-specific nucleases designed to cleave at a unique site within the DNA is now centerstage in the development of effective molecular therapies. Once viewed as being impossible, this technology now has great potential and, while cellular and molecular barriers persist to clinical implementations, there is little doubt that these barriers will be crossed, and human beings will soon be treated with gene editing tools. The most ambitious of these desires is the correction of genetic mutations resident within the human genome that are responsible for oncogenesis and a wide range of inherited diseases. The process by which gene editing activity could act to reverse these mutations to wild-type and restore normal protein function has been generally categorized as homology directed repair. This is a catch-all basket term that includes the insertion of short fragments of DNA, the replacement of long fragments of DNA, and the surgical exchange of single bases in the correction of point mutations. The foundation of homology directed repair lies in pioneering work that unravel the mystery surrounding genetic exchange using single-stranded DNA oligonucleotides as the sole gene editing agent. Single agent gene editing has provided guidance on how to build combinatorial approaches to human gene editing using the remarkable programmable nuclease complexes known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their closely associated (Cas) nucleases. In this manuscript, we outline the historical pathway that has helped evolve the current molecular toolbox being utilized for the genetic re-engineering of the human genome.

Coaction of Electrostatic and Hydrophobic Interactions: Dynamic Constraints on Disordered TrkA Juxtamembrane Domain

2019 ◽  
Author(s):  
Zichen Wang ◽  
Huaxun Fan ◽  
Xiao Hu ◽  
John Khamo ◽  
Jiajie Diao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Hydrophobic Interactions ◽  
Transmembrane Helix ◽  
Point Mutations ◽  
Salt Bridge ◽  
Receptor Activation ◽  
Membrane Dynamics ◽  
Juxtamembrane Domain ◽  
Joint Work

<p>The receptor tyrosine kinase family transmits signals into cell via a single transmembrane helix and a flexible juxtamembrane domain (JMD). Membrane dynamics makes it challenging to study the structural mechanism of receptor activation experimentally. In this study, we employ all-atom molecular dynamics with Highly Mobile Membrane-Mimetic to capture membrane interactions with the JMD of tropomyosin receptor kinase A (TrkA). We find that PIP<sub>2 </sub>lipids engage in lasting binding to multiple basic residues and compete with salt bridge within the peptide. We discover three residues insertion into the membrane, and perturb it through computationally designed point mutations. Single-molecule experiments indicate the contribution from hydrophobic insertion is comparable to electrostatic binding, and in-cell experiments show that enhanced TrkA-JMD insertion promotes receptor ubiquitination. Our joint work points to a scenario where basic and hydrophobic residues on disordered domains interact with lipid headgroups and tails, respectively, to restrain flexibility and potentially modulate protein function.</p>

scholarly journals mtDNAcombine: tools to combine sequences from multiple studies

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Eleanor F. Miller ◽  
Andrea Manica
Keyword(s):  
Sequence Data ◽  
Data Extraction ◽  
Bayesian Skyline Plot ◽  
Model Organisms ◽  
Data Sets ◽  
Data Handling ◽  
Online Database ◽  
Genetic Studies ◽  
Wide Range ◽  
Existing Data

Abstract Background Today an unprecedented amount of genetic sequence data is stored in publicly available repositories. For decades now, mitochondrial DNA (mtDNA) has been the workhorse of genetic studies, and as a result, there is a large volume of mtDNA data available in these repositories for a wide range of species. Indeed, whilst whole genome sequencing is an exciting prospect for the future, for most non-model organisms’ classical markers such as mtDNA remain widely used. By compiling existing data from multiple original studies, it is possible to build powerful new datasets capable of exploring many questions in ecology, evolution and conservation biology. One key question that these data can help inform is what happened in a species’ demographic past. However, compiling data in this manner is not trivial, there are many complexities associated with data extraction, data quality and data handling. Results Here we present the mtDNAcombine package, a collection of tools developed to manage some of the major decisions associated with handling multi-study sequence data with a particular focus on preparing sequence data for Bayesian skyline plot demographic reconstructions. Conclusions There is now more genetic information available than ever before and large meta-data sets offer great opportunities to explore new and exciting avenues of research. However, compiling multi-study datasets still remains a technically challenging prospect. The mtDNAcombine package provides a pipeline to streamline the process of downloading, curating, and analysing sequence data, guiding the process of compiling data sets from the online database GenBank.

scholarly journals Growth Promotion or Osmotic Stress Response: How SNF1-Related Protein Kinase 2 (SnRK2) Kinases Are Activated and Manage Intracellular Signaling in Plants

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1443
Author(s):  
Yoshiaki Kamiyama ◽  
Sotaro Katagiri ◽  
Taishi Umezawa
Keyword(s):  
Protein Kinase ◽  
Plant Growth ◽  
Osmotic Stress ◽  
Protein Function ◽  
Intracellular Signaling ◽  
Growth Promotion ◽  
Research Field ◽  
Dependent Manner ◽  
Related Protein ◽  
Wide Range

Reversible phosphorylation is a major mechanism for regulating protein function and controls a wide range of cellular functions including responses to external stimuli. The plant-specific SNF1-related protein kinase 2s (SnRK2s) function as central regulators of plant growth and development, as well as tolerance to multiple abiotic stresses. Although the activity of SnRK2s is tightly regulated in a phytohormone abscisic acid (ABA)-dependent manner, recent investigations have revealed that SnRK2s can be activated by group B Raf-like protein kinases independently of ABA. Furthermore, evidence is accumulating that SnRK2s modulate plant growth through regulation of target of rapamycin (TOR) signaling. Here, we summarize recent advances in knowledge of how SnRK2s mediate plant growth and osmotic stress signaling and discuss future challenges in this research field.

scholarly journals Molecular variation in chloroplast DNA regions in ancestral species of wheat.

Genetics ◽  
1994 ◽  
Vol 137 (3) ◽  
pp. 883-889 ◽  
Author(s):  
N T Miyashita ◽  
N Mori ◽  
K Tsunewaki
Keyword(s):  
Chloroplast Dna ◽  
Restriction Enzymes ◽  
Triticum Dicoccoides ◽  
The Other ◽  
Major Type ◽  
Aegilops Speltoides ◽  
Triticum Timopheevi ◽  
Restriction Sites ◽  
D Values ◽  
Chloroplast Dna Regions

Abstract Restriction map variation in two 5-6-kb chloroplast DNA regions of five diploid Aegilops species in the section Sitopsis and two wild tetraploid wheats, Triticum dicoccoides and Triticum araraticum, was investigated with a battery of four-cutter restriction enzymes. A single accession each of Triticum durum, Triticum timopheevi and Triticum aestivum was included as a reference. More than 250 restriction sites were scored, of which only seven sites were found polymorphic in Aegilops speltoides. No restriction site polymorphisms were detected in all of the other diploid and tetraploid species. In addition, six insertion/deletion polymorphisms were detected, but they were mostly unique or species-specific. Estimated nucleotide diversity was 0.0006 for A. speltoides, and 0.0000 for all the other investigated species. In A. speltoides, none of Tajima's D values was significant, indicating no clear deviation from the neutrality of molecular polymorphisms. Significant non-random association was detected for three combinations out of 10 possible pairs between polymorphic restriction sites in A. speltoides. Phylogenetic relationship among all the plastotypes (plastid genotype) suggested the diphyletic origin of T. dicoccoides and T. araraticum. A plastotype of one A. speltoides accession was identical to the major type of T. araraticum (T. timopheevi inclusively). Three of the plastotypes found in the Sitopsis species are very similar, but not identical, to that of T. dicoccoides, T. durum and T. aestivum.

scholarly journals An In Vitro Cell Culture Model for Pyoverdine-Mediated Virulence

Pathogens ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 9
Author(s):  
Donghoon Kang ◽  
Natalia V. Kirienko
Keyword(s):  
Cell Culture ◽  
Virulence Factors ◽  
Model Organisms ◽  
Cell Culture Model ◽  
Chronic Infections ◽  
In Vitro Cell Culture ◽  
Mitochondrial Homeostasis ◽  
Culture Model ◽  
Wide Range

Pseudomonas aeruginosa is a multidrug-resistant, opportunistic pathogen that utilizes a wide-range of virulence factors to cause acute, life-threatening infections in immunocompromised patients, especially those in intensive care units. It also causes debilitating chronic infections that shorten lives and worsen the quality of life for cystic fibrosis patients. One of the key virulence factors in P. aeruginosa is the siderophore pyoverdine, which provides the pathogen with iron during infection, regulates the production of secreted toxins, and disrupts host iron and mitochondrial homeostasis. These roles have been characterized in model organisms such as Caenorhabditis elegans and mice. However, an intermediary system, using cell culture to investigate the activity of this siderophore has been absent. In this report, we describe such a system, using murine macrophages treated with pyoverdine. We demonstrate that pyoverdine-rich filtrates from P. aeruginosa exhibit substantial cytotoxicity, and that the inhibition of pyoverdine production (genetic or chemical) is sufficient to mitigate virulence. Furthermore, consistent with previous observations made in C. elegans, pyoverdine translocates into cells and disrupts host mitochondrial homeostasis. Most importantly, we observe a strong correlation between pyoverdine production and virulence in P. aeruginosa clinical isolates, confirming pyoverdine’s value as a promising target for therapeutic intervention. This in vitro cell culture model will allow rapid validation of pyoverdine antivirulents in a simple but physiologically relevant manner.

scholarly journals Molecular Subtyping of Blastocystis sp. Isolated from Farmed Animals in Southern Italy

2021 ◽  
Vol 9 (8) ◽  
pp. 1656
Author(s):  
Simona Gabrielli ◽  
Marialetizia Palomba ◽  
Federica Furzi ◽  
Emanuele Brianti ◽  
Gabriella Gaglio ◽  
...  
Keyword(s):  
Current Knowledge ◽  
Pcr Amplification ◽  
Epidemiological Studies ◽  
Fallow Deer ◽  
Small Subunit ◽  
Zoonotic Transmission ◽  
Ssu Rrna ◽  
Molecular Approaches ◽  
Wide Range ◽  
Blastocystis Sp

Blastocystis is a common intestinal protist distributed worldwide, infecting humans and a wide range of domestic and wild animals. It exhibits an extensive genetic diversity and, so far, 25 distinct small subunit ribosomal RNA (SSU rRNA) lineages termed subtypes (STs)) have been characterized; among them, 12 have thus far been reported in humans. The aims of the present study were to detect and genetically characterize Blastocystis sp. in synantropic animals to improve our current knowledge on the distribution and zoonotic transmission of Blastocystis STs in Italy. Samples were collected from N = 193 farmed animals and submitted to DNA extraction and PCR amplification of the SSU rRNA. Blastocystis was detected in 60 samples (31.08%) and successfully subtyped. Phylogenetic analysis evidenced that the isolates from fallow deer, goats, and pigs (N = 9) clustered within the ST5; those from pheasants (N = 2) in the ST6; those from chickens (N = 8) in the ST7; those from sheep (N = 6) in the ST10; and those from water buffaloes (N = 9) in the ST14 clade. The comparison between the present isolates from animals and those previously detected in humans in Italy suggested the animal-to-human spillover for ST6 and ST7. The present study represents the widest Blastocystis survey performed thus far in farmed animals in Italy. Further epidemiological studies using molecular approaches are required to determine the occurrence and distribution of Blastocystis STs in other potential animal reservoirs in Italy and to define the pathways of zoonotic transmission.

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