scholarly journals An Efficient DNA Sequencing Strategy Based on the Bacteriophage Mu in Vitro DNA Transposition Reaction

1999 ◽  
Vol 9 (3) ◽  
pp. 308-315 ◽  
Author(s):  
Saija Haapa ◽  
Sini Suomalainen ◽  
Simo Eerikäinen ◽  
Matti Airaksinen ◽  
Lars Paulin ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Sequence Data ◽  
Sequence Information ◽  
Bacteriophage Mu ◽  
Reaction Products ◽  
Dna Transposition ◽  
Sequencing Strategy ◽  
Transposon Insertions ◽  
Transposition Reaction

A highly efficient DNA sequencing strategy was developed on the basis of the bacteriophage Mu in vitro DNA transposition reaction. In the reaction, an artificial transposon with a chloramphenicol acetyltransferase (cat) gene as a selectable marker integrated into the target plasmid DNA containing a 10.3-kb mouse genomic insert to be sequenced. Bacterial clones carrying plasmids with the transposon insertions in different positions were produced by transforming transposition reaction products into Escherichia coli cells that were then selected on appropriate selection plates. Plasmids from individual clones were isolated and used as templates for DNA sequencing, each with two primers specific for the transposon sequence but reading the sequence into opposite directions, thus creating a minicontig. By combining the information from overlapping minicontigs, the sequence of the entire 10,288-bp region of mouse genome including six exons of mouse Kcc2 gene was obtained. The results indicated that the described methodology is extremely well suited for DNA sequencing projects in which considerable sequence information is on demand. In addition, massive DNA sequencing projects, including those of full genomes, are expected to benefit substantially from the Mu strategy.[The sequence data reported in this paper have been submitted to the GenBank data library under accession no. AJ011033.]

scholarly journals Rigorous Plasma Microbiome Analysis Method Enables Disease Association Discovery in Clinic

2021 ◽  
Vol 11 ◽  
Author(s):  
Zhenwu Luo ◽  
Alexander V. Alekseyenko ◽  
Elizabeth Ogunrinde ◽  
Min Li ◽  
Quan-Zhen Li ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Lupus Erythematosus ◽  
Sequence Data ◽  
Microbial Infection ◽  
Disease Pathogenesis ◽  
Host Interaction ◽  
Association Discovery ◽  
Low Levels ◽  
Quality Filtering

Blood microbiome is important to investigate microbial-host interactions and the effects on systemic immune perturbations. However, this effort has met with major challenges due to low microbial biomass and background artifacts. In the current study, microbial 16S DNA sequencing was applied to analyze plasma microbiome. We have developed a quality-filtering strategy to evaluate and exclude low levels of microbial sequences, potential contaminations, and artifacts from plasma microbial 16S DNA sequencing analyses. Furthermore, we have applied our technique in three cohorts, including tobacco-smokers, HIV-infected individuals, and individuals with systemic lupus erythematosus (SLE), as well as corresponding controls. More than 97% of total sequence data was removed using stringent quality-filtering strategy analyses; those removed amplicon sequence variants (ASVs) were low levels of microbial sequences, contaminations, and artifacts. The specifically enriched pathobiont bacterial ASVs have been identified in plasmas from tobacco-smokers, HIV-infected individuals, and individuals with SLE but not from control subjects. The associations between these ASVs and disease pathogenesis were demonstrated. The pathologic activities of some identified bacteria were further verified in vitro. We present a quality-filtering strategy to identify pathogenesis-associated plasma microbiome. Our approach provides a method for studying the diagnosis of subclinical microbial infection as well as for understanding the roles of microbiome-host interaction in disease pathogenesis.

scholarly journals Efficient Insertion Mutagenesis Strategy for Bacterial Genomes Involving Electroporation of In Vitro-Assembled DNA Transposition Complexes of Bacteriophage Mu

2002 ◽  
Vol 68 (2) ◽  
pp. 705-712 ◽  
Author(s):  
Arja Lamberg ◽  
Sari Nieminen ◽  
Mingqiang Qiao ◽  
Harri Savilahti
Keyword(s):  
Divalent Cations ◽  
Erwinia Carotovora ◽  
Insertion Mutagenesis ◽  
Bacterial Cells ◽  
Divalent Metal Ions ◽  
Bacterial Genomes ◽  
Dna Transposition ◽  
Bacterial Chromosomes ◽  
Transposition Reaction

ABSTRACT An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 104 to 106 CFU/μg of input transposon DNA in the four species tested, i.e., Escherichia coli, Salmonella enterica serovar Typhimurium, Erwinia carotovora, and Yersinia enterocolitica. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.

scholarly journals CirBiTree: Citrullination Site Inference Based on a Fuzzy Neural Network and Flexible Neural Tree

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Chuandong Song ◽  
Haifeng Wang
Keyword(s):  
Neural Network ◽  
Fuzzy Neural Network ◽  
Classification Model ◽  
Peptide Sequence ◽  
Sequence Information ◽  
Post Translational Modification ◽  
Fuzzy Neural ◽  
Human Complex ◽  
Better Than

Emerging evidence demonstrates that post-translational modification plays an important role in several human complex diseases. Nevertheless, considering the inherent high cost and time consumption of classical and typical in vitro experiments, an increasing attention has been paid to the development of efficient and available computational tools to identify the potential modification sites in the level of protein. In this work, we propose a machine learning-based model called CirBiTree for identification the potential citrullination sites. More specifically, we initially utilize the biprofile Bayesian to extract peptide sequence information. Then, a flexible neural tree and fuzzy neural network are employed as the classification model. Finally, the most available length of identified peptides has been selected in this model. To evaluate the performance of the proposed methods, some state-of-the-art methods have been employed for comparison. The experimental results demonstrate that the proposed method is better than other methods. CirBiTree can achieve 83.07% in sn%, 80.50% in sp, 0.8201 in F1, and 0.6359 in MCC, respectively.

scholarly journals Bacteriophage Mu DNA replication in vitro.

1983 ◽  
Vol 258 (7) ◽  
pp. 4293-4297
Author(s):  
N P Higgins ◽  
D Moncecchi ◽  
P Manlapaz-Ramos ◽  
B M Olivera
Keyword(s):  
Dna Replication ◽  
Bacteriophage Mu

scholarly journals DNA sonification for public engagement in bioinformatics

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Heleen Plaisier ◽  
Thomas R. Meagher ◽  
Daniel Barker
Keyword(s):  
Dna Sequence ◽  
Public Engagement ◽  
Sequence Data ◽  
Sensory Perception ◽  
Data Representation ◽  
Sequence Information ◽  
Dna Sequence Data ◽  
Public Events ◽  
Dna Base ◽  
Alternative Means

Abstract Objective Visualisation methods, primarily color-coded representation of sequence data, have been a predominant means of representation of DNA data. Algorithmic conversion of DNA sequence data to sound—sonification—represents an alternative means of representation that uses a different range of human sensory perception. We propose that sonification has value for public engagement with DNA sequence information because it has potential to be entertaining as well as informative. We conduct preliminary work to explore the potential of DNA sequence sonification in public engagement with bioinformatics. We apply a simple sonification technique for DNA, in which each DNA base is represented by a specific note. Additionally, a beat may be added to indicate codon boundaries or for musical effect. We report a brief analysis from public engagement events we conducted that featured this method of sonification. Results We report on use of DNA sequence sonification at two public events. Sonification has potential in public engagement with bioinformatics, both as a means of data representation and as a means to attract audience to a drop-in stand. We also discuss further directions for research on integration of sonification into bioinformatics public engagement and education.

Purification of Chalcone Synthase from Tulip Anthers and Comparison with the Synthase from Cosmos Petals

1981 ◽  
Vol 36 (1-2) ◽  
pp. 30-34 ◽  
Author(s):  
Rainer Sütfeld ◽  
Rolf Wiermann
Keyword(s):  
Chalcone Synthase ◽  
In Vitro Assay ◽  
Flavonoid Biosynthesis ◽  
Chalcone Isomerase ◽  
Gel Chromatography ◽  
Purification Procedure ◽  
Reaction Products ◽  
Complete Elimination ◽  
Vitro Assay

Abstract Chalcone synthase was isolated from both anthers of Tulipa cv. “Apeldoorn” and petals of Cosmos sulphureus Cav. After certain prepurification steps, the enzymes were further purified using gel chromatography on Sephadex G-200 followed by repeated hydroxylapatite absorption chromatography. Both the enzymes showed the same chromatographic properties. After gel chromatography as well as after the first hydroxylapatite fractionation, the reaction products appeared as flavanones. However, after the second hydroxylapatite step, production of chalcones was observed. Like the enzyme from tulip anthers, the synthase from Cosmos petals produced the correspondingly substituted chalcones when p-coumaroyl-CoA, caffeoyl-CoA and feruloyl-CoA, respectively, were used as substractes. In both the cases, the ratios of the different chalcones produced were found to be about the same. The appearance of chalcone synthesis in this in vitro assay is caused by the complete elimination of chalcone isomerase in the purification procedure. The importance of the isomerase for flavonoid biosynthesis, particularly in plant systems which are accumulating chalcones, is discussed.

scholarly journals Identification and Characterization of Novel Human Endogenous Retrovirus Families by Phylogenetic Screening of the Human Genome Mapping Project Database

2000 ◽  
Vol 74 (8) ◽  
pp. 3715-3730 ◽  
Author(s):  
Michael Tristem
Keyword(s):  
Human Genome ◽  
Genome Mapping ◽  
Sequence Data ◽  
Endogenous Retrovirus ◽  
Endogenous Retroviruses ◽  
Human Endogenous Retrovirus ◽  
Sequence Information ◽  
Class Iii ◽  
Genome Mapping Project ◽  
Human Genome Mapping Project

ABSTRACT Human endogenous retroviruses (HERVs) were first identified almost 20 years ago, and since then numerous families have been described. It has, however, been difficult to obtain a good estimate of both the total number of independently derived families and their relationship to each other as well as to other members of the familyRetroviridae. In this study, I used sequence data derived from over 150 novel HERVs, obtained from the Human Genome Mapping Project database, and a variety of recently identified nonhuman retroviruses to classify the HERVs into 22 independently acquired families. Of these, 17 families were loosely assigned to the class I HERVs, 3 to the class II HERVs and 2 to the class III HERVs. Many of these families have been identified previously, but six are described here for the first time and another four, for which only partial sequence information was previously available, were further characterized. Members of each of the 10 families are defective, and calculation of their integration dates suggested that most of them are likely to have been present within the human lineage since it diverged from the Old World monkeys more than 25 million years ago.

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