Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

Regulation of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> body size and male tail development by the novel gene <Emphasis Type="Italic">lon-8</Emphasis>

Background  

In C. elegans and other nematode species, body size is determined by the composition of the extracellular cuticle as well as by the nuclear DNA content of the underlying hypodermis. Mutants that are defective in these processes can exhibit either a short or a long body size phenotype. Several mutations that give a long body size (Lon) phenotype have been characterized and found to be regulated by the DBL-1/TGF-β pathway, that controls post-embryonic growth and male tail development.     

RNA mutagenesis yields highly diverse mRNA libraries for <Emphasis Type="Italic">in vitro</Emphasis>protein evolution

Background  

In protein drug development, in vitro molecular optimization or protein maturation can be used to modify protein properties. One basic approach to protein maturation is the introduction of random DNA mutations into the target gene sequence to produce a library of variants that can be screened for the preferred protein properties. Unfortunately, the capability of this approach has been restricted by deficiencies in the methods currently available for random DNA mutagenesis and library generation. Current DNA based methodologies generally suffer from nucleotide substitution bias that preferentially mutate particular base pairs or show significant bias with respect to transitions or transversions. In this report, we describe a novel RNA-based random mutagenesis strategy that utilizes Qβ replicase to manufacture complex mRNA libraries with a mutational spectrum that is close to the ideal.     

A Combined test using both cell sediment and supernatant cell‐free DNA in pleural effusion shows increased sensitivity in detecting activating EGFR mutation in lung cancer patients

Introduction

The aim of this study was to examine whether a combined test using both cell sediment and supernatant cytology cell‐free DNA (ccfDNA) is more useful in detecting EGFR mutation than using cell sediment DNA or supernatant ccfDNA alone in pleural effusion of lung cancer patients.

Methods

A total of 74 lung adenocarcinoma patients with paired samples between primary tumour and corresponding metastatic tumour with both cell sediment and supernatant ccfDNA of pleural effusion cytology were enrolled in this study. Cell sediment and supernatant ccfDNA were analysed separately for EGFR mutations by polymerase chain reaction.

Results

Out of 45 patients with mutant EGFR in primary tumours, EGFR mutations were detected in 23 cell sediments of corresponding metastases (sensitivity; 51.1%) and 20 supernatant ccfDNA corresponding metastases (sensitivity; 44.4%). By contrast, the combined test detected EGFR mutations in 27 corresponding metastases (sensitivity; 60.0%), and had a higher sensitivity than the cell sediment or the supernatant ccfDNA alone (P < .05). Out of 45 patients with mutant EGFR, 24, three and 18 were cytologically diagnosed as positive, atypical or negative, respectively. The detection rate in the combined test was highest (95.8%) in the positive group, and mutant EGFR was also detected in four of 18 samples (22.2%) in the negative group.

Conclusions

A combined test using both cell sediment DNA and supernatant ccfDNA samples increases the concordance rate of EGFR mutations between primary tumour and corresponding metastases. Our findings indicate that supernatant ccfDNA is useful even in cases where the cytological diagnosis is negative.     

Molecular and biochemical characterisation of a novel mutation in <Emphasis Type="Italic">POLG</Emphasis>associated with Alpers syndrome

Background  

DNA polymerase γ (POLG) is the only known mitochondrial DNA (mtDNA) polymerase. It mediates mtDNA replication and base excision repair. Mutations in the POLG gene lead to reduction of functional mtDNA (mtDNA depletion and/or deletions) and are therefore predicted to result in defective oxidative phosphorylation (OXPHOS). Many mutations map to the polymerase and exonuclease domains of the enzyme and produce a broad clinical spectrum. The most frequent mutation p.A467T is localised in the linker region between these domains. In compound heterozygote patients the p.A467T mutation has been described to be associated amongst others with fatal childhood encephalopathy. These patients have a poorer survival rate compared to homozygotes.     

Genome-wide target profiling of <Emphasis Type="Italic">piggyBac</Emphasis> and <Emphasis Type="Italic">Tol2</Emphasis>in HEK 293: pros and cons for gene discovery and gene therapy

Background  

DNA transposons have emerged as indispensible tools for manipulating vertebrate genomes with applications ranging from insertional mutagenesis and transgenesis to gene therapy. To fully explore the potential of two highly active DNA transposons, piggyBac and Tol2, as mammalian genetic tools, we have conducted a side-by-side comparison of the two transposon systems in the same setting to evaluate their advantages and disadvantages for use in gene therapy and gene discovery.     

Evaluation of the <Emphasis Type="Italic">OPTC</Emphasis> gene in primary open angle glaucoma: functional significance of a silent change

Background  

We investigated the molecular basis of primary open-angle glaucoma (POAG) using Opticin (OPTC) as a candidate gene on the basis of its expression in the trabecular meshwork cells involved in the disease pathogenesis. Two hundred POAG patients and 100 controls were enrolled in this study. The coding sequence of OPTC was amplified by PCR from genomic DNA of POAG patients, followed by SSCP, DHPLC and DNA sequencing. Subsequent bioinformatic analysis, site-directed mutagenesis, quantitative RT-PCR and western blot experiments were performed to address the functional significance of a 'silent' change in the OPTC coding region while screening for mutations in POAG patients.     

A simplified,robust, and streamlined procedure for the production of <Emphasis Type="Italic">C. elegans</Emphasis>transgenes via recombineering

Background  

The nematode Caenorhabditis elegans has emerged as a powerful system to study biologic questions ranging from development to aging. The generation of transgenic animals is an important experimental tool and allows use of GFP fusion proteins to study the expression of genes of interest or generation of epitope tagged versions of specific genes. Transgenes are often generated by placing a promoter upstream of a reporter gene or cDNA. This often produces a representative expression pattern, but important exceptions have been observed. To better capture the genuine expression pattern and timing, several investigators have modified large pieces of DNA carried by BACs or fosmids for use in the construction of transgenic animals via recombineering. However, these techniques are not in widespread use despite the advantages when compared to traditional approaches. Additionally, some groups have encountered problems with employing these techniques. Hence, we sought identify ways to improve the simplicity and reliability of the procedure.     

In vitro selection of resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella</Emphasis> spp. at <Emphasis Type="Italic">in vivo</Emphasis> fluoroquinolone concentrations

Background  

Fluoroquinolones are potent antimicrobial agents used for the treatment of a wide variety of community- and nosocomial- infections. However, resistance to fluoroquinolones in Enterobacteriaceae is increasingly reported. Studies assessing the ability of fluoroquinolones to select for resistance have often used antimicrobial concentrations quite different from those actually acquired at the site of infection. The present study compared the ability to select for resistance of levofloxacin, ciprofloxacin and prulifloxacin at concentrations observed in vivo in twenty strains of Escherichia coli and Klebsiella spp. isolated from patients with respiratory and urinary infections. The frequencies of spontaneous single-step mutations at plasma peak and trough antibiotic concentrations were calculated. Multi-step selection of resistance was evaluated by performing 10 serial cultures on agar plates containing a linear gradient from trough to peak antimicrobial concentrations, followed by 10 subcultures on antibiotic-free agar. E. coli resistant strains selected after multi-step selection were characterized for DNA mutations by sequencing gyrA, gyrB, parC and parE genes.     

Phenotypic instability of <Emphasis Type="Italic">Arabidopsis</Emphasis> alleles affecting a disease <Emphasis Type="Italic">Resistance</Emphasis>gene cluster

Background  

Three mutations in Arabidopsis thaliana strain Columbia – cpr1, snc1, and bal – map to the RPP5 locus, which contains a cluster of disease Resistance genes. The similar phenotypes, gene expression patterns, and genetic interactions observed in these mutants are related to constitutive activation of pathogen defense signaling. However, these mutant alleles respond differently to various conditions. Exposure to mutagens, such as ethyl methanesulfonate (EMS) and γ-irradiation, induce high frequency phenotypic instability of the bal allele. In addition, a fraction of the bal and cpr1 alleles segregated from bal × cpr1 F1 hybrids also show signs of phenotypic instability. To gain more insight into the mechanism of phenotypic instability of the bal and cpr1 mutations, we systematically compared the behavior of these unusual alleles with that of the missense gain-of-function snc1 allele in response to DNA damage or passage through F1 hybrids.     

Remobilization of <Emphasis Type="Italic">Tol2</Emphasis> transposons in <Emphasis Type="Italic">Xenopus tropicalis</Emphasis>

Background  

The Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring Tol2 element from Oryzias latipes efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, Xenopus tropicalis. Tol2 transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency.     

A strong constitutive ethylene-response phenotype conferred on Arabidopsis plants containing null mutations in the ethylene receptors <Emphasis Type="Italic">ETR1</Emphasis> and <Emphasis Type="Italic">ERS1</Emphasis>

Background  

The ethylene receptor family of Arabidopsis consists of five members, falling into two subfamilies. Subfamily 1 is composed of ETR1 and ERS1, and subfamily 2 is composed of ETR2, ERS2, and EIN4. Although mutations have been isolated in the genes encoding all five family members, the only previous insertion allele of ERS1 (ers1-2) is a partial loss-of-function mutation based on our analysis. The purpose of this study was to determine the extent of signaling mediated by subfamily-1 ethylene receptors through isolation and characterization of null mutations.     

Low density DNA microarray for detection of most frequent <Emphasis Type="Italic">TP53</Emphasis> missense point mutations

Background  

We have developed an oligonucleotide microarray (genosensor) utilizing a double tandem hybridization technique to search for 9 point mutations located in the most frequently altered codons of the TP53 gene. Isolated and multiplexed PCR products, 108 and 92 bp long, from exons 7 and 8, respectively, were obtained from 24 different samples. Single-stranded target DNA was then prepared from isolated or multiplexed PCR products, through cyclic DNA synthesis. Independent ssDNA's were annealed with the corresponding pairs of labeled stacking oligonucleotides to create partially duplex DNA having a 7-nt gap, which contains the sequence that will be interrogated by the capture probes forming double tandem hybridization. In the case of multiplexed ssPCR products, only two stacking oligonucleotides were added per target, therefore the gap for the PCR products having two consecutive codons to be interrogated in exon 7 was 12 nt long, so only single tandem hybridization was produced with these respective probes.     

Identification of ochratoxin A producing Aspergillus carbonarius and A. niger clade isolated from grapes using the loop‐mediated isothermal amplification (LAMP) reaction

Aims

To develop two assays based on the loop‐mediated isothermal amplification (LAMP) of DNA for the quick and specific identification of Aspergillus carbonarius and ochratoxigenic strains of the Aspergillus niger clade isolated from grapes.

Methods and Results

Two sets of primers were designed based on the polyketide synthase genes involved or putatively involved in ochratoxin A (OTA) biosynthesis in A. carbonarius and A. niger clade. Hydroxynaphthol blue was used as indirect method to indicate DNA amplification. The limit of detection of both assays was comparable to that of a PCR reaction. Specificities of the reactions were tested using DNA from different black aspergilli isolated from grapes. The two LAMP assays were then used to identify A. carbonarius and ochratoxigenic A. niger and A. awamori grown in pure cultures without a prior DNA extraction.

Conclusions

The two LAMP assays permitted to quickly and specifically identify DNA from OTA‐producing black aspergilli, as well as isolates grown in pure culture.

Significance and Impact of the Study

Monitoring vineyards for the presence of OTA‐producing strains is part of the measures to minimize the occurrence of OTA in grape products. The two LAMP assays developed here could be potentially used to speed the screening process of vineyards for the presence of OTA‐producing black aspergilli.     

Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Background  

Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of γ-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair.     

Rapid identification and mapping of insertion sequences in <Emphasis Type="Italic">Escherichia coli</Emphasis> genomes using vectorette PCR

Background  

Insertion sequences (IS) are small DNA segments capable of transposing within and between prokaryotic genomes, often causing insertional mutations and chromosomal rearrangements. Although several methods are available for locating ISs in microbial genomes, they are either labor-intensive or inefficient. Here, we use vectorette PCR to identify and map the genomic positions of the eight insertion sequences (IS1, 2, 3, 4, 5, 30, 150, and 186) found in E. coli strain CGSC6300, a close relative of MG1655 whose genome has been sequenced.     

Efficient assembly of <Emphasis Type="Italic">de novo</Emphasis>human artificial chromosomes from large genomic loci

Background  

Human Artificial Chromosomes (HACs) are potentially useful vectors for gene transfer studies and for functional annotation of the genome because of their suitability for cloning, manipulating and transferring large segments of the genome. However, development of HACs for the transfer of large genomic loci into mammalian cells has been limited by difficulties in manipulating high-molecular weight DNA, as well as by the low overall frequencies of de novo HAC formation. Indeed, to date, only a small number of large (>100 kb) genomic loci have been reported to be successfully packaged into de novo HACs.     

Detection of excretory <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> DNA in the urine,and detection of <Emphasis Type="Italic">E. histolytica</Emphasis> DNA and lectin antigen in the liver abscess pus for the diagnosis of amoebic liver abscess

Background  

Amoebic liver abscess (ALA) and pyogenic liver abscesses (PLA) appear identical by ultrasound and other imaging techniques. Collection of blood or liver abscess pus for diagnosis of liver abscesses is an invasive procedure, and the procedure requires technical expertise and disposable syringes. Collection of urine is a noninvasive procedure. Therefore, there has been much interest shown towards the use of urine as an alternative clinical specimen for the diagnosis of some parasitic infections. Here, we report for the first time the detection of E. histolytica DNA excreted in the urine for diagnosis of the cases of ALA.     

Genomic organization of Tropomodulins 2 and 4 and unusual intergenic and intraexonic splicing of YL-1 and Tropomodulin 4

Background  

The tropomodulins (TMODs) are a family of proteins that cap the pointed ends of actin filaments. Four TMODs have been identified in humans, with orthologs in mice. Mutations in actin or actin-binding proteins have been found to cause several human diseases, ranging from hypertrophic cardiomyopathy to immunodefiencies such as Wiskott-Aldrich syndrome. We had previously mapped Tropomodulin 2 (TMOD2) to the genomic region containing the gene for amyotrophic lateral sclerosis 5 (ALS5). We determined the genomic structure of Tmod2 in order to better analyze patient DNA for mutations; we also determined the genomic structure of Tropomodulin 4 (TMOD4).     

A non-sense mutation in the putative anti-mutator gene <Emphasis Type="Italic">ada/alkA</Emphasis> of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> and <Emphasis Type="Italic">M. bovis</Emphasis> isolates suggests convergent evolution

Background  

Previous studies have suggested that variations in DNA repair genes of W-Beijing strains may have led to transient mutator phenotypes which in turn may have contributed to host adaptation of this strain family. Single nucleotide polymorphism (SNP) in the DNA repair gene mutT1 was identified in MDR-prone strains from the Central African Republic. A Mycobacteriumtuberculosis H37Rv mutant inactivated in two DNA repair genes, namely ada/alkA and ogt, was shown to display a hypermutator phenotype. We then looked for polymorphisms in these genes in Central African Republic strains (CAR).