Analysis of the Phenotype and the Restriction Enzyme Mapping Level of Mutations Induced by the New Mutagen Glycidyl Methacrylate

Glycidyl methaerylate (GMA) is a recently recognized chemical mutagen. In order to explore the mutagenicity and mutagenic process of GMA, plasmid pBR322 was used for in vitro binding, mutant screening, and restriction enzyme mapping. The binding between GMA and DNA in vitro has been verified by means of a spectrophotometric method. When pBR322 and GMAbound pBR322 were used to transform Eschenchia coli HB101, the following results were obtained: (1) The transformation efficiency of GMA-bound pBR322 was much lower than that of pBR322 alone. (2) GMA-bound pBR322 induced phenotype changes in competent cells (i.e., tetracycline-resistance inactivation or ampicillin-resistance inactivation). There were two mutants of pBR322, Ap~RTc~S and Ap~STc~R, in the transformants and a deductive mutant Ap~STc~S in the nontranstormants. (3) All of the selected mutants were stable and heritable. (4) When restriction enzyme maps were used to analyze the mutant Ap~RTc~S, four of seven maps were changed. some sites were shifted to other resistant gene regions, for example, sites of Bgll, EcoRl, Ilindlll. Hinclll, etc., and there was a new recognition site for Hindi (252). We did not observe any DNA fragment insertion or deletion on any maps. Our results suggest that when GMA is covalently linked to the plasmid DNA, it gives rise to a premutagenic lesion of DNA that is converted in vivo into a point mutation. (C)1990 Academic Press, Inc.

Revolutionizing Non-Invasive Biomarker Discoveries: The Power of Methylation Screening Analysis in Cell-Free DNA Liquid Biopsy

Epigenetic changes of DNA, including methylation, have long been recognized as key indicators of various diseases, including aging, cancer, and neurological disorders. Biomarker discoveries based on distinct methylation patterns for both hypermethylation and hypomethylation lead the way in discovery of novel diagnosis and treatment targets. Many different approaches are present to detect the level of methylation in whole genome (whole genome bisulfite sequencing, microarray) as well as at specific loci (methylation specific PCR). Cell-free DNA (cf-DNA) found in body fluids like blood provides information about DNA methylation and serves as a less invasive approach for genetic screening. Cell-free DNA and methylation screening technologies, when combined, have the potential to transform the way we approach genetic screening and personalized therapy. These technologies can help enhance disease diagnostic accuracy and inform the development of targeted therapeutics by providing a non-invasive way for acquiring genomic information and identifying disease-associated methylation patterns. We highlight the clinical benefits of using cell-free DNA (cf-DNA) liquid biopsy analysis and available methylation screening technologies that have been crucial in identifying biomarkers for disease from patients using a non-invasive way. Powering such biomarker discoveries are various methods of cf-DNA methylation analysis such as Bisulfite Sequencing and most recently, Methylation-Specific Restriction Enzyme (MSRE-seq) Analysis, paving the way for novel epigenetic biomarker discoveries for more robust diagnosis such as early disease detection, prognosis, monitoring of disease progression and treatment response as well as discovery of novel drug targets.

Evaluation of the Method Based on Restriction Fragment Length Polymorphism Analysis as Simple Analysis Method of Lactic Acid Bacteria in Foods

Lactic acid bacteria have not only been used to produce various kinds of fermented food, but also used as probiotic products. As lactic acid bacterial group was consisted from diverse genera, a simple inspection method by which numbers and contained microorganisms could be automatically analyzed without any preliminary information was required to use them more effectively. In this manuscript, lactic acid bacterial groups in commercial products of kimuchi, komekouji-miso, and yoghurt were identified and enumerated by our newly developed method [1]-[3], to evaluate whether the method could be used as an inspection method of various food samples. In kimuchi, numerically dominant bacteria were Lactobacillus sakei, and L. casei (1.4 × 104 MPN g-1) and Leuconostoc spp. (l.4 × 104 MPN). In kouji-miso, numerically dominant bacteria was Bacillus spp. (3 × 103 MPN), which mainly included B. subtilis group and B. cereus group. Lactic acid bacteria such as Lactobacillus spp., or Lactococcus spp., included in the komekouji-miso, could be enumerated after 3 days incubation (1.24 × 104 MPN), but not detected after 7 days incubation. In yoghurt A and C, Lactococcus lactis was detected as numerically dominant lactic acid bacteria (3.0 × 105 MPN). In yoghurt B, Lactobacillus spp., or Lactococcus spp., was detected not only by a culturebased method but also by an unculture-based method, although there was a difference between the both estimated numbers. The present results suggested that the method might become useful as a simple inspection method of food microorganisms, because time and labor of the analysis could be reduced by using an unculture-based method and MCE-202 MultiNA. In this study, Bifidobacteriium spp. was not detected in B and C yoghurt, in spite of indicating their existence, and numbers of lactic acid bacteria were lower than the level of the daily product regulation, because 16S rDNA of Bifidobacteriium spp. might not be amplified by the used PCR condition. The PCR condition must be changed so as to amplify Bifidobacterium spp., before the method will be used as an inspection method for lactic acid bacteria.

Identification and Enumeration Method of Both Eukaryotic and Prokaryotic Microorganisms in Food Sample

The method to analyze both eukaryotic and prokaryotic microorganisms without preliminary microbial information of sample seemed to be useful not only for research and investigation of microorganisms but also for industry using microorganisms. In the present manuscript, preparation of a new DNA primers, new reference database for 18S rDNA for our newly developed method [1]- [3], and analyses of eukaryotic and prokaryotic microorganisms in fermentation products were presented. In komekouji, Aspergillus spp., was enumerated to be 46.5 × 106 MPN g-1, and Penicillium spp., was enumerated to be 1.5 × 106 MPN g-1. In dry yeast, Saccharomyces group, were enumerated to be 8600 × 106 MPN g-1. In komekouji-miso, no eukaryotic microorganism was detected, while the other Bacillus spp., was numerically dominant (21.5 × 106 MPN g-1) as prokaryotic microorganisms, followed by B. subtilis group (4.65 × 106 MPN g-1), and the other Firmicutes (3.7 × 106 MPN g-1). The komekouji-miso included lower number of Actinobacteria (0.15 × 106 MPN g-1), Burkhokderia sp. (1.5 × 106 MPN g-1), and the other α,β,γ-proteobacteria (0.12 × 106 MPN g-1). In sake-kasu, both prokaryote and eukaryote were not detected by the method. Present results indicated that using both universal primers for eukaryotic and prokaryotic microorganisms, each groups of prokaryotic and eukaryotic microorganisms were enumerated without any preliminary information nor setting up standard curve, which were required for real time PCR.

A Simple Evaluation System for Microbial Property in Soil and Manure

Analyses of microbial properties in soil and manure had always included the problem that there was no available standard method to evaluate microbial property. The one of the major problems was the vast diversity and the enormous population of soil microorganisms [1], the other was an existence of numerically dominant unculturable microorganisms which comprise 99% of soil habitat [2]. We evaluated whether our newly developed method, by which taxonomies and their number of each bacterial groups were estimated, could be used as evaluation method of microbial properties of soils and manures. In the forest soil, β-Proteobacteria, which included Burkholderia sp., Ralstonia sp., and Alcaligenes sp., was numerically dominant bacteria (3.64 × 106 MPN g-1 dry soil), followed by γ-Proteobacteria (1.32 × 106 MPN), δ-Proteobacteria (0.006 × 106 MPN), and the other gram negative bacteria (0.006 × 106 MPN). In the commercial manure, Actinobacteria, which included Streptoverticillium salmonis, Mycrococcus sp., Streptomyces bikiniensis, and Microbacterium ulmi, was numerically dominant bacterial group (30.8 × 106 MPN), followed by α-Proteobacteria (26.0 × 106 MPN), β-Proteobacteria (17.1 × 106 MPN), δ-Proteobacteria (11.2 × 106 MPN), the other Firmicutes (1.71 × 106 MPN), γ-Proteobacteria (0.5 × 106 MPN), and the other gram negative bacteria (0.05 × 106 MPN). In the upland field, the other Firmicutes, which included Paenibacillus sp., was numerically dominant bacteria (4.41 × 106 MPN), followed by Actinobacteria (2.14 × 106 MPN), Bacillus sp. (2.14 × 106 MPN), and γ-Proteobacteria (0.35 × 106 MPN). Although the precision of the affiliations became lower because of higher diversity of samples and the number of some Antinobacteria and Firmicutes might be underestimated by the used PCR condition, the method was found suitable as a candidate of a new evaluation system of soil and manure.

A New Evaluation Method for Antibiotic-Resistant Bacterial Groups in Environment

In the present manuscript it was presented whether spreading of antibiotic resistant bacterial groups in environment could be monitored by our newly developed method by enumerating antibiotic resistant bacterial groups in various biological wastes and composts. Although the numbers were not so high, diverse kinds of colistin resistant bacteria (25 mg·L-1) were included in row cattle feces (1.78 × 104 MPN g-1) and cattle feces manure (>3.84 × 104 MPN g-1). Compost originated from leftover food (>44.8 × 104 MPN g-1) and shochu lee (>320 × 104 MPN g-1) included higher numbers of chlortetracycline resistant Pseudomonas sp., (25 mg·L-1), and row cattle feces included higher numbers of chlortetracycline resistant Enterobacteriacea (15.7 × 104 MPN g-1), which mostly consisted from Pantoea sp. or Xenorhobdus doucetiae. Numbers of multi drug resistant bacteria, resistant to 25 mg·L-1 of ciprofloxacin, streptomycin, chloramphenicol, and ampicillin, were the highest in row cattle feces (>143.6 × 104 MPN g-1), followed by cattle feces manure (4.19 × 104 MPN g-1), and shochu lee (0.36 × 104 MPN g-1), which included diverse kinds of bacterial group. The present results indicated that higher numbers of multi drug resistant bacteria were typically found in row cattle feces, and the method was found suitable to enumerate and identify them. These results suggested that the method might become their environmental risk evaluation method.

Pitfalls of restriction enzyme analysis in identifying, characterizing,typing, and naming viral pathogens in the era of whole genome data, as illustrated by HAdV type 55

Restriction endonuclease analysis(REA),or restriction fragment length polymorphism(RFLP),was useful for identifying and determining the relatedness and putative identities of microbial strains(Tang et al.,1997)and for characterizing and discriminating large numbers of samples inexpensively in the past。

Point mutations of muscle mitochondrial DNA from patients with mitochondrial encephalomyopathies

Objective To study the relation between point mutations at nt3243 and nt8344 of muscle mitochondrial DNA from patients with mitochondrial encephalomyopathies and phenotypes. Methods DNA was extracted from muscle specimens from 5 patients with mitochondrial encephalomyopathies and amplified by PCR method, using corresponding oligonucleotide primers. DNA fragments were digested with restriction enzymes BglⅠ and ApaⅠ, then the digested DNA fragments were analyzed with an electrophoresis method.Results The point mutation at nt3243 of mtDNA was found in 2 patients, one with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) and another with myoclonic epilepsy with ragged red fibers (MERRF). The point mutation at nt8344 was found in 2 patients with MERRF, including the one with point mutation at nt3243.Conclusion The point mutation of DNA at nt3243 correlated with MELAS and nt8344 correlated with MERRF. In addition, the detection of point mutations at both nt3243 and nt8344 in a patient with MERRF shows the association of mutation with diversity in clinical manifestations of mitochondrial encephalomyopathies.