Chromosomal inversion between rrn operons among Streptococcus mutans serotype c oral and blood isolates

Streptococcus mutans causes dental caries and infective endocarditis. The aim of this study was to determine genomic diversity among serotype c S. mutans laboratory and clinical strains and to characterize the genetic events involved. A genome-based approach using PFGE coupled with Southern hybridization was employed to examine a total of 58 serotype c oral and blood isolates and seven laboratory strains and to compare them with S. mutans UA159. No significant differences were found in the phenotypic characteristics of the strains tested, except that some of the strains exhibited smooth rather than rough colony morphology. In contrast, PFGE profiles of clinical isolates, from either diseased or healthy subjects, exhibited diverse patterns, suggesting that recombination or point mutations occurred frequently in vivo. Diverse PFGE patterns, with various lengths of insertions and deletions, could be detected even within a localized chromosomal region between rRNA operons. Comparative analysis using Southern hybridization with specific markers revealed that a large chromosomal inversion had also occurred between rrn operons in 25 strains.

Download Full-text

Tight Genetic Linkage of a Glucosyltransferase and Dextranase of Streptococcus mutans GS-5

Journal of Dental Research ◽

10.1177/00220345860650120301 ◽

1986 ◽

Vol 65 (12) ◽

pp. 1392-1401 ◽

Cited By ~ 14

Author(s):

R.A. Burne ◽

B. Rubinfeld ◽

W.H. Bowen ◽

R.E. Yasbin

Keyword(s):

Streptococcus Mutans ◽

Genetic Linkage ◽

Southern Hybridization ◽

Plasmid Vector ◽

Average Insert Size ◽

Insert Size ◽

Ph Optimum ◽

Detectable Activity

A genetic library consisting of over 5000 clones with an average insert size of 6.9 kilobasepairs (kbp) of Streptococcus mutans GS-5 has been constructed in a bivalent plasmid vector pMK3, which is capable of replicating in Escherichia coli and Bacillus subtilis. The recombinant plasmid pSUCRI, containing a 6.0 kbp fragment of S. mutans GS-5 DNA, was the focus of this study. Using Southern hybridization, in vitro and in vivo gene expression techniques, and biochemical analysis, this clone was shown to encode the 55 kiloDalton (kDal) GS-5 gtfA gene product, as well as a 38 and a 66 kDal polypeptide. In addition to the gtfA gene, pSUCRI encodes a dextranase activity with specificity for α(1→6)-linked glucans, and with no detectable activity on mutan. The dextranase enzyme had an apparent molecular weight of 66 kDal as demonstrated by SDS-PAGE analysis of the proteins produced by a dextranase-negative deletion derivative. The pH optimum of the enzyme was approximately 6.0, and there was no detectable activity below pH 5.0. By subcloning various combinations of DNA fragments from pSUCRI, it was demonstrated that the dextranase gene (designated dexB) can be separated from the gtfA gene and still be efficiently expressed in both E. coli and B. subtilis. The dexB gene contained its own promoter and ribosome-binding site. The genetic linkage of the gtfA and dexB genes in the S. mutans GS-5 chromosome was confirmed by Southern hybridization and by the independent isolation of four distinct clones containing the gtfA gene and common flanking sequences. In addition to a glucosyltransferase and dextranase, an invertase-like activity is also encoded on pSUCRI, indicating that there is a cluster of genes on the S. mutans GS-5 chromosome which is devoted to the dissimilation of sucrose and concomitant synthesis or modification of glucans into a water-insoluble form, perhaps constituting an operon for glucan modification which can be coordinately regulated in response to environmental alterations.

Download Full-text

Genetic Diversity and Identification of Homozygosity-Rich Genomic Regions in Seven Italian Heritage Turkey (Meleagris gallopavo) Breeds

Genes ◽

10.3390/genes12091342 ◽

2021 ◽

Vol 12 (9) ◽

pp. 1342

Author(s):

Francesca Bernini ◽

Alessandro Bagnato ◽

Stefano Paolo Marelli ◽

Luisa Zaniboni ◽

Silvia Cerolini ◽

...

Keyword(s):

Genetic Variation ◽

Target Selection ◽

Meleagris Gallopavo ◽

Principal Component ◽

Genomic Diversity ◽

Effective Population ◽

A Genome ◽

Founder Populations ◽

Genomic Regions

Italian autochthonous turkey breeds are an important reservoir of genetic biodiversity that should be maintained with an in vivo approach. The aim of this study, part of the TuBAvI national project on biodiversity, was to use run of homozygosity (ROH), together with others statistical approaches (e.g., Wright’s F-statistics, principal component analysis, ADMIXTURE analysis), to investigate the genomic diversity in several heritage turkey breeds. We performed a genome-wide characterization of ROH-rich regions in seven autochthonous turkey breeds, i.e., Brianzolo (Brzl), Bronzato Comune Italiano (BrCI), Bronzato dei Colli Euganei (CoEu), Parma e Piacenza (PrPc), Nero d’Italia (NeIt), Ermellinato di Rovigo (ErRo) and Romagnolo (Roma). ROHs were detected based on a 650K SNP genotyping. ROH_islands were identified as homozygous ROH regions shared by at least 75% of birds (within breed). Annotation of genes was performed with DAVID. The admixture analyses revealed that six breeds are unique populations while the Roma breed consists in an admixture of founder populations. Effective population size estimated on genomic data shows a numeric contraction. ROH_islands harbour genes that may be interesting for target selection in commercial populations also. Among them the PTGS2 and PLA2G4A genes on chr10 were related to reproduction efficiency. This is the first study mapping genetic variation in autochthonous turkey populations. Breeds were genetically different among them, with the Roma breed proving to be a mixture of the other breeds. The ROH_islands identified harboured genes peculiar to the selection that occurred in heritage breeds. Finally, this study releases previously undisclosed information on existing genetic variation in the turkey species.

Download Full-text

Global Analysis of Furfural-Induced Genomic Instability Using a Yeast Model

Applied and Environmental Microbiology ◽

10.1128/aem.01237-19 ◽

2019 ◽

Vol 85 (18) ◽

Cited By ~ 2

Author(s):

Lei Qi ◽

Ke Zhang ◽

Yu-Ting Wang ◽

Jian-Kun Wu ◽

Yang Sui ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Global Analysis ◽

Chromosomal Rearrangements ◽

Point Mutations ◽

Mitotic Recombination ◽

Whole Genome ◽

Wild Type ◽

Content Type ◽

A Genome

ABSTRACT Furfural is an important renewable precursor for multiple commercial chemicals and fuels; a main inhibitor existing in cellulosic hydrolysate, which is used for bioethanol fermentation; and a potential carcinogen, as well. Using a genetic system in Saccharomyces cerevisiae that allows detection of crossover events, we observed that the frequency of mitotic recombination was elevated by 1.5- to 40-fold when cells were treated with 0.1 g/liter to 20 g/liter furfural. Analysis of the gene conversion tracts associated with crossover events suggested that most furfural-induced recombination resulted from repair of DNA double-strand breaks (DSBs) that occurred in the G1 phase. Furfural was incapable of breaking DNA directly in vitro but could trigger DSBs in vivo related to reactive oxygen species accumulation. By whole-genome single nucleotide polymorphism (SNP) microarray and sequencing, furfural-induced genomic alterations that range from single base substitutions, loss of heterozygosity, and chromosomal rearrangements to aneuploidy were explored. At the whole-genome level, furfural-induced events were evenly distributed across 16 chromosomes but were enriched in high-GC-content regions. Point mutations, particularly the C-to-T/G-to-A transitions, were significantly elevated in furfural-treated cells compared to wild-type cells. This study provided multiple novel insights into the global effects of furfural on genomic stability. IMPORTANCE Whether and how furfural affects genome integrity have not been clarified. Using a Saccharomyces cerevisiae model, we found that furfural exposure leads to in vivo DSBs and elevation in mitotic recombination by orders of magnitude. Gross chromosomal rearrangements and aneuploidy events also occurred at a higher frequency in furfural-treated cells. In a genome-wide analysis, we show that the patterns of mitotic recombination and point mutations differed dramatically in furfural-treated cells and wild-type cells.

Download Full-text

Genome Plasticity among RelatedLactococcus Strains: Identification of Genetic Events Associated with Macrorestriction Polymorphisms

Journal of Bacteriology ◽

10.1128/jb.182.9.2481-2491.2000 ◽

2000 ◽

Vol 182 (9) ◽

pp. 2481-2491 ◽

Cited By ~ 30

Author(s):

Pascal Le Bourgeois ◽

Marie-Line Daveran-Mingot ◽

Paul Ritzenthaler

Keyword(s):

Physical Map ◽

Genomic Diversity ◽

Enzyme Analysis ◽

Genetic Lineage ◽

Length Polymorphisms ◽

A Genome ◽

Genome Map ◽

High Degree ◽

Oligopeptide Transport System ◽

Genetic Events

ABSTRACT The genomic diversity of nine strains of the Lactococcus lactis subsp. cremoris (NCDO712, NCDO505, NCDO2031, NCDO763, MMS36, C2, LM0230, LM2301, and MG1363) was studied by macrorestriction enzyme analysis using pulsed-field gel electrophoresis. These strains were considered adequate for the investigation of genomic plasticity because they have been described as belonging to the same genetic lineage. Comparison of ApaI and SmaI genome fingerprints of each strain revealed the presence of several macrorestriction fragment length polymorphisms (RFLPs), despite a high degree of similarity of the generated restriction patterns. The physical map of the MG1363 chromosome was used to establish a genome map of the other strains and allocate the RFLPs to five regions. Southern hybridization analysis correlated the polymorphic regions with genetic events such as chromosomal inversion, integration of prophage DNA, and location of the transposon-like structures carrying conjugative factor or oligopeptide transport system.

Download Full-text

Compensatory mechanisms in resistant Anopheles gambiae AcerKis and KdrKis neurons modulate insecticide-based mosquito control

Communications Biology ◽

10.1038/s42003-021-02192-0 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Stéphane Perrier ◽

Eléonore Moreau ◽

Caroline Deshayes ◽

Marine El-Adouzi ◽

Delphine Goven ◽

...

Keyword(s):

Sodium Channel ◽

Anopheles Gambiae ◽

Calcium Concentration ◽

Mosquito Control ◽

Point Mutations ◽

Resting Membrane Potential ◽

Compensatory Mechanisms ◽

Pyrethroid Insecticides

AbstractIn the malaria vector Anopheles gambiae, two point mutations in the acetylcholinesterase (ace-1R) and the sodium channel (kdrR) genes confer resistance to organophosphate/carbamate and pyrethroid insecticides, respectively. The mechanisms of compensation that recover the functional alterations associated with these mutations and their role in the modulation of insecticide efficacy are unknown. Using multidisciplinary approaches adapted to neurons isolated from resistant Anopheles gambiae AcerKis and KdrKis strains together with larval bioassays, we demonstrate that nAChRs, and the intracellular calcium concentration represent the key components of an adaptation strategy ensuring neuronal functions maintenance. In AcerKis neurons, the increased effect of acetylcholine related to the reduced acetylcholinesterase activity is compensated by expressing higher density of nAChRs permeable to calcium. In KdrKis neurons, changes in the biophysical properties of the L1014F mutant sodium channel, leading to enhance overlap between activation and inactivation relationships, diminish the resting membrane potential and reduce the fraction of calcium channels available involved in acetylcholine release. Together with the lower intracellular basal calcium concentration observed, these factors increase nAChRs sensitivity to maintain the effect of low concentration of acetylcholine. These results explain the opposite effects of the insecticide clothianidin observed in AcerKis and KdrKis neurons in vitro and in vivo.

Download Full-text

Characterization of LDD-2633 as a Novel RET Kinase Inhibitor with Anti-Tumor Effects in Thyroid Cancer

Pharmaceuticals ◽

10.3390/ph14010038 ◽

2021 ◽

Vol 14 (1) ◽

pp. 38

Author(s):

Hyo Jeong Lee ◽

Pyeonghwa Jeong ◽

Yeongyu Moon ◽

Jungil Choi ◽

Jeong Doo Heo ◽

...

Keyword(s):

Thyroid Cancer ◽

Thyroid Carcinoma ◽

Kinase Inhibitor ◽

Point Mutations ◽

Carcinoma Cells ◽

Medullary Thyroid ◽

Potent Inhibition ◽

Kinase Inhibitory Activity

Rearranged during transfection (RET), a receptor tyrosine kinase, is activated by glial cell line-derived neurotrophic factor family ligands. Chromosomal rearrangement or point mutations in RET are observed in patients with papillary thyroid and medullary thyroid carcinomas. Oncogenic alteration of RET results in constitutive activation of RET activity. Therefore, inhibiting RET activity has become a target in thyroid cancer therapy. Here, the anti-tumor activity of a novel RET inhibitor was characterized in medullary thyroid carcinoma cells. The indirubin derivative LDD-2633 was tested for RET kinase inhibitory activity. In vitro, LDD-2633 showed potent inhibition of RET kinase activity, with an IC50 of 4.42 nM. The growth of TT thyroid carcinoma cells harboring an RET mutation was suppressed by LDD-2633 treatment via the proliferation suppression and the induction of apoptosis. The effects of LDD-2633 on the RET signaling pathway were examined; LDD-2633 inhibited the phosphorylation of the RET protein and the downstream molecules Shc and ERK1/2. Oral administration of 20 or 40 mg/kg of LDD-2633 induced dose-dependent suppression of TT cell xenograft tumor growth. The in vivo and in vitro experimental results supported the potential use of LDD-2633 as an anticancer drug for thyroid cancers.

Download Full-text

Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL

International Journal of Molecular Sciences ◽

10.3390/ijms22010434 ◽

2021 ◽

Vol 22 (1) ◽

pp. 434

Author(s):

Yuria Jang ◽

Hong Moon Sohn ◽

Young Jong Ko ◽

Hoon Hyun ◽

Wonbong Lim

Keyword(s):

Nuclear Translocation ◽

Critical Role ◽

Osteoclast Differentiation ◽

Point Mutations ◽

Tartrate Resistant Acid Phosphatase ◽

Mice Model ◽

Gsk 3Β ◽

Rank Signaling

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK–RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3β phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.

Download Full-text

Identification of Resistance Determinants for a Promising Antileishmanial Oxaborole Series

Microorganisms ◽

10.3390/microorganisms9071408 ◽

2021 ◽

Vol 9 (7) ◽

pp. 1408

Author(s):

Magali Van den Kerkhof ◽

Philippe Leprohon ◽

Dorien Mabille ◽

Sarah Hendrickx ◽

Lindsay B. Tulloch ◽

...

Keyword(s):

Drug Exposure ◽

In Vitro Selection ◽

Selection Procedure ◽

Cosmid Library ◽

Neglected Diseases ◽

Chromosome 2 ◽

Resistance Determinants ◽

A Genome

Current treatment options for visceral leishmaniasis have several drawbacks, and clinicians are confronted with an increasing number of treatment failures. To overcome this, the Drugs for Neglected Diseases initiative (DNDi) has invested in the development of novel antileishmanial leads, including a very promising class of oxaboroles. The mode of action/resistance of this series to Leishmania is still unknown and may be important for its further development and implementation. Repeated in vivo drug exposure and an in vitro selection procedure on both extracellular promastigote and intracellular amastigote stages were both unable to select for resistance. The use of specific inhibitors for ABC-transporters could not demonstrate the putative involvement of efflux pumps. Selection experiments and inhibitor studies, therefore, suggest that resistance to oxaboroles may not emerge readily in the field. The selection of a genome-wide cosmid library coupled to next-generation sequencing (Cos-seq) was used to identify resistance determinants and putative targets. This resulted in the identification of a highly enriched cosmid, harboring genes of chromosome 2 that confer a subtly increased resistance to the oxaboroles tested. Moderately enriched cosmids encompassing a region of chromosome 34 contained the cleavage and polyadenylation specificity factor (cpsf) gene, encoding the molecular target of several related benzoxaboroles in other organisms.

Download Full-text

New homozygous gpt delta transgenic rat strain improves an efficiency of the in vivo mutagenicity assay

Genes and Environment ◽

10.1186/s41021-021-00195-1 ◽

2021 ◽

Vol 43 (1) ◽

Author(s):

Kenichi Masumura ◽

Tomoko Ando ◽

Akiko Ukai ◽

Sho Fujiwara ◽

Shigeo Yokose ◽

...

Keyword(s):

Bone Marrow ◽

Gene Mutation ◽

Point Mutations ◽

Chromosome 1 ◽

Transgenic Rat ◽

Target Tissue ◽

Sequencing Analysis ◽

Rat Strain ◽

Packaging Efficiency

Abstract Background Gene mutation assays in transgenic rodents are useful tools to investigate in vivo mutagenicity in a target tissue. Using a lambda EG10 transgene containing reporter genes, gpt delta transgenic mice and rats have been developed to detect point mutations and deletions. The transgene is integrated in the genome and can be rescued through an in vitro packaging reaction. However, the packaging efficiency is lower in gpt delta rats than in mice, because of the transgene in gpt delta rats being heterozygous and in low copy number. To improve the packaging efficiency, we herein describe a newly developed homozygous gpt delta rat strain. Results The new gpt delta rat has a Wistar Hannover background and has been successfully maintained as homozygous for the transgene. The packaging efficiency in the liver was 4 to 8 times higher than that of existing heterozygous F344 gpt delta rats. The frequency of gpt point mutations significantly increased in the liver and bone marrow of N-nitroso-N-ethylurea (ENU)- and benzo[a]pyrene (BaP)-treated rats. Spi− deletion frequencies significantly increased in the liver and bone marrow of BaP-treated rats but not in ENU-treated rats. Whole genome sequencing analysis identified ≥ 30 copies of lambda EG10 transgenes integrated in rat chromosome 1. Conclusions The new homozygous gpt delta rat strain showed a higher packaging efficiency, and could be useful for in vivo gene mutation assays in rats.

Download Full-text

Genetic Events Inhibiting Apoptosis in Diffuse Large B Cell Lymphoma

Cancers ◽

10.3390/cancers13092167 ◽

2021 ◽

Vol 13 (9) ◽

pp. 2167

Author(s):

Etienne Leveille ◽

Nathalie Johnson

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

Point Mutations ◽

B Cell Lymphoma ◽

Malignant Cells ◽

Large B Cell Lymphoma ◽

Apoptotic Proteins ◽

Apoptotic Pathways ◽

Genetic Events ◽

Large B Cell

Diffuse large B cell lymphoma (DLBCL) is curable with chemoimmunotherapy in ~65% of patients. One of the hallmarks of the pathogenesis and resistance to therapy in DLBCL is inhibition of apoptosis, which allows malignant cells to survive and acquire further alterations. Inhibition of apoptosis can be the result of genetic events inhibiting the intrinsic or extrinsic apoptotic pathways, as well as their modulators, such as the inhibitor of apoptosis proteins, P53, and components of the NF-kB pathway. Mechanisms of dysregulation include upregulation of anti-apoptotic proteins and downregulation of pro-apoptotic proteins via point mutations, amplifications, deletions, translocations, and influences of other proteins. Understanding the factors contributing to resistance to apoptosis in DLBCL is crucial in order to be able to develop targeted therapies that could improve outcomes by restoring apoptosis in malignant cells. This review describes the genetic events inhibiting apoptosis in DLBCL, provides a perspective of their interactions in lymphomagenesis, and discusses their implication for the future of DLBCL therapy.

Download Full-text