Transposition of pGh9:ISS1 is random and efficient in Streptococcus thermophilus CNRZ368

2002 ◽  
Vol 48 (5) ◽  
pp. 473-478 ◽  
Author(s):  
Annabelle Thibessard ◽  
Annabelle Fernandez ◽  
Brigitte Gintz ◽  
Bernard Decaris ◽  
Nathalie Leblond-Bourget
Keyword(s):  
Homologous Recombination ◽  
Insertional Mutagenesis ◽  
Physical Map ◽  
Insertion Site ◽  
Streptococcus Thermophilus ◽  
Good Tool ◽  
Gram Positive Bacteria ◽  
Multiple Copies ◽  
Homologue Recombination ◽  
Random Transposition

Streptococcus thermophilus bacteria are used as a starter in the fermentation of yogurts and many cheeses. To construct mutants of S. thermophilus CNRZ368, the use of the plasmid pGh9:ISS1 was considered. This plasmid is known to be a good tool for insertional mutagenesis in gram-positive bacteria, owing to its ability to integrate in the genome by a mechanism of replicative transposition. However, the presence of three endogenous ISS1 copies in the genome of S. thermophilus CNRZ368 and the possible occurrence of homologous recombination could reduce the efficiency of pGh9:ISS1 as a tool for generating mutants. To address this question, the ability of pGh9:ISS1 to transpose randomly in the genome of strain CNRZ368 was investigated. The results of our experiments indicated that: (i) the frequency of transposition of ISS1 was high, approximately 2 × 10–2, in S. thermophilus CNRZ368; (ii) the integration of multiple tandem copies of the plasmid was frequent; (iii) homologous recombination events between ISS1 were not predominant; and (iv) plasmid pGh9:ISS1 transposed randomly around the S. thermophilus CNRZ368 chromosome. In addition, we describe the strategy used to localize the pGh9:ISS1 insertion locus on the physical map of strain CNRZ368 and the method used to clone the regions flanking this insertion site, especially when multiple copies of the plasmid were integrated in tandem.Key words: insertional mutagenesis, random transposition, Streptococcus thermophilus, homologue recombination, ISS1.

scholarly journals Antibacterial Activity of Some Lactic Acid Bacteria Isolated from an Algerian Dairy Product

2009 ◽  
Vol 2009 ◽  
pp. 1-6 ◽  
Author(s):  
Abdelkader Mezaini ◽  
Nour-Eddine Chihib ◽  
Abdelkader Dilmi Bouras ◽  
Naima Nedjar-Arroume ◽  
Jean Pierre Hornez
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Antibacterial Effect ◽  
Dairy Product ◽  
Streptococcus Thermophilus ◽  
Fermented Food ◽  
Bacteriocin Production ◽  
Gram Positive ◽  
Bacteria Growth ◽  
Gram Positive Bacteria

In the present study, the antibacterial effect of 20 lactic acid bacteria isolates from a traditional cheese was investigated. 6 isolates showed antibacterial effect against Gram positive bacteria.Streptococcus thermophilusT2 strain showed the wide inhibitory spectrum against the Gram positive bacteria. Growth and bacteriocin production profiles showed that the maximal bacteriocin production, byS. thermophilusT2 cells, was measured by the end of the late-log phase (90 AUml−1) with a bacteriocine production rate of 9.3 (AUml−1)h−1. In addition, our findings showed that the bacteriocin, produced byS. thermophilusT2, was stable over a wide pH range (4–8); this indicates that such bacteriocin may be useful in acidic as well as nonacidic food. This preliminarily work shows the potential application of autochthonous lactic acid bacteria to improve safety of traditional fermented food.

Efficient insertional mutagenesis in Streptococcus thermophilus

Gene ◽  
2000 ◽  
Vol 258 (1-2) ◽  
pp. 9-14 ◽  
Author(s):  
Loredana Baccigalupi ◽  
Gino Naclerio ◽  
Maurilio De Felice ◽  
Ezio Ricca
Keyword(s):  
Insertional Mutagenesis ◽  
Streptococcus Thermophilus

An integrative vector exploiting the transposition properties of Tn1545 for insertional mutagenesis and cloning of genes from Gram-positive bacteria

Gene ◽  
1991 ◽  
Vol 106 (1) ◽  
pp. 21-27 ◽  
Author(s):  
Patrick Trieu-Cuot ◽  
Cécile Carlier ◽  
Claire Poyart-Salmeron ◽  
Patrice Courvalin
Keyword(s):  
Insertional Mutagenesis ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Integrative Vector

Automated Retroviral Insertion Site Sequence Analysis and Mapping Tool Followed by Database Analysis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5528-5528
Author(s):  
Stephanie Laufs ◽  
Frank A. Giordano ◽  
Agnes Hotz-Wagenblatt ◽  
Uwe Appelt ◽  
Daniel Lauterborn ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Integration Site ◽  
Query Sequence ◽  
Cpg Islands ◽  
Insertion Site ◽  
High Throughput Analysis ◽  
Site Analysis ◽  
Conventional Analysis ◽  
Integration Site Analysis ◽  
Insertion Sites

Abstract Increasing use of retroviral vector-mediated gene transfer and recent reports on insertional mutagenesis in mice and humans created intense interest to characterize vector integrations on the genomic level. Techniques to determine insertion sites, mainly based on time consuming manual data processing and compilation, are thus commonly applied in gene therapy laboratories. Since a high variability in processing methods hampers further data comparison, there is an urgent need to systematically process the data arising from such analysis. The obtained sequences from the integration site analysis are judged to be authentic only if the matching part of the genomic query sequence is surrounded by the 5′LTR-sequence on the one side and the adapter-sequence on the other side. Therefore we developed an Integrationseq tool. In this task, different methods for converting the ABI sequence trace files to high quality sequences and for recognizing and deleting the LTR and adaptor parts of the isolated clones were implemented. If neither a primer nor a LTR could be found, the sequence is discarded. If the LTR is found on the complementary strand, the integration sequence is reversed. The remaining sequence between primer and LTR positions are taken as the n integration sequence and written to a sequence output file. We validated the Integrationseq tool using 259 trace files originating from integration site analysis (LM-PCR). Sequences can be trimmed by IntegrationSeq, leading to an increased yield of valid integration sequence detection, which has shown to be more sensitive (100%) than conventional analysis (94.3%) and 15 times faster than conventional analysis, while the specifities are equal (both 100%). Valid integration sequences get further processed with IntegrationMap for automatic genomic mapping. IntegrationMap runs 50 times faster than conventional methods and retrieves detailed information about whether integrations are located in or close to genes, the name of the gene, the exact localization in the transcriptional units and further parameters like the distance from the transcription start site to the integration. Further information, e.g. data about CpG-Islands, LINEs or SINEs, and their distances to the integration is also displayed. Output files generated by the task were found to be 99.8% identical with results retrieved by conventional mapping with the Ensembl alignment tool. Using both tools, IntegrationSeq and IntegrationMap, a validated, fast and standardized high-throughput analysis of insertion sites can be achieved for the first time.

Cooperating Oncogenes and Their Targets in LMO2-Induced T-Cell Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2458-2458
Author(s):  
Stephen B. Smith ◽  
Rati Tripathi ◽  
Susan Cleveland ◽  
Elizabeth Mathias ◽  
Andrew Yi ◽  
...  
Keyword(s):  
T Cell ◽  
Insertional Mutagenesis ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Insertion Site ◽  
Gene Expression Signature ◽  
Treatment Resistance ◽  
Macromolecular Complex ◽  
Helix Loop Helix ◽  
E Boxes

Abstract Abstract 2458 LIM domain Only 2 (LMO2) is an important driver in acute T-cell lymphoblastic leukemia. Enforced expression of LMO2 in mouse models induces T-ALL with long latency suggesting that it requires cooperating oncogenic mutations for leukemia to fully manifest. To identify cooperating oncogenes, we analyzed spontaneous T-ALLs induced by retroviral insertional mutation, and T-ALLs that developed in B6.CD2-Lmo2 transgenic mice. First, we noted that the CD2-Lmo2 transgenic T-ALLs exclusively upregulated the class II basic helix-loop-helix transcription factor, Lyl1, whereas the retrovirally-induced T-ALLs showed upregulation of Tal1. We attributed this difference to the stage at which the Lmo2 activating mutations occurred. Nevertheless, we found that LYL1 protein upregulation is functionally important, as it is part of a macromolecular complex containing LMO2 that binds tandem E boxes as described for TAL1. LMO2 and LYL1 are known to be co-expressed in human T-ALL, but we found that they are part of a larger gene expression signature comprised of NMYC, HHEX, MEF2C, and BCL2 genes. The latter genes are remarkable because they are oncogenic when overexpressed; and, Mef2c and Nmyc are concordant insertions in independent tumors driven by Lmo2 or Hhex in retroviral insertional mutagenesis models. In fact, insertion site analysis suggests that Lmo2 and Hhex may actually operate in the same pathway and that Hhex may be a target of Lmo2. We tested this genetic result by chromatin immunoprecipitation and reporter assays and discovered that Lmo2 and its binding partners, Ldb1 and Lyl1, occupy an enhancer in the first intron of Hhex in T-ALL cells. Furthermore, LMO2 or LYL1 knockdown downregulated expression of HHEX in T-ALL. HHEX activation has significant functional consequences in T-ALL. We found that its upregulation is associated with T-ALL treatment resistance, clinical latency, and differential activation of Notch1 targets. These data offer important insight into the pathogenesis of Lmo2-induced T-ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification of Streptococcus thermophilus CNRZ368 Genes Involved in Defense against Superoxide Stress

2004 ◽  
Vol 70 (4) ◽  
pp. 2220-2229 ◽  
Author(s):  
Annabelle Thibessard ◽  
Frédéric Borges ◽  
Annabelle Fernandez ◽  
Brigitte Gintz ◽  
Bernard Decaris ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Insertional Mutagenesis ◽  
Iron Homeostasis ◽  
Defense Mechanism ◽  
Streptococcus Thermophilus ◽  
Rna Modification ◽  
Cell Wall Metabolism ◽  
Iron Sulfur Clusters ◽  
Iron Sulfur ◽  
Oxidative Stress Defense

ABSTRACT To better understand the defense mechanism of Streptococcus thermophilus against superoxide stress, molecular analysis of 10 menadione-sensitive mutants, obtained by insertional mutagenesis, was undertaken. This analysis allowed the identification of 10 genes that, with respect to their putative functions, were classified into five categories: (i) those involved in cell wall metabolism, (ii) those involved in exopolysaccharide translocation, (iii) those involved in RNA modification, (iv) those involved in iron homeostasis, and (v) those whose functions are still unknown. The behavior of the 10 menadione-sensitive mutants exposed to heat shock was investigated. Data from these experiments allowed us to distinguish genes whose action might be specific to oxidative stress defense (tgt, ossF, and ossG) from those whose action may be generalized to other stressful conditions (mreD, rodA, pbp2b, cpsX, and iscU). Among the mutants, two harbored an independently inserted copy of pGh9:ISS1 in two loci close to each other. More precisely, these two loci are homologous to the sufD and iscU genes, which are involved in the biosynthesis of iron-sulfur clusters. This region, called the suf region, was further characterized in S. thermophilus CNRZ368 by sequencing and by construction of ΔsufD and iscU97 nonpolar mutants. The streptonigrin sensitivity levels of both mutants suggest that these two genes are involved in iron metabolism.

The development of APE-PCR for the cloning of genomic insertion sites of DNA elements

Biologia ◽  
2013 ◽  
Vol 68 (4) ◽  
Author(s):  
Zhongjuan Xu ◽  
Yanli Li ◽  
Zhengwei Mao ◽  
Bin Yin
Keyword(s):  
Insertional Mutagenesis ◽  
Magnetic Particles ◽  
Magnetic Beads ◽  
Genetic Regulation ◽  
Insertion Site ◽  
Primer Extension ◽  
Successful Implementation ◽  
Nano Scale ◽  
Dna Elements ◽  
Insertion Sites

AbstractInsertional mutagenesis is a productive strategy for the exploration of genetic regulation of important biological and pathological processes, such as tumorigenesis. Successful implementation of this strategy depends heavily on an efficient approach to the identification of insertion sites present in the host genome. Here, we have introduced an easy and efficient protocol, called Adenosine-ended Primer Extension Polymerase Chain Reaction (APE-PCR), which represents several advantages, including the Addition technique we previously developed, primer extension approach coupled with biotin-streptavidin based purification, introduction of nano-scale magnetic particles, and digestion of DNA with a combination of enzymes. We have demonstrated that APE-PCR is able to amplify more and larger specific proviral insertion site (PIS)-derived fragments, with a lower non-specific background produced, fewer steps and less DNA samples required, flexibility in choice of restriction enzymes applied, at a lower cost. Replacement of regular magnetic beads with nano-scale ones in the protocol can further increase its power. Moreover, even with small amount of sample DNA, PISs can be recovered and analyzed. Thus, based on the results provided from this study, we believe that APE-PCR represents an efficient method in mapping of PISs and likely, the insertion sites of other types of DNA elements as well.

scholarly journals Expression of a foreign gene in a line of transgenic mice is modulated by a chromosomal position effect.

1990 ◽  
Vol 10 (3) ◽  
pp. 1192-1198 ◽  
Author(s):  
R al-Shawi ◽  
J Kinnaird ◽  
J Burke ◽  
J O Bishop
Keyword(s):  
Transgenic Mice ◽  
Position Effect ◽  
Insertion Site ◽  
Foreign Gene ◽  
Aberrant Expression ◽  
Transgenic Mouse Line ◽  
Chromosomal Position ◽  
Multiple Copies ◽  
Chromosomal Position Effect ◽  
Or Gene

Unusual aberrant expression of a foreign gene in a particular transgenic mouse line is often attributed to chromosomal position effect, although proof of this is lacking. An alternative explanation is that expression has been modified by the arrangement of multiple copies of the foreign gene at the insertion site or by mutation or gene rearrangement. We have distinguished between these explanations in the case of one particular transgenic line by recovering the aberrantly expressed foreign DNA and reintroducing it into the mouse genome to produce secondary transgenic mice. The expression pattern of the gene in the secondary transgenic mice was normal, showing that this case of aberrant expression is due to a chromosomal position effect.

scholarly journals Chromosomal Location Determines the Rate of Intrachromosomal Homologous Recombination in Salmonella

mBio ◽  
2021 ◽  
Author(s):  
Eva Garmendia ◽  
Gerrit Brandis ◽  
Lionel Guy ◽  
Sha Cao ◽  
Diarmaid Hughes
Keyword(s):  
Homologous Recombination ◽  
Chromosomal Location ◽  
Content Type ◽  
Bacterial Chromosomes ◽  
Multiple Copies

Bacterial chromosomes frequently carry multiple copies of genes at separate chromosomal locations. In Salmonella , these include the 7 rrn operons and the duplicate tuf genes.

scholarly journals The zebrafishspiel-ohne-grenzen(spg) gene encodes the POU domain protein Pou2 related to mammalianOct4and is essential for formation of the midbrain and hindbrain, and for pre-gastrula morphogenesis

Development ◽  
2002 ◽  
Vol 129 (4) ◽  
pp. 905-916 ◽  
Author(s):  
Shawn Burgess ◽  
Gerlinde Reim ◽  
Wenbiao Chen ◽  
Nancy Hopkins ◽  
Michael Brand
Keyword(s):  
Insertional Mutagenesis ◽  
Inner Cell Mass ◽  
Insertion Site ◽  
Cell Mass ◽  
Brain Regions ◽  
Chemical Mutagenesis ◽  
Gene Encoding ◽  
Specific Expression ◽  
Pou Domain ◽  
Similar Phenotype

In early embryonic development, the brain is divided into three main regions along the anteroposterior axis: the forebrain, midbrain and hindbrain. Through retroviral insertional mutagenesis and chemical mutagenesis experiments in zebrafish, we have isolated mutations that cause abnormal hindbrain organization and a failure of the midbrain-hindbrain boundary (MHB) to form, a region that acts as an organizer for the adjacent brain regions. The mutations fail to complement the spiel-ohne-grenzen (spg) mutation, which causes a similar phenotype, but for which the affected gene is unknown. We show through genetic mapping, cloning of the proviral insertion site and allele sequencing that spg mutations disrupt pou2, a gene encoding the Pou2 transcription factor. Based on chromosomal synteny, phylogenetic sequence comparison, and expression and functional data, we suggest that pou2 is the zebrafish ortholog of mouse Oct3/Oct4 and human POU5F1. For the mammalian genes, a function in brain development has so far not been described. In the absence of functional pou2, expression of markers for the midbrain, MHB and the hindbrain primordium (pax2.1, wnt1, krox20) are severely reduced, correlating with the neuroectoderm-specific expression phase of pou2. Injection of pou2 mRNA restores these defects in spg mutant embryos, but does not activate these markers ectopically, demonstrating a permissive role for pou2. Injections of pou2-morpholinos phenocopy the spg phenotype at low concentration, further proving that spg encodes pou2. Two observations suggest that pou2 has an additional earlier function: higher pou2-morpholino concentrations specifically cause a pre-gastrula arrest of cell division and morphogenesis, and expression of pou2 mRNA itself is reduced in spg-homozygous embryos at this stage. These experiments suggest two roles for pou2. Initially, Pou2 functions during early proliferation and morphogenesis of the blastomeres, similar to Oct3/4 in mammals during formation of the inner cell mass. During zebrafish brain formation, Pou2 then functions a second time to activate gene expression in the midbrain and hindbrain primordium, which is reflected at later stages in the specific lack in spg embryos of the MHB and associated defects in the mid- and hindbrain.

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