In vitro transposition of ISY100, a bacterial insertion sequence belonging to the Tc1/mariner family

The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix–turn–helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3′ ends and two nucleotides inside the 5′ ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3′ end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes

Cross talk between hedgehog and epithelial–mesenchymal transition pathways in gastric pit cells and in diffuse-type gastric cancers

We previously reported hedgehog (Hh) signal activation in the mucus-secreting pit cell of the stomach and in diffuse-type gastric cancer (GC). Epithelial–mesenchymal transition (EMT) is known to be involved in tumour malignancy. However, little is known about whether and how both signallings cooperatively act in diffuse-type GC. By microarray and reverse transcription–PCR, we investigated the expression of those Hh and EMT signalling molecules in pit cells and in diffuse-type GCs. How both signallings act cooperatively in those cells was also investigated by the treatment of an Hh-signal inhibitor and siRNAs of Hh and EMT transcriptional key regulator genes on a mouse primary culture and on human GC cell lines. Pit cells and diffuse-type GCs co-expressed many Hh and EMT signalling genes. Mesenchymal-related genes (WNT5A, CDH2, PDGFRB, EDNRA, ROBO1, ROR2, and MEF2C) were found to be activated by an EMT regulator, SIP1/ZFHX1B/ZEB2, which was a target of a primary transcriptional regulator GLI1 in Hh signal. Furthermore, we identified two cancer-specific Hh targets, ELK1 and MSX2, which have an essential role in GC cell growth. These findings suggest that the gastric pit cell exhibits mesenchymal-like gene expression, and that diffuse-type GC maintains expression through the Hh–EMT pathway. Our proposed extensive Hh–EMT signal pathway has the potential to an understanding of diffuse-type GC and to the development of new drugs

Linking Human Diseases to Animal Models Using Ontology-Based Phenotype Annotation

A novel method for quantifying the similarity between phenotypes by the use of ontologies can be used to search for candidate genes, pathway members, and human disease models on the basis of phenotypes alone

Present state and future perspectives of using pluripotent stem cells in toxicology research

The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro–cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative. The article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed

Site-specific transposition of insertion sequence IS630

IS630 is a 1.15-kilobase sequence in Shigella sonnei that, unlike many mobile elements, seems not to mediate cointegration between different replicons. To assess its transposition, we constructed composite elements containing inverted copies of IS630 flanking a drug resistance gene. We found that these composite elements transposed to plasmid ColE1 in Escherichia coli. DNA sequencing showed that transposition was, in all cases, to the dinucleotide sequence 5'-TA-3'. There were two preferred insertion sites which corresponded to the TA sequences in the inverted repeats of a 13-base-pair stem region of the [rho]-dependent transcription terminator. IS630 is flanked by TA, and nucleotide substitution by in vitro mutagenesis at these ends did not affect transposition activity of a composite element or its ability to insert preferentially into TA within the 13-base-pair inverted repeat sequences or to duplicate the target sequence.</jats:p

Mutagenesis via IS transposition in Deinococcus radiodurans

Analysis of the complete genome indicates that insertion sequences (ISs) are abundant in the radio-resistant bacterium Deinococcus radiodurans. By developing a forward mutagenesis assay to detect any inactivation events in D. radiodurans, we found that in the presence of an active mismatch repair system 75% of the mutations to trimethoprim-resistance (Tmp(R)) resulted from an IS insertion into the thyA coding region. Analysis of their distribution among the spontaneous Tmp(R) mutants indicated that five different ISs were transpositionally active. A type II Miniature Inverted-repeat Transposable Element (MITE), related to one of the deinococcal ISs, was also discovered as an insertion into thyA. Seven additional genomic copies of this MITE element were identified by BLASTN. Gamma-ray irradiation of D. radiodurans led to an increase of up to 10-fold in the frequency of Tmp(R) mutants. Analysis of the induced mutations in cells exposed to 10 kGy indicated that gamma-irradiation induced transposition of ISDra2 approximately 100-fold. A 50-fold induction of ISDra2 transposition was also observed in cells exposed to 600 J m(-2) UV-irradiation. Point mutations to rifampicin resistance (Rif(R)) were also induced by gamma-irradiation to reach a plateau at 2 kGy. The plateau value represented a 16-fold increase in the mutant frequency over the background. Although error-free repair strategies predominate in D. radiodurans, an upregulation of transposition, as well as induction of point mutations in cells recovering from DNA damage, provide a genetic variability that may have long-term evolutionary consequences on the fitness of this organism in its habitat