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Results for “mapping insertion sites”

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PubMed2002

Localization of the ventricular insertion site of concealed left-sided accessory pathways using ventricular pace mapping.

The aim of the present study was to localize the ventricular insertion site of concealed accessory pathway (APs) by using the ventricular pace mapping and examined if the analysis of the timing of retrograde atrial electrogram recorded at the ventricular side of the mitral annulus is useful in identifying the ventricular insertion site of the AP. In 39 patients with concealed left-sided APs, ventricular pacing was delivered along the mitral annulus at a cycle length of 500 ms while measuring the conduction interval from the pacing stimulus to the earliest retrograde atrial electrogram recorded in the coronary sinus (St-A). The ventricular insertion site of the AP was localized by identifying the shortest St-A. Also the interval between the onsets of QRS and atrial electrograms (QRS-A) and presence of continuous electrical activity (CEA) between the ventricular and atrial electrograms were evaluated at each mapping site during atrioventricular reciprocating tachycardia. Initial radiofrequency energy application to the site with the shortest St-A (46 +/- 15 ms) eliminated the AP conduction in all patients, suggesting the accurate localization of the ventricular insertion site by ventricular pace mapping. The QRS-A and the percentage of the presence of CEA at the shortest St-A site were 79 +/- 19 ms and 64%, respectively. However, the earliest retrograde atrial activation site did not coincide with the shortest St-A site in 19 of 39 patients, suggesting an oblique course of AP. Thus, in these 19 patients, there was a significant difference in St-A (47 +/- 16 vs 59 +/- 15 ms, P < 0.0001), QRS-A (83 +/- 13 vs 72 +/- 12 ms, P < 0.0001) and the presence of CEA (32 vs 74%, P < 0.01) between the shortest St-A site and the earliest retrograde atrial activation site, respectively. These indicate that the earliest retrograde atrial activation is not necessarily indicative of the ventricular insertion site of AP. However, ventricular pace mapping was considered to be useful for identifying and ablating the ventricular insertion site of AP, irrespective of the course of AP.

Adolescent↗

PubMed1987

Identification and nucleotide sequence of the regions of Autographa californica nuclear polyhedrosis virus genome carrying insertion elements derived from Spodoptera frugiperda.

A morphology mutant of the baculovirus Autographa californica nuclear polyhedrosis virus called M5 was previously shown to synthesize two size classes of viral DNA, one of which had a deletion of 42% of the genome. It was hypothesized that the presence of the smaller M5 circular DNA resulted from the specific deletion of the region located between two sites carrying short DNA insertions. These sites have now been identified by DNA-DNA hybridization using cloned EcoRI fragments of M5 DNA. One cloned M5 EcoRI fragment was found to correspond to the deletion junction fragment where sequences from the insertion site in the 2.6-map unit region were covalently linked to sequences from the insertion site in the 46-map unit region. The 2.6- and 46-map unit regions of Wt AcMNPV DNA were sequenced. Potential long open reading frames which would be disrupted by the M5 inserts were detected. Nucleotide sequence analysis of the same regions of M5 DNA revealed the presence of almost identical inserts of 290 bp. The primary sequence of the inserts revealed characteristics similar to the termini of transposons. Hybridization studies suggested that the inserts were derived from repetitive elements of the Spodoptera frugiperda host cell genome.

Animals↗

PubMed2006

A gain-of-function screen identifying genes required for vein formation in the Drosophila melanogaster wing.

The formation of the Drosophila wing involves developmental processes such as cell proliferation, pattern formation, and cell differentiation that are common to all multicellular organisms. The genes controlling these cellular behaviors are conserved throughout the animal kingdom, and the genetic analysis of wing development has been instrumental in their identification and functional characterization. The wing is a postembryonic structure, and most loss-of-function mutations are lethal in homozygous flies before metamorphosis. In this manner, loss-of-function genetic screens aiming to identify genes affecting wing formation have not been systematically utilized. As an alternative, a number of genetic searches have utilized the phenotypic consequences of gene gain-of-expression, as a method more efficient to search for genes required during imaginal development. Here we present the results of a gain-of-function screen designed to identify genes involved in the formation of the wing veins. We generated 13,000 P-GS insertions of a P element containing UAS sequences (P-GS) and combined them with a Gal4 driver expressed mainly in the developing pupal veins. We selected 500 P-GSs that, in combination with the Gal4 driver, result in modifications of the veins, changes in the morphology of the wing, or defects in the differentiation of the trichomes. The P-element insertion sites were mapped to the genomic sequence, identifying 373 gene candidates to participate in wing morphogenesis and vein formation.

Animals↗

PubMed2000

Developmental characterization and chromosomal mapping of a LacZ transgene expressed in the mouse apical ectodermal ridge.

The apical ectodermal ridge (AER) has an essential role in limb morphogenesis involving the specification of the proximal-distal axis of the limb. During the analysis of transgenic mice that harbor a LacZ transgene, we detected strong expression of beta-galactosidase within the AER of developing embryos. In this mouse line, called Z16, the bacterial LacZ gene is linked to a Herpes simplex virus immediate early promoter that is normally silent in mice. Embryos from other independent mouse lines harboring the same DNA construct exhibited no AER specific staining. Thus, it appears that the LacZ transgene in the Z16 line is expressed in the AER in response to regulatory influences from genomic DNA flanking the integration site. By fluorescent in situ hybridization, the transgene insertion site was mapped to chromosome 12. Hemizygous and homozygous transgenic mice appear normal and are fertile. AER specific beta-galactosidase staining was detected by 9.5 days post coitum in the forelimb and hindlimb bud. beta-galactosidase staining could be seen throughout the development of the limbs up to 14.5 days post coitum when expression was restricted to the distal-most regions of the digits of the hindlimbs. The loss of beta-galactosidase staining between digits correlated with the onset of programmed cell death, or apoptosis, in the digit interzones. LacZ expression in this transgenic line represents a useful marker for studying AER function in limb specification during mouse embryogenesis.

Animals↗

PubMed1995

Is there a Brachyury the Second? Analysis of a transgenic mutation involved in notochord maintenance in mice.

A new phenotype mapping to the t-complex, which is designated Brachyury the Second (T2), is characterized by a slightly shortened tail in heterozygotes and homozygous failure to form an organized notochord with subsequent abnormal development of posterior somites and neural tube. The phenotype of T2 superficially resembles that of Brachyury; however, there are several important differences. Brachyury homozygotes fail to make posterior somites, notochord, floor plate, and a placental connection, resulting in death by 10.5 days of development. In contrast, T2 homozygotes make posterior somites, scattered notochord cells, and floorplate and achieve an allantoic connection. However, despite making a maternal connection, T2 homozygotes cease development at E11.5 and die soon after. We have cloned and analyzed the transgene insertion site, which maps within 100 kb of the Brachyury gene, but does not seem to physically interrupt nor affect transcription from that locus. The existence of a second gene mapping near Brachyury and affecting the same developmental processes was alluded to over 50 years ago and has been debated ever since. An embryological description of T2 is presented, as is a discussion of the implications of a single, larger Brachyury locus versus two closely linked genes coordinately regulating axial development.

Animals↗

PubMed2005

Pinocchio, a novel protein expressed in the antenna, contributes to olfactory behavior in Drosophila melanogaster.

Most organisms depend on chemoreception for survival and reproduction. In Drosophila melanogaster multigene families of chemosensory receptors and putative odorant binding proteins have been identified. Here, we introduce an additional distinct protein, encoded by the CG4710 gene, that contributes to olfactory behavior. Previously, we identified through P[lArB]-element mutagenesis a smell impaired (smi) mutant, smi21F, with odorant-specific defects in avoidance responses. Here, we show that the smi21F mutant also exhibits reduced attractant responses to some, but not all, of a select group of odorants. Furthermore, electroantennogram amplitudes are increased in smi21F flies. Characterization of flanking sequences of the P[lArB] insertion site, complementation mapping, phenotypic reversion through P-element excision, and expression analysis implicate a predicted gene, CG4710, as the candidate smi gene. CG4710 produces two transcripts that encode proteins that contain conserved cysteines and which are reduced in the smi21F mutant. Furthermore, in situ hybridization reveals CG4710 expression in the third antennal segment. We have named this gene of previously unknown function and its product "Pinocchio (Pino)".

Amino Acid Sequence↗

PubMed1978

Conjugal transfer system of plasmid RP4: analysis by transposon 7 insertion.

We have begun an analysis in Escherichia coli of the conjugal transfer functions of the broad-host-range plasmid RP4. We have isolated 19 tra mutants of RP4, generated by insertion of transposon 7, and mapped their insertion sites by restriction endonuclease analysis. These sites fall into two separate regions on either side of the kanamycin resistance determinant. The transfer rates of the mutants range from 10% of that of RP4 to an undetectable level. Spot tests with the P-1 pilus-specific phages PRR1, Pf3, and PR4 and electron microscopic examination for pili have classified the mutants into several phenotypes consistent with their having normal, retracted, or no pili. Analysis of transient plasmid heterozygotes, created by P1 transduction, divided the tra mutants into a minimum of five complementation groups. Some of these groups contain more than one phenotypic class and may represent more than one gene because of the possible polar and deletion effects of Tn7 insertion.

Chromosome Mapping↗

PubMed2025

Genome-wide profiling the integration patterns with T7-PCR.

Integration of exogenous gene fragments into the host genomes is a widely used and powerful method for studying gene functions, advancing molecular breeding, and conducting gene therapy. Accurately identifying the integration sites is essential for ensuring both the safety and efficacy of genome engineering efforts. However, current mapping techniques are constrained by high costs and a low signal-to-noise ratio. In this study, we developed an innovative tool for mapping integration sites, leveraging T7 polymerase-mediated in vitro transcription (T7-IVT) to capture the junction fragments surrounding integration loci. This approach converts genomic flanking sequences into RNA, enabling the simultaneous enrichment of junction fragments and the elimination of background genomic DNA, thereby significantly enhancing the signal-to-noise ratio. We have validated the efficiency of this method, named T7-PCR, across yeast, plant, and human cells under diverse integration scenarios. T7-PCR outperforms current next-generation sequencing (NGS)-based mapping strategies in terms of efficiency and accuracy, with minimal positional effects. This method is highly applicable for high-throughput transgene screening and also supports the development of next-generation tools for targeted integration of large fragments.

Humans↗

PubMed1994

Deregulation of temperature-dependent transcription of the invasion regulatory gene, virB, in Shigella by rho mutation.

Expression of the virB gene, the transcriptional regulator for the invasion genes encoded by the large plasmid of Shigella flexneri, is temperature-regulated. virB transcription is under the control of VirF and H-NS, which act as positive and negative regulators, respectively, and is highly responsive to changes in DNA superhelicity. To further investigate the molecular mechanisms underlying the thermoregulation of virB transcription, a mutant which expressed an invasion phenotype at both 30 degrees C and 37 degrees C was isolated using miniTn10-kan (miniKAN) random insertion mutagenesis. The insertion site was mapped to the rho gene, and resulted in the addition of 11 amino acids to the C-terminus of the Rho protein. Consequently, decreased transcription termination activity at a rho-dependent terminator, lambda tL1, was observed. In the rho mutant, both the transcription of virB and expression of invasion genes were activated at 30 degrees C and were less responsive to changes in temperature. The deregulation of virB expression by the mutation was dependent upon the virB promoter, since the effects of the mutation on virB transcription were abolished when its promoter region was replaced by the tac promoter. Temperature-responsive changes in DNA topology, as determined by linking numbers of a reporter plasmid, showed that changes in DNA superhelicity in the rho mutant were smaller than that in the wild type. Furthermore, when the mutant was grown in medium containing novobiocin, an inhibitor of DNA gyrase, virB transcription at 30 degrees C as well as at 37 degrees C was greatly diminished. These results indicated that Rho protein could have a profound effect on topological temperature-dependent changes in DNA structure, thus contributing to thermoregulation of virB transcription.

Bacterial Outer Membrane Proteins↗

PubMed2003

Heterologous transposition in Ustilago maydis.

The phytopathogenic basidiomycete Ustilago maydis has become a model system for the analysis of plant-pathogen interactions. The genome sequence of this organism will soon be available, increasing the need for techniques to analyse gene function on a broad basis. We describe a heterologous transposition system for U. maydis that is based on the Caenorhabditis transposon Tc1, which is known to function independently of host factors and to be active in evolutionarily distant species. We have established a nitrate reductase based two-component counterselection system to screen for Tc1 transposition. The element was shown to be functional and transposed to several different locations in the genome of U. maydis. The insertion pattern observed was consistent with the proposed general mechanism of Tc1/mariner integration and constitutes a proof of principle for the first heterologous transposition system in a basidiomycete species. By mapping the insertion site context to known genomic sequences, Tc1 insertion events were shown to occur on different chromosomes, but exhibit a preference for non-coding regions. Only 20% of the insertions were found in putative open reading frames. The establishment of this system will permit efficient gene tagging in U. maydis and possibly also in other fungi.

Animals↗

PubMed1996

The amb2 locus from Serratia entomophila confers anti-feeding effect on larvae of Costelytra zealandica (Coleoptera: Scarabaeidae).

Serratia entomophila (Se) causes amber disease in the soil-dwelling pest, Costelytra zealandica (Cz). The disease presents two main signs: anti-feeding effect (AFE) and development of amber coloration (AC). To identify the genetic loci involved in pathogenicity, non-pathogenic (Path-) Se mutants were created by transposon (TnphoA) mutagenesis [Upadhyaya et al., J. Bacteriol. 174 (1992) 1020-1028]. The mutant UC24 lost the ability to produce amber disease signs and it was shown to contain a single TnphoA insertion. The TnphoA insertion site was mapped in a 5.3-kb DNA fragment, which was named amb2 locus. Cosmids containing amb2 fully restored AFE and partially restored AC in UC24. Escherichia coli (Ec) HB101 bearing the amb2 locus was able to cause AFE in a multiple-dose bioassay. SDS-PAGE analysis of the amb2 gene products produced in minicells showed the synthesis of two proteins of 16 and 19.5 kDa, named AnfA and AnfB. The genes encoding these proteins were mapped by deletion analysis. Pathogenicity tests with insect larvae fed with bacteria carrying the anfA and anfB gene regions separately showed that both regions are essential for AFE. It is proposed that the AnfA and AnfB proteins are virulence factors (toxin-like proteins) causing AFE in Cz.

Amino Acid Sequence↗

PubMed2001

Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron.

The adhC1 gene from Acinetobacter baumannii 8399, which encodes a glutathione-dependent formaldehyde dehydrogenase (GSH-FDH), was identified and cloned after mapping the insertion site of Tn3-HoHo1 in a recombinant cosmid isolated from a gene library. Sequence analysis showed that this gene encodes a protein exhibiting significant similarity to alcohol dehydrogenases in bacterial, yeast, plant and animal cells. The expression of the adhC1 gene was confirmed by the detection of GSH-FDH enzyme activity in A. baumannii and Escherichia coli cells that expressed the cloned gene. However, the construction and analysis of an A. baumannii 8399 adhC1::Tn3-HoHo1 isogenic derivative revealed the presence of adhC2, a second copy of the gene encoding GSH-FDH activity. Enzyme assays and immunoblot analysis showed that adhC2 encodes a 46.5 kDa protein that is produced in similar amounts under iron-rich and iron-limited conditions. In contrast, the expression of adhC1, which encodes a 45 kDa protein with GSH-FDH activity, is induced under iron limitation and repressed when the cells are cultured in the presence of free inorganic iron. The differential expression of adhC1 is controlled at the transcriptional level and mediated through the Fur iron-repressor protein, which has potential binding sites within the promoter region of this adhC copy. The expression of both adhC copies is significantly enhanced by the presence of sub-inhibitory concentrations of formaldehyde in the culture media. Examination of different A. baumannii isolates indicates that they can be divided into two groups based on the type of GSH-FDH they produce. One group contains only the constitutively expressed 46.5 kDa protein, whilst the other produces this GSH-FDH type in addition to the iron-regulated isoenzyme. Further analysis showed that the presence and expression of the two adhC genes does not confer resistance to exogenous formaldehyde, nor does it enable it to utilize methylated compounds as a sole carbon source when cultured under iron-rich as well as iron-deficient conditions.

Acinetobacter↗

PubMed1992

Identification and characterization of novel low-temperature-inducible promoters of Escherichia coli.

Escherichia coli promoters that are more active at low temperature (15 to 20 degrees C) than at 37 degrees C were identified by using the transposon Tn5-lac to generate promoter fusions expressing beta-galactosidase (beta-Gal). Tn5-lac insertions that resulted in low-temperature-regulated beta-Gal expression were isolated by selecting kanamycin-resistant mutants capable of growth on lactose minimal medium at 15 degrees C but which grew poorly at 37 degrees C on this medium. Seven independent mutants were selected for further studies. In one such strain, designated WQ11, a temperature shift from 37 degrees C to either 20 or 15 degrees C resulted in a 15- to 24-fold induction of beta-Gal expression. Extended growth at 20 or 15 degrees C resulted in 36- to 42-fold-higher beta-Gal expression over that of cells grown at 37 degrees C. Treatment of WQ11 with streptomycin, reported to induce a response similar to heat shock, failed to induce beta-Gal expression. In contrast, treatment with either chloramphenicol or tetracycline, which mimics a cold shock response, resulted in a fourfold induction of beta-Gal expression in strain WQ11. Hfr genetic mapping studies complemented by physical mapping indicated that in at least three mutants (WQ3, WQ6, and WQ11), Tn5-lac insertions mapped at unique sites where no known cold shock genes have been reported. The Tn5-lac insertions of these mutants mapped to 81, 12, and 34 min on the E. coli chromosome, respectively. The cold-inducible promoters from two of the mutants (WQ3 and WQ11) were cloned and sequenced, and their temperature regulation was examined. Comparison of the nucleotide sequences of these two promoters with the regulatory elements of other known cold shock genes identified the sequence CCAAT as a putative conserved motif.

Anti-Bacterial Agents↗

PubMed1996

The use of an improved transposon mutagenesis system for DNA sequencing leads to the characterization of a new insertion sequence of Streptomyces lividans 66.

A DNA sequencing strategy was developed based on the tetracycline resistance transposon Tn1721. A universal M13 primer binding site (UP) for DNA sequencing and restriction sites for mapping were inserted near one end of Tn1721 and the new derivative, Tn5491, introduced onto a conjugative F' plasmid. The target sequence is inserted between two inverted resolution sites (res) of Tn1721 present on the high-copy plasmid pJOE2114. Due to the inviability of long palindromic sequences in Escherichia coli insertions between the inversely orientated res sites of pJOE2114 are positively selected. Transposition of Tn5491 into the target sequence is selected by cointegrate formation of Tn5491 during transposition, mating and transfer of the nonconjugative sequencing vector. After cointegrate resolution, the additional res sites in the vector result in a second site-specific recombination removing most of the transposon (except of 136 bp) and part of the target sequence. The reduced plasmid sizes and the use of the universal primer improved the quality of the sequencing results obtained on an automated fluorescent sequencer. A 3.35-kb EcoRI fragment from the 30-kb terminal inverted repeats (TIR) of the Streptomyces lividans chromosome was sequenced by this method. A 1304-bp sequence was found on this fragment with the features of insertion elements. The element called IS1372 had 27-bp IR and two potential open reading frames. The predicted gene products had similar sizes and high similarity to gene products encoded by insertion sequences of the IS3 family. Furthermore, a potential signal stimulating ribosomal shifts and typical for members of the IS3 family was identified. Five to seven copies of IS1372 were found in different strains of S. lividans but none in other Streptomyces species tested.

Amino Acid Sequence↗

PubMed1991

Repeated DNA sequence involved in mutations affecting transport of sucrose into Streptococcus mutans V403 via the phosphoenolpyruvate phosphotransferase system.

Mutants of Streptococcus mutans V403 defective in the intracellular sucrose-6-phosphate hydrolase (product of the scrB gene) are sensitive to sucrose because of the intracellular accumulation of the phosphorylated sugar. Using a scrB mutant prepared by allelic exchange, we have isolated and characterized a number of sucrose-resistant revertants. One such mutant was found to lack the ability to transport sucrose into the cell via the phosphoenolpyruvate-dependent sucrose phosphotransferase system (PTS). Genetic analysis of this strain revealed this lesion to be linked to the scrB gene. This was corroborated by the physical demonstration of an insertion mutation very near scrB. Taken together with DNA sequence information (Y. Sato, F. Poy, G. R. Jacobson, and H. K. Kuramitsu, J. Bacteriol. 171:263-271, 1989), our results indicated that all of the mutations characterized were located in the adjoining scrA gene which encodes the membrane-associated, sugar-specific enzyme II (EIIsucrose) component of the sucrose PTS in S. mutans. Biochemically, such a genetic lesion disables the sucrose PTS and prevents sucrose from entering the cell by this system. In this paper, we detail the nature of two independent insertion mutations and conclude them to be the result of duplicative transposition events into the scrA gene. This region of the chromosome was amplified and purified in large quantities by using the polymerase chain reaction. Examination of the amplified DNA revealed that the two independent insertion mutations were composed of sequences that were indistinguishable by size and by restriction site endonuclease maps. Their insertion points in the scrA gene were approximately 200 bp apart. The amplified DNA fragment was also used as a probe to demonstrate the presence of five copies of this element on the S. mutans V403 chromosome. A second strain, S. mutans V310, also was found to carry similarly arranged, multiple copies of this sequence on its chromosome, suggesting a clonal origin of V403 and V310. The small size of this sequence, its presence in multiple copies on the V403 chromosome, and its ability to duplicate itself semiconservatively into remote sites argue compellingly that it is an insertion sequence element. One such insertion mutant, with a defective sucrose PTS, was tested for virulence in rats and was found to cause caries at levels similar to those of the wild-type strain.

Animals↗

PubMed1998

Mapping of Drosophila mutations using site-specific male recombination.

Although recombination does not usually occur in the male Drosophila germline, site-specific recombination can be induced at the ends of P elements. This finding suggested that male recombination could be used to map Drosophila mutations. In this article, we describe the general method and its application to the mapping of two EMS-induced female-sterile mutations, grauzone and cortex. Within two months, the grauzone gene was mapped relative to seven different P-element insertion sites, and cortex was mapped relative to 23 different P-elements. The results allowed us to map grauzone to a region of about 50 kb, and cortex distal to the chromosomal region 33E. These experiments demonstrate that P-element-induced site-specific male recombination is an efficient and general method to map Drosophila autosomal mutations.

Animals↗

PubMed1993

Identification of cpsD, a gene essential for type III capsule expression in group B streptococci.

We showed previously that a mutant strain of group B Streptococcus (GBS) defective in capsule production was avirulent. This study describes the derivation of an unencapsulated mutant from a highly encapsulated wild-type strain of type III GBS, COH1, by transposon mutagenesis with Tn916 delta E. The mutant, COH1-13, was sensitive to phagocytic killing by human leukocytes in vitro and was relatively avirulent in a neonatal rat sepsis model compared with the wild-type strain. No capsular polysaccharide was evident in the cytoplasm or on the cell surface of the mutant strain. The Tn916 delta E insertion site in COH1-13 was mapped to the same chromosomal location as the Tn916 insertion site in the unencapsulated type III mutant COH31-15 reported previously. Nucleotide sequencing of DNA flanking the insertion site in COH1-13 revealed an open reading frame, designated cpsD, with significant homology to the rfbP gene of Salmonella typhimurium. RfbP encodes a galactosyl transferase enzyme that catalyses the transfer of galactose to undecaprenol phosphate, the initial step in O-polysaccharide synthesis. A particulate fraction of a lysate of wild-type strain GBS COH1 mediated the transfer of galactose from UDP-galactose to an endogenous acceptor. The galactose-acceptor complex partitioned into organic solvents, suggesting it is lipid in nature or membrane-associated. Galactosyl transferase activity was significantly reduced in the unencapsulated mutant strain COH1-13. These results, together with the similarity in deduced amino acid sequence between cpsD and rfbP suggest that cpsD encodes a galactosyl transferase essential for assembly of the GBS type III capsular polysaccharide.

Amino Acid Sequence↗

PubMed1990

Lysogenic phages of Clostridium perfringens: mapping of the chromosomal attachment sites.

The sites of insertion for two lysogenic bacteriophages have been mapped on the chromosome of Clostridium perfringens strain CPN50 using two techniques based on pulsed field gel electrophoresis. Phage phi 29 was mapped to the 1 Mb region of the 3.6 Mb genome, near nanH which encodes a potential virulence factor, while phi 59 was found to have inserted at 2.9 Mb.

Attachment Sites, Microbiological↗