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PubMed1991

Chromosomal insertion of TOL transposons in Pseudomonas aeruginosa PAO.

Insertions of the TOL plasmid transposons Tn4651 and Tn4653 into the Pseudomonas aeruginosa PAO chromosome were isolated by a temperature selection technique. The locations and orientations of 16 insertions were determined by pulsed field gel electrophoresis and Southern hybridization with genomic and TOL DNA probes. All insertions occurred within a 334 kb region of the chromosome (representing less than 6% of the genome) with nine of the inserts clustered within a 10 kb area. Each transposon was able to insert in either orientation. An internal duplication of the 39 kb excisable region of pWW0 was seen in two independent insertions.

Chromosome Mapping↗
PubMed1992

An additional exon in the human vinculin gene specifically encodes meta-vinculin-specific difference peptide. Cross-species comparison reveals variable and conserved motifs in the meta-vinculin insert.

We have analyzed the structure, origin and expression of the high-molecular-mass muscle-specific variant of vinculin, called meta-vinculin. The meta-vinculin-specific inserts from the human and avian molecules have been isolated and sequenced and the sequences confirmed via cloning of the corresponding cDNA. Comparison of the human, avian and determined porcine sequences revealed cross-species identity in the C-terminal half of the insert. Human and porcine meta-vinculin were highly similar in the insert region, showing only five amino acid exchanges; avian meta-vinculin showed 22 exchanges in the same region compared to human meta-vinculin and exhibited, in addition, one extra amino acid, making 69 in all. Each insert was flanked by characteristic KWSSK motifs. Evidence for two vinculin mRNA species in human uterus smooth muscle was provided by reverse transcription combined with the polymerase chain reaction, as well as by ribonuclease-mapping analysis of cDNA/mRNA hybrids. One of the mRNA species contained an additional 204-nucleotide insert that precisely encoded the meta-vinculin-specific peptide. Sequence analysis of the appropriate portion of the human vinculin gene showed that the section coding for the meta-vinculin-specific insert is present as a discrete exon. Thus, meta-vinculin and vinculin mRNA are generated by alternative splicing.

Amino Acid Sequence↗
PubMed1975

Electron microscope heteroduplex studies of sequence relations among bacterial plasmids: identification and mapping of the insertion sequences IS1 and IS2 in F and R plasmids.

Heteroduplex experiments between the plasmid R6 and one strand of the deoxyribonucleic acid (DNA) of a lambda phage carrying the insertion sequence IS1 show that IS1 occurs on R6 at the two previously mapped junctions of resistance transfer factor (RTF) DNA with R-determinant DNA. From previous heteroduplex experiments, it then follows that IS1 occurs at the same junctions in R6-5, R100-1, and R1 plasmids. Heteroduplex experiments with the DNA from a lambda phage carrying the insertion sequence IS2 show that one copy of IS2 occurs in R6, R6-5, and R100-1 (but not R1) at a point within the RTF with coordinates 67.5 TO 68.9 kilobase units (kb). In an accompanying paper, Ptashne and Cohen (1975) show that the insertion sequence IS3 occurs on R6 and R6-5. R100-25, a traC mutant, differs from its parent R100-1 only in that it contains an additional copy of IS1 inserted within the tra gene region of 82.1 kb. R100-31, atraX, TC-s mutant of R100-1, is deleted in R100-1 sequences starting at one of the IS3 termini (46.9 kb) and extending with RTF to 61.0 kb. Heteroduplex studies of F plasmids with the DNA of a lambda phage bearing insertion sequence IS2 show that the sequence of F with coordinates 16.3-17.6F is IS2. The occurrence of IS1 at the two junctions of R-determinant DNA and RTF DNA in R plasmids provides a structural basis to explain the mechanism of the previously observed formation of molecules containing one RTF unit and several tandem copies of the R-determinant unit, when R plasmids in Proteus mirabilis are grown in the presence of antibiotics, and the segregation of an R plasmid into an RTF unit and an R-determinant unit. In general, correlation of our results with previous studies shows that insertion sequences play a role in a variety of F- and R-related intra- and intermolecular recombination phenomena.

Base Sequence↗
PubMed1978

Insertion of bacteriophage lambda into the deo operon of Escherichia coli K-12 and isolation of plaque-forming lambdadeo+ transducing bacteriophages.

A procedure has been devised to isolate plaque-forming lambda cI857S7 transducing bacteriophage which carry the internal promoter, P3, of the deo operon of Escherichia coli and the deoB and deoD genes, while lacking the deoP and cytP promoters of the same operon, in order to study, specifically, regulation at the P3 site. This has been accomplished by selecting for the insertion of bacteriophage lambda into the deoA gene in a strain deleted for the normal lambda attachment site (delta att lambda) and isolating from this lysogen lambda spi- and lambda EDTAr phage. Among these, lambda pdeoB+D+ phage were identified by their transducing abilities. From in vivo enzyme induction experiments performed on a delta deo strain lysogenized with such phage, they were shown to carry the P3 promoter while lacking the deoP and cytP promoters. A lambdapdeo B+D+ phage phage was used to lysogenize a deo+ delta att lambda strain, integration of lambda occurring within the region of homology, and, from a heat-induced lysate of this strain, a plaque-forming lambda+ phage carrying the complete deo operon was obtained. Phage lambda was also inserted into the deoB and deoD genes and into the tdk gene. By isolating lambdaspi- and lambdaEDTAr phage from the deo::(lambda) mutants and determining which bacterial genes they carried and whether they retained the int gene of lambda, it was found that lambda had inserted into deoD with the same orientation as lambda inserted into attlambda, whereas lambda inserted into deoA and deoB had the opposite orientation. Deletions extending from the site of lambda insertion into the bacterial chromosome were isolated by selecting for heat-resistant revertants. These confirmed the order of markers to be deo-serB-trpR-thr and also placed a locus, msp, determining sensitivity or resistance of male strains to male-specific phages, between trpR and thr. For some reason unknown, but which may be related to the orientation of the lambda prophages, short deletions rendering the bacterium Ser- Thr+ were of much lower frequency from the deoD::(lambda) lysogen than from the other two lysogens. From an examination of the residual deoD enzyme levels in deoB::(lambda) mutants, it was deduced that there may be two promoter sites within the deoB::(lambda) mutants, it was deduced that there may be two promoter sites within the deoB gene, transcription from one of these being sufficient to account for the noncoordinate nature of the induction of deoB and deoD gene products.

Chromosome Mapping↗
PubMed1991

Novel insertion sequence IS1380 from Acetobacter pasteurianus is involved in loss of ethanol-oxidizing ability.

Acetobacter pasteurianus NCI1380, a thermophilic strain isolated from the surface culture of acetic acid fermentation, showed genetic instability to produce at high frequency spontaneous mutants which were deficient in ethanol oxidation because of the loss of alcohol dehydrogenase activity. Southern hybridization experiments with the cloned alcohol dehydrogenase-cytochrome c gene cluster as the probe showed insertion of an unknown DNA fragment into a specific position in the cytochrome c gene in most of the mutant strains. Cloning and sequencing analyses revealed that the inserted sequence was 1,665 bp in length and had a terminal inverted repeat of 15 bp. In addition, this inserted sequence was found to generate a 4-bp duplication at the inserted site upon transposition. The target site specificity was not very strict, but a TCGA sequence appeared to be preferentially used. The inserted sequence contains two long open reading frames of 461 and 222 amino acids which are overlapped and encoded by different strands. Although these open reading frames showed no homology to any protein registered in the DNA data bases, the longer open reading frame contained many basic amino acids (87 of 461), as was observed with transposases of so-called insertion sequence (IS) elements. All of these characteristics are typical of IS elements, and the sequence was named IS1380. The copy number of IS1380 in a cell of A. pasteurianus NCI1380 was estimated to be about 100. Several strains of acetic acid bacteria also contained IS1380 at high copy numbers. These results suggest that IS1380 is associated with the genetic loss of ethanol-oxidizing ability as well as the genetic instability of acetic acid bacteria in general.

Acetobacter↗
PubMed1992

Hyperhemolytic phenomena associated with insertions of Tn916 into the hemolysin determinant of Enterococcus faecalis plasmid pAD1.

Members of the Tn916 family of conjugative transposons are able to insert themselves into Enterococcus faecalis hemolysin/bacteriocin plasmid pAD1 (and related elements) in such a way as to generate hyperexpression of the hemolysin/bacteriocin. To examine this phenomenon in more detail, E. faecalis (pAD1::Tn916) derivatives defective or altered in hemolysin expression were isolated and characterized with respect to production of the L (lytic) or A (activator) component (also known as CylA) and the specific location of the transposon. The mutants fell into five classes. Class 1 strains were nonhemolytic, and the related insertions mapped in a location known to affect expression of the L component. The other four classes varied from an inability to express hemolysin (class 2) to different degrees of hyperhemolytic expression (classes 3 to 5); the insertions in these classes mapped in a similar place within cylA, near the 3' end of the determinant. A previous study provided evidence that CylA is also necessary for bacteriocin immunity; however, these insertions did not destroy this function. (A Tn917 insertion in the 5' half of the determinant eliminates immunity.) In mutant classes 3 to 5, the presence of tetracycline enhanced hemolysin expression. In late-exponential-phase broth cultures, hemolysin could not be detected in supernatants of classes 2 to 5, in contrast to a wild-type control strain; however, different amounts of the L component could be detected, with the lowest in class 2 and greater-than-normal amounts in classes 3 to 5. Although nucleotide sequencing showed that the Tn916 insertions in classes 2 to 5 were at identical sites, the transposon junction sequences differed in some cases. The data indicated that cylA translation into the transposon would result in different truncation sites, and these differences were probably related to phenotype differences.

Amino Acid Sequence↗
PubMed1992

Propofol induction for laryngeal mask airway insertion: dose requirement and cardiorespiratory effects.

The dosage, haemodynamic and respiratory effects of propofol for laryngeal mask airway (LMA) insertion were investigated. Fifty patients (ASA I-II) were randomly assigned one of four induction doses of propofol (1.5-2.5 mg/kg) delivered over 30 seconds and the first attempt at LMA insertion was made at 90 seconds. The LMA was inserted at 90 seconds in 35 patients and by 300 seconds in 13 others (mean plasma concentration at 90 seconds was 7.7 mcg/ml (no delay) versus 5.2 mcg/ml (insertion delayed), P < 0.01). Insertion was less successful after 1.5 mcg/kg (failed at 90 seconds in 6 of 12 patients), but did not vary with the other doses. Additional propofol (0.5 mg/kg/30s) was required in 22 patients for LMA insertion or to prevent movement, resulting in propofol concentrations at 120-180 seconds above 7 mcg/ml. Respiratory effects were minor, but MAP decreased by 18 +/- 1.4 mmHg at 90 seconds. Cardiovascular effects did not differ significantly between dosage groups or with the use of additional propofol.

Adult↗
PubMed1978

Genetic aspects of intracellular motility: cortical localization and insertion of trichocysts in Paramecium tetraurelia.

Wild-type and selected mutants of Paramecium tetraurelia were subjected to detailed analysis of the trichocyst phenotype. The characteristics of morphology, saltatory motility, and cortical insertion of trichocysts were found to be genetically distinguishable. In mutants with defective trichocyst saltatory motility, other intracellular motility systems (i.e. mitochondrial saltatory motility, and cyclosis) appeared to be unaffected. Saltatory motility appears to be an essential intermediate step in the functional development of the trichocyst, since it serves to transport the trichocyst from its site of assembly, deep in the cytoplasm, to the cortex, where it is inserted and becomes discharge-competent. If trichocyst saltatory motility is lost, as in the mutants ndA and tam8, then trichocysts are not transported to the cortex and are therefore not inserted. However, the successful transport of a trichocyst to the cortex does not guarantee the proper insertion of that trichocyst, as in the case of the mutant am. Apparently there are independent steps involved specifically with trichocyst insertion. The gross morphology of the trichocyst appears to be independent of its saltatory motility, and to a degree, independent of cortical insertion.

Animals↗
PubMedDate not supplied

Three-dimensional theoretical SAR and temperature distributions created in brain tissue by 915 and 2450 MHz dipole antenna arrays with varying insertion depths.

Theoretical three-dimensional power deposition and temperature distributions were calculated for interstitial hyperthermia microwave antenna arrays driven at 915 and 2450 MHz in brain tissue. Four dipole antennas were assumed to be placed in a 2 x 2 cm array with varying insertion depths in cylindrical tumour models. The bioheat transfer equation was solved for the three-dimensional steady-state temperature distributions using a finite element method. Homogeneous and non-homogeneous blood flow models were considered. As a basis of comparison of the various temperature distributions, the volume of the tumour heated to greater than or equal to 43 degrees C was calculated. SAR distributions calculated for the 915 MHz antenna arrays in brain tissue were very similar to those calculated for muscle. The 2450 MHz arrays showed similar behaviour to the 915 MHz arrays; however, as the insertion depth increased from slightly less than a full-wavelength there was a single hotspot centred at the antenna junction. For the 2450 MHz arrays, the predicted therapeutic tumour volumes were relatively constant over the entire range of insertion depths considered, and in fact, for most insertion depths considered, the model predicted the 2450 MHz arrays would heat larger therapeutic volumes than the 915 MHz arrays. For the 915 MHz array, at insertion depths between 7.8 and 14.6 cm there was a sharp decrease in the predicted therapeutic volume due to a proximal secondary hotspot in the normal tissue causing overheating. However, when the same size tumour at the same insertion depth was heated with the 2450 MHz array, the hotspot was in the tumour, adding to the volume of tumour that was heated to therapeutic temperatures.

Body Temperature↗
PubMed1975

[Genetic counseling in cases of chromosome insertions].

The principles of the estimation of the risk of repeated birth of malformed child and spontaneous abortion in families of balanced carriers of insertions are presented. A risk for a carrier is formed from two parts: a risk for a carrier of reciprocal translocation and a risk for a carrier of insertion of the came length as inseried segment. The first component of the risk is a constant, the latter one is a variable. It depends on the length of the inserted segment, the type of its inclusion (direct or inverted) and sites of break points. An estimation of the risk in hypothetical insertion 46, XX, inv ins (4; 2) (q24; q22q34) is described as a model. The same method of counselling may be used for the families with triple translocations t(a-, b-, c+) with "insertion" of fragment of chromosome "a" between the parts of "b" and "c" chromosomes. A tentative empirical mean value of repeated birth of malformed child and spontaneous abortion in families with insertions is 29% and 45% for the female carrier, and about 37% and 25% for the male carrier.

Abortion, Spontaneous↗
PubMed1992

Membrane insertion of the mannitol permease of Escherichia coli occurs under conditions of impaired SecA function.

The membrane insertion of the mannitol permease (MtlA protein) of Escherichia coli, a polytopic cytoplasmic membrane protein possessing an uncleaved amphiphilic signal sequence, was studied using a cell-free protein synthesis system. The MtlA protein synthesized in the presence of inside-out cytoplasmic membrane vesicles was shown to insert into the membranes based on the following criteria: (a) co-sedimentation of the majority of the MtlA protein with the vesicles; (b) selective extraction of the membrane-associated MtlA by doxycholate but not by urea treatment; and (c) protease resistance of a defined MtlA fragment observed exclusively in the presence of membranes. Post-translational addition of membrane vesicles allowed membrane association of MtlA but did not allow efficient integration. In cell-free systems having reduced levels of the export factors SecA and SecB and exhibiting defective translocation of preOmpA and preLamB, insertion of the in vitro synthesized MtlA apparently occurred normally. In contrast, when membranes from the secY24ts mutant or trypsin-treated membranes were used, insertion of MtlA was reduced. In vivo experiments monitoring the permease activity of MtlA in the secA and secY mutants supported the conclusions of the in vitro results. Thus, the insertion of MtlA is essentially SecA- and SecB-independent but may be dependent on SecY and/or an as yet unidentified membrane protein. The requirements for the insertion of the mannitol permease are therefore clearly different from those for the translocation of most proteins having a cleavable hydrophobic signal sequence.

Adenosine Triphosphatases↗
PubMed1992

Clinical evaluation of percutaneous insertion and long-term usage of a new cuffed polyurethane catheter for central venous access.

A new, long-term venous access catheter was evaluated in clinical practice and the insertion time, complication rate and prospective follow-up recorded. Fifty novel polyurethane catheters (Cuff-Cath) were inserted in 48 patients, for cytotoxic chemotherapy in 36, long-term total parenteral nutrition in five and miscellaneous indications in seven. All catheters were inserted by a percutaneous technique under local anaesthesia. The mean insertion time was 18 min. There were three insertion complications; failure to cannulate, pneumothorax and malposition. Seven catheters required removal (sepsis in five, subclavian vein thrombosis in two) and one catheter fell out. Total catheter days to date has been 6607 (mean 132, range 18-831 days). Eleven catheters are still in use a mean of 154 days (range 38-490 days) after insertion. Furthermore, a new technique has been described which prevents inadvertent displacement. This new catheter combines the mechanical advantages of polyurethane, together with those of a Dacron cuff. Early results suggest that this catheter may be a useful alternative to silicone catheters of the Hickman/Broviac type for long-term central venous access.

Antineoplastic Agents↗
PubMed1992

Transduction of circular membrane ruffling by the platelet-derived growth factor beta-receptor is dependent on its kinase insert.

The platelet-derived growth factor (PDGF) alpha- and beta-receptors both mediate a mitogenic response, but only the beta-receptor mediates circular actin reorganization and chemotaxis. The tyrosine kinase domains of the receptors contain noncatalytic inserts of about 100 residues. In order to determine the role of these domains in the differential signaling of the two receptors, we constructed chimeric PDGF receptors and expressed the constructs in porcine aortic endothelial cells. The chimeric receptors were similar to the wild-type receptors in their ability to induce mitogenicity in response to ligand. Examination of receptor-associated substrates by in vitro kinase assays revealed that phosphoproteins of 72 and 110 kilodaltons were associated with the kinase insert of the alpha-receptor, whereas a phosphoprotein of 130 kilodaltons was associated with the kinase insert of the beta-receptor. Actin reorganization in the form of circular membrane ruffling was seen after ligand stimulation of the beta-receptor and the alpha-receptor containing the beta-receptor kinase insert but not after stimulation of the alpha-receptor or the beta-receptor containing the alpha-receptor kinase insert. These data indicate that the PDGF beta-receptor kinase insert has an essential function in the signal transduction pathway leading to circular membrane ruffling.

Actins↗
PubMed1992

Nodule DNA in the (GA)37.(CT)37 insert in superhelical plasmids.

Di- or trivalent metal ions stabilize a supercoil-dependent transition in pGA37, which contains the (GA)37.(CT)37 insert, at neutral and basic pH. The structure formed is different from the well known protonated triplexes (H-DNA) adopted at low pH by polypurine.polypyrimidine (Pur.Pyr) inserts in plasmids. DNA samples must be preincubated in the presence of multivalent ions at 50 degrees C for the new transition to occur. At neutral pH in the presence of Co hexamine, both strands of the insert have modification maxima situated at one-third of the distance from both ends. We propose the formation of a new structure called nodule DNA which consists of both Pyr.Pur.Pyr and Pur.Pur.Pyr triplexes and does not contain continuous single-stranded regions. At basic pH (greater than 8.5) in the presence of magnesium ions, the modification pattern corresponds to Pur.Pur.Pyr triplex formation in the whole insert. At neutral pH in the presence of magnesium, both nodule DNA and the Pur.Pur.Pyr triplex can be formed in the insert. We also observed a magnesium-dependent transition at neutral pH in the other Pur.Pyr insert containing plasmids. These data demonstrate that Pur.Pyr sequences can adopt several non-B conformations at close to in vivo conditions.

Base Sequence↗
PubMed1992

Insertional mutations in mammals and mammalian cells.

The retroposon sequences, their mechanisms of transposition and the occurrence of insertional mutation in the mammalian genome are reviewed. Insertional mutations fall into two broad categories: those due to the disruption of a gene following the physical integration of a foreign DNA sequence result in loss of gene product and would be expected to be associated with a recessive mutation. A second class of insertional mutation is well documented in which upon integration the promoter/enhancer activities inherent in the retroposon genome exert their influence on neighboring genes. This promoter/enhancer activity of integrated retroposons may have effects over relatively long distances and thus limit the possibilities of establishing an association between retroposon integration and mutation. It is emphasized that a systematic search for insertional mutations in the mammalian genome involves an extensive two-dimensional array of possible retroposon sequences and mutant alleles. Present results represent only a small portion of the total array. Future studies promise to be fruitful in efforts to isolate genes through insertional tagging, to characterize the mechanisms of retroposon transposition, as well as to study the stability of the mammalian genome.

Animals↗
PubMed1992

Analysis of the bovine viral diarrhea virus genome for possible cellular insertions.

Mucosal disease is the most severe disease resulting from bovine viral diarrhea virus (BVDV) infection in cattle. Two biotypes of BVDV may be isolated from animals with mucosal disease: cytopathic (cp) and noncytopathic (ncp). These "pairs" of cp/ncp viruses are often closely related and it has been suggested that the cp virus arises from a ncp virus by insertion of cellular RNA in the p125 region of the BVDV genome. We have used four pairs of cp/ncp BVDV isolated from cattle with mucosal disease, to examine the genomic sequence of the region of the genome coding for the nonstructural protein p125 (processed to p54/p80 in cp viruses) by PCR analysis and sequencing. We did not detect any cellular gene insertions in any of the four ncp viruses; however, we found a large duplication of the p80 gene and a ubiquitin gene insertion in three of the four cp isolates. Our results suggest that cellular RNA insertions in the p125 region may contribute significantly to the cytopathogenicity of BVDV. However, this does not appear to be the only mechanism of cytopathogenicity as we did not detect any insertions or duplications in one of the cp viruses. Comparison of the DNA sequence in the p80 region revealed greater homology within the "pairs" than to NADL, which lend further support to the hypothesis that a cp virus is originated from a ncp virus.

Amino Acid Sequence↗
PubMed1992

Genomic structure of the locus associated with an insertional mutation in line 4 transgenic mice.

In line 4 transgenic mice, six to eight copies of a 50-kb lambda recombinant clone are arranged in a head-to-tail tandem array on chromosome 3. Embryos homozygous for the transgene become arrested in their development on Day 5 of gestation shortly after implantation. The insertion site was cloned using a small segment of the transgene as a probe. Comparison of the insertion site with the wildtype locus indicates that a 22-kb deletion of host DNA has occurred in line 4 mice. Restriction enzyme analyses showed that neither the tandem array nor the flanking chromosomal DNA had any detectable rearrangements. Sequencing of the junctions between host and foreign DNA, however, revealed insertions of small fragments of DNA of unknown origin as well as an insertion of a DNA segment derived from another region of the transgene. Therefore, disruption of an essential gene in the line 4 transgenic mouse may have been caused by the insertion of 300-400 kb of foreign DNA or a deletion of sequences in the host genome.

Animals↗
PubMed1992

Interactions between intrauterine contraceptive device use and breast-feeding status at time of intrauterine contraceptive device insertion: analysis of TCu-380A acceptors in developing countries.

OBJECTIVE: This paper is a reassessment of earlier findings from a preliminary analysis of data from a multicenter international trial regimen on breast-feeding and non-breast-feeding women in which events related to insertion, expulsion, and removal of the TCu-380A intrauterine contraceptive device (ParaGard 380) were investigated. STUDY DESIGN: Performance of the TCu-380A through 12 months after insertion was compared with life-table rate analysis, chi 2, Fisher exact test, or Student's t test. Variables were events reported during intrauterine contraceptive device insertion and events throughout the 12 months of study participation by breast-feeding status. RESULTS: Breast-feeding among intrauterine contraceptive device users was associated with fewer insertion-related complaints and lower removal rates for bleeding and pain. No uterine perforations were reported throughout the study. CONCLUSION: Differences in the performance of the TCu-380A intrauterine contraceptive device suggest physiologic effects associated with lactational amenorrhea. The TCu-380A intrauterine contraceptive device is a viable option for women breast-feeding at the time of intrauterine contraceptive device insertion.

Adult↗